Sourcebook of Models for Biomedical Research

SOURCEBOOK OF MODELS FOR BIOMEDICAL RESEARCH

SOURCEBOOK OF

MODELS FOR BIOMEDICAL RESEARCH EDITED BY

P. MICHAEL CONN,

PhD

ASSOCIATE DIRECTOR AND SENIOR SCIENTIST, OREGON NATIONAL PRIMATE RESEARCH CENTER, BEAVERTON, OR; PROFESSOR OF PHYSIOLOGY AND PHARMACOLOGY, PROFESSOR OF CELL AND DEVELOPMENTAL BIOLOGY, OREGON HEALTH AND SCIENCE UNIVERSITY, PORTLAND, OR

© 2008 Humana Press Inc., a part of Springer Science+Business Media, LLC 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 humanapress.com All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. The content and opinions expressed in this book are the sole work of the authors and editors, who have warranted due diligence in the creation and issuance of their work. The publisher, editors, and authors are not responsible for errors or omissions or for any consequences arising form the information or opinions presented in this book and make no warranty, express or implied, with respect to its contents. This publication is printed on acid-free paper. ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials. Cover Illustration: We are indebted to Mr. Joel Ito, Medical Illustrator at the Oregon National Primate Research Center, Oregon Health & Sciences University for the design of this image that combines many of the elements of research models described in this book. For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel.: 973-256-1699; Fax: 973-256-8341; or visit our Website: www.humanapress.com Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $30.00 per copy is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license form the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [978-1-58829-933-8/08 $30.00]. 10 9 8 7 6 5 4 3 2 1 e-ISBN: 978-1-59745-285-4 Library of Congress Control Number: 2007926774

Preface

The collection of systems represented in Sourcebook of Models for Biomedical Research is an effort to reflect the diversity and utility of models that are used in biomedicine. That utility is based on the consideration that observations made in particular organisms will provide insight into the workings of other, more complex, systems. Even the cell cycle in the simple yeast cell has similarities to that in humans and regulation with similar proteins occurs. Some models have the advantage that the reproductive, mitotic, development or aging cycles are rapid compared with those in humans; others are utilized because individual proteins may be studied in an advantageous way and that have human homologs. Other organisms are facile to grow in laboratory settings or lend themselves to convenient analyses, have defined genomes or present especially good human models of human or animal disease. We have made an effort not to be seduced into making the entire book homage to the remarkable success of the

genomic programs, although this work is certainly well represented and indexed. Some models have been omitted due to page limitations and we have encouraged the authors to use tables and figures to make comparisons of models so that observations not available in primary publications can become useful to the reader. We thank Richard Lansing and the staff at Humana for guidance through the publication process. As this book was entering production, we learned of the loss of Tom Lanigan, Sr. Tom was a leader and innovator in scientific publishing and a good friend and colleague to all in the exploratory enterprise. We dedicate this book to his memory. We will miss him greatly.

P. Michael Conn

Contents

PREFACE CONTRIBUTORS LIST OF COLOR PLATES

I

INTRODUCTION

1

Animal Models for Human Diseases: An Overview

10

V XI

Selection of Biomedical Animal Models

XV

11

Improved Models for Animal Research

12

9 17

Rolf Sprengel, Leonid Eshkind, Jan Hengstler, and Ernesto Bockamp

II 4

13 27

14

Bibliographic Searching Tools on Disease Models to Locate Alternatives for Animals in Research: A Website Companion 35 NIH Policies on Sharing of Model Organisms and Related Research Resources

15

Zebrafish as a Model for Development

103

Zebrafish as a Model for Studying Adult Effects of Challenges to the Embryonic Nervous System

113

Modeling Cognitive and Neurodegenerative Disorders in Drosophila melanogaster

121

Junjiro Horiuchi and Minoru Saitoe

43

J.P. Kim, Charlene Cho, and Norka Ruiz Bravo

7

91

Nicole Marie Roy and Elwood A. Linney

Mary W. Wood and Lynette A. Hart

6

Caenorhabditis elegans Models of Human Neurodegenerative Diseases: A Powerful Tool to Identify Molecular Mechanisms and Novel Therapeutic Targets

Brian A. Link and Sean G. Megason

James Parker

5

85

Richard Nass and Lihsia Chen

GENERAL CONSIDERATIONS The Ethical Basis for Animal Use in Research

Sea Urchin Embryo: A Model System for Analyzing Cellular Activities During Early Development Tetsuya Kominami and Hiromi Takata

3

Michael S. Rand

3

75

Xavier Fernàndez-Busquets

Jann Hau

2

The Sponge as a Model of Cellular Recognition

16

Databases for Biomedical Animal Resources 49

Biomedical Research with Honey Bees

129

Michelle M. Elekonich

Richard L. Crawford and Tim Allen

8

Psychological Enrichment for Animals in Captivity

17 55

WELL-ESTABLISHED MODELS

18

A 9

YEAST, WORMS, FLIES, SEA ANIMALS, AND BIRDS Integrated Network Modeling of Molecular and Genetic Interactions

139

Punita Koustubhan, Debra Sorocco, and Michael S. Levin

Kristine Coleman

III

Establishing and Maintaining a Xenopus laevis Colony for Research Laboratories

The Chicken as a Model Organism

161

Janice M. Bahr

19

67

B RODENTS Rat Knockout and Mutant Models Bart M.G. Smits, Jill D. Haag, Michael N. Gould, and Edwin Cuppen

Gregory W. Carter, Vesteinn Thorsson, and Timothy Galitski vii

171

viii

20

CONTENTS

Rodent Genetics, Models, and Genotyping Methods

34 179

Sheryl J. Wildt, Andrew I. Brooks, and Robert J. Russell

21

The House Mouse in Biomedical Research Mouse Model for Alzheimer’s Disease

35 187

Guinea Pigs as Models for Human Cholesterol and Lipoprotein Metabolism

Transgenic Animal Models of Neurodegenerative Diseases

323

Anthony W.S. Chan and Yuksel Agca

191

36

M. Azhar Chishti and Shaheen M. Nakeeb

23

313

Emma L. Lane, Thomas Carlsson, Deniz Kirik, and Stephen B. Dunnett

John G. Vandenbergh

22

Animal Models of Parkinson’s Disease

Animal Models of Nociception and Pain

333

James D. Rose and C. Jeffrey Woodbury

37 201

Nonmammalian Models for the Study of Pain

341

Craig W. Stevens

Maria Luz Fernandez and Richard J. Wood

24

Reliability of Rodent Models

213

Dileep Kumar Rohra and Yureeda Qazi

25

C CATS, DOGS, AND PIGS The Domestic Cat, Felis catus, as a Model of Hereditary and Infectious Disease

38

Swine in Biomedical Research

39

27

The Minipig as an Animal Model in Biomedical Stem Cell Research

40 241

Petr Vodicka, Jana Hlucilova, Jiri Klima, Radek Prochazka, Jitka Ourednik, Vaclav Ourednik, and Jan Motlik

28

D NONHUMAN PRIMATES The Nonhuman Primate as a Model for Biomedical Research Primates as Models of Behavior in Biomedical Research Primate Models for Understanding Brain Mechanisms of Cognitive Behavior

251 42 259

267

369

Transgenic Mouse Models of HIV-1/AIDS and Cardiac Performance

385

B REPRODUCTION Primate Models for the Assisted Reproductive Technologies and Embryonic Stem Cell Biology

397

43

Rat Models of Polycystic Ovary Syndrome

44

Murine Models for Reproduction

45

Pig Model to Study Dynamics of Steroids During Ovarian Follicular Growth and Maturation

405

Krishna B. Singh

411

Damayanti Chakraborty and T. Rajendra Kumar

IV MODELS FOR SPECIFIC PURPOSES A VISUAL AND AUDITORY DISEASE Animal Models for Eye Diseases and Therapeutics

Animal Models for Atherosclerosis, Restenosis, and Endovascular Aneurysm Repair

Don P. Wolf, Shoukhrat Mitalipov, and James A. Byrne

Ronald E. Kettner, M. Lysetskiy, and M. Suh

31

361

James J. Kohler and William Lewis

Steven J. Schapiro

30

Models of Behavior: Cardiovascular

András Kónya, Kenneth C. Wright, Matthew Gounis, and Krishna Kandarpa

41

David M. Anderson

29

355

Lina Badimon, Laura Casaní, and Gemma Vilahur

233

M. Michael Swindle and Alison C. Smith

A CARDIOVASCULAR Animal Models of Vascular Development and Endothelial Cell Biology Victoria L. Bautch

221

Marilyn Menotti-Raymond and Stephen J. O’Brien

26

V MODELS OF BEHAVIOR

279

425

Damodar K. Mahajan

Takeshi Iwata and Stanislav Tomarev

32

Animal Models of Noise-Induced Hearing Loss

289

Richard Salvi and Flint A. Boettcher

33

B TRAUMA, PAIN, AND NEUROLOGY Human and Animal Models for the Study of Muscle Pain Norman F. Capra and Jin Y. Ro

305

46

C DRUG DEVELOPMENT AND RESEARCH MODELS Molecular Genetic Approach to Identify Inhibitors of Signal Transduction Pathways: Fission Yeast as a Model System for Drug Discovery 439 Shunji Ishiwata, Takayoshi Kuno, Hirofumi Takada, Atsushi Koike, and Reiko Sugiura

CONTENTS

47

Yeast as a Model System to Study DNA Damage and DNA Repair

61 445

Antonio Conconi

48

D PHYSIOLOGY Human Models of Space Physiology Developmental Space Biology of Mammals: Concepts and Methods of Study

457

A Practical Approach to Animal Models of Sepsis

465

62

Animal Models in Functional Magnetic Resonance Imaging

53

Animal Models in Aging Research: A Critical Examination

483

499 66

E GENETICS Gene Targeting in Human Somatic Cells

67

Genetic Models of Alzheimer’s Disease

56

509 68

Animal Models in Virology

527

69

Nonhuman Primate Models for AIDS

539

70 71

547

72

557 565

Animal Models of Sudden Infant Death Syndrome Jane Blood-Siegfried

Animal Models of Diabetes

651

Animal Models of Kidney Diseases

657

Animal Models of Multiple Sclerosis

665

Canine and Feline Models for Cancer

677

Obese Mouse Models

683

Study of Polycystic Kidney Disease in the Nematode Caenorhabditis elegans

703

73

Animal Models of Myelofibrosis

713

Alessandro M. Vannucchi, Jean-Luc Villeval, Orianne Wagner-Ballon, Paola Guglielmelli, and Anna Rita Migliaccio

Animal Models for Bone Tissue Engineering Purposes

725

Véronique Viateau, Delphine Logeart-Avramoglou, Geneviève Guillemin, and Hervé Petite

575

VII

Ute Christine Rogner and Philip Avner

60

639

Matthew J. Buechner

MODELS OF OTHER HUMAN DISEASES Use of Congenic Mouse Strains for Candidate Disease Gene Identification in Complex Traits

Animal Models of Prostate Cancer

Deborah J. Good

74

59

631

Gionata De Vico and Paola Maiolino

Nancy L. Haigwood, Raymond R. O’Neill, and Vanessa M. Hirsch

VI

Rat Models of Skin Wound Healing

Colin R. Young and C. Jane Welsh

Joseph L. Bryant

58

623

Chunsun Dai, Lawrence P. Kiss, and Youhua Liu

Erik Wallström and Tomas Olsson

57

Mouse Models for Experimental Cancer Therapy

Willy J. Malaisse and Abdullah Sener

Yann S. Mineur

F IMMUNOLOGY AND VIROLOGY Rat Models of Experimental Autoimmune Encephalomyelitis

615

Adebusola A. Alagbala and Barbara A. Foster

John B. Mailhes and Mary Ann Handel

55

Modeling Drug and Alcohol Abuse: Experimental Examples of the Utility of Zebrafish

Wanda A. Dorsett-Martin and Annette B. Wysocki

Rochelle Buffenstein, Yael H. Edrey, and Pamela L. Larsen

Animal Models for Investigating the Causes and Mechanisms of Mammalian Germ Cell Aneuploidy

603

Sara Rockwell and Karen R. Rockwell

65

Eric A. Hendrickson

54

Animal Models for Studying Fetal Alcohol Syndrome, Alcohol-Related Birth Defects, and Alcohol-Related Neurodevelopmental Disorder

Robert T. Gerlai

64

Afonso C. Silva and Bojana Stefanovic

52

63

473

Bryan Belikoff and Jon A. Buras

51

591

Timothy A. Cudd

April E. Ronca

50

Animal Models of Posttraumatic Stress Disorder Hagit Cohen, Michael A. Matar, and Joseph Zohar

Victor A. Convertino and Caroline A. Rickards

49

ix

75 583

OTHER TOOLS Markov Processes for Biomedical Data Analysis Ricardo Ocaña-Riola

739

x

CONTENTS

76

Software Tools for Modeling Biomedical Systems

78 747

Karl Thomaseth

77

Developing Websites for Biomedical Research and Training Krishna B. Singh

Building Virtual Research Communities Using Web Technology

759

Krishna B. Singh

753

Index

763

Contributors

JOSEPH L. BRYANT, DVM, Animal Models Division, University of Maryland Biotechnology Institute, The Institute of Human Virology, Baltimore, MD MATTHEW J. BUECHNER, PhD, Department of Molecular Biosciences, University of Kansas, Lawrence, KS ROCHELLE BUFFENSTEIN, Department of Biology, City College of New York, The City University of New York, New York, NY JON A. BURAS, MD, PhD, New England Inflammation and Tissue Protection Institute Consortium at Northeastern University; and Departments of Biology and Pharmaceutical Sciences, Boston, MA JAMES A. BYRNE, PhD, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR NORMAN F. CAPRA, PhD, Department of Biomedical Sciences, School of Dentistry, University of Maryland Baltimore, Baltimore, MD THOMAS CARLSSON, PhD student, Brain Repair Group, School of Biosciences, Cardiff University, Cardiff, UK GREGORY W. CARTER, PhD, Institute for Systems Biology, Seattle, WA LAURA CASANÍ, DVM, PhD, Centro de Investigacion Cardiovascular, CSIC-Institut Catala de Ciences Cardiovasculars, Hospital de la Santa Creu I Sant Pau, Barcelona, Spain DAMAYANTI CHAKRABORTY, BS, Integrated Graduate Program in Biomedical Sciences, University of Kansas Medical Center, Kansas, City, KS ANTHONY W.S. CHAN, DVM, PhD, Division of Neuroscience, Yerkes National Primate Research Center, Department of Human Genetics, Emory University School of Medicine, Atlanta, GA LIHSIA CHEN, PhD, Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN M. AZHAR CHISHTI, DVM, PhD, University of Toronto, Mississauga, ONT, Canada CHARLENE CHO, PhD, Office of Extramural Research, National Institutes of Health, Bethesda, MD HAGIT COHEN, PhD, Ministry of Health Mental Health Center, Anxiety and Stress Research Unit, Faculty of Health Sciences, Gen-Gurion University of the Negev, Beer-Sheva, Israel KRISTINE COLEMAN, PhD, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR

YUKSEL AGCA, DVM, PhD, College of Veterinary Medicine, Comparative Medicine Center, University of MissouriColumbia, Columbia, MO ADEBUSOLA A. ALAGBALA, BS, Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY TIM ALLEN, MS, US Department of Agriculture, National Agricultural Library, Animal Welfare Information Center, Beltsville, MD DAVID M. ANDERSON, DVM, Washington National Primate Research Center, Department of Comparative Medicine, University of Washington, Seattle, WA PHILIP AVNER, PhD, Departement de Biologie du Développement, Unite de Genetique Moleculaire Murine, Institut Pasteur, Paris, France LINA BADIMON, PhD, FAHA, FESC, Centro de Investigacion Cardiovascular, CSIC-Institut Catala de Ciencies Cardiovasculars, Hosptial de la Santa Creu i Sant Pau, Barcelona, Cardiovascular Research Center, Pavello del Convent, Barcelona, Spain JANICE M. BAHR, PhD, Department of Animal Sciences, University of Illinois, Urbana, IL VICTORIA L. BAUTCH, PhD, Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC BRYAN BELIKOFF, BS, MA, MS, New England Inflammation and Tissue Protection Institute Consortium at Northeastern University; and Departments of Biology and Pharmaceutical Sciences, Boston, MA JANE BLOOD-SIEGFRIED, DNSc, CPNP, Department of Nursing, Duke University School of Nursing, Durham, NC ERNESTO BOCKAMP, Dr. rer.nat., Institute of Toxicology/Mouse Genetics, Johannes Gutenberg-Universitat Mainz, Mainz, Germany FLINT A. BOETTCHER, PhD, Department of Communicative Sciences and Disorders, University of South Alabama, Mobile, AL NORKA RUIZ BRAVO, PhD, Deputy Director for Extramural Research, National Institutes of Health, Department of Health and Human Services, Bethesda, MD ANDREW I. BROOKS, PhD, Bionomics and Research Technology Center, Environmental and Occupational Health Science Institute, Piscataway, NJ

xi

xii

CONTRIBUTORS

ANTONIO CONCONI, PhD, Departement de Microbiologie et d’Infectiologie, Faculte de Medecine, Universite de Sherbrooke, Sherbrooke, Québec, Canada P. MICHAEL CONN, PhD, Oregon National Primate Research Center, Beaverton, OR; Oregon Health and Science University, Portland, OR VICTOR A. CONVERTINO, PhD, US Army Institute of Surgical Research, Fort Sam Houston, TX RICHARD L. CRAWFORD, DVM, US Department of Agriculture, National Agricultural Library, Animal Welfare Information Center, Beltsville, MD TIMOTHY A. CUDD, DVM, PhD, Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, The Texas A&M University System Health Science Center, College Station, TX EDWIN CUPPEN, PhD, Hubrecht Laboratory, Utrecht, The Netherlands CHUNSUN DAI, MD, PhD, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA GIONATA DE VICO, DVM, Faculty of Mathematics, Physics, and Natural Sciences, Department of Biological Sciences, Naples, Italy WANDA A. DORSETT-MARTIN, DVM, Division of Plastic Surgery, Department of Surgery, The University of Mississippi Medical Center, School of Medicine, Jackson, MS STEPHEN B. DUNNETT, DSc, Brain Repair Group, School of Biosciences, Cardiff University, Cardiff, UK YAEL H. EDREY, Department of Biology, City College of New York, The City University of New York, New York, NY MICHELLE M. ELEKONICH, PhD, School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV LEONID ESHKIND, PhD, Institute of Toxicology/Mouse Genetics, Johannes Gutenberg-Universitat Mainz, Mainz, Germany MARIA LUZ FERNANDEZ, PhD, Department of Nutritional Sciences, University of Connecticut, Storrs, CT XAVIER FERNÀNDEZ-BUSQUETS, PhD, Bioengineering Institute of Catalonia and Nanoscience and Nanotechnology Institute, Barcelona Science Park, University of Barcelona, Barcelona, Spain BARBARA A. FOSTER, PhD, Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY TIMOTHY GALITSKI, PhD, Institute for Systems Biology, Seattle, WA ROBERT T. GERLAI, PhD, Department of Psychology, University of Toronto at Mississauga, Mississauga, Ontario, Canada DEBORAH J. GOOD, PhD, Department of Human Nutrition, Foods and Exercise, Institute for Biomedical and Public Health Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA MICHAEL N. GOULD, PhD, McArdle Lab for Cancer Research, University of Wisconsin-Madison, Madison, WI MATTHEW GOUNIS, PhD, The University of Massachusetts, New England Center for Stroke Research, Boston, MA PAOLA GUGLIELMELLI, MD, Department of Hematology, University of Florence, Florence, Italy GENEVIÈVE GUILLEMIN, PhD, Laboratoire de Recherches Orthopediques, Université Denis Diderot, Paris, France JILL D. HAAG, MS, McArdle Lab for Cancer Research, University of Wisconsin-Madison, Madison, WI

NANCY L. HAIGWOOD, PhD, Viral Vaccines Program, Seattle Biomedical Research Institute, Seattle, WA MARY ANN HANDEL, PhD, The Jackson Laboratory, Bar Harbor, ME LYNETTE A. HART, PhD, UC Center for Animal Alternatives, School of Veterinary Medicine, University of California at Davis, Davis, CA JANN HAU, MD, Chair of Comparative Medicine, Head of Department of Experimental Medicine, University of Copenhagen and Rigshospitalet, The Panum Institute, Copenhagen, N Denmark ERIC A. HENDRICKSON, PhD, Cancer Center Research Program, Genetic Mechanisms of Cancer, Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Medical School, Minneapolis, MN JAN HENGSTLER, MD, Center for Toxicology, Institute of Legal Medicine and Rudolf-Boehm Institute of Pharmacology and Toxicology, Leipzig, Germany VANESSA M. HIRSCH, DVM, DSc, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD JANA HLUCILOVA, DVM, Institute of Animal Physiology and Genetics, The Academy of Sciences of the Czech Republic, Libechov, Czech Republic; and Center for Cell Therapy and Tissue Repair, Charles University, Praha, Czech Republic JUNJIRO HORIUCHI, PhD, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan SHUNJI ISHIWATA, Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Higashi-Osaka, Japan TAKESHI IWATA, PhD, National Institute of Sensory Organs, National Hospital Organzation Tokyo Medical Center, Tokyo, Japan KRISHNA KANDARPA, MD, PhD, Radiologist-in-Chief, The University of Massachusetts Memorial Medical Center, Professor and Chairman, Department of Radiology, The University of Massachusetts, Worcester, MA RONALD E. KETTNER, PhD, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL J.P. KIM, JD, MBA, MSc, MALS, Office of Extramural Research, National Institutes of Health, Bethesda, MD DENIZ KIRIK, MD, PhD, CNS Disease Modeling Unit, Department of Experimental Medical Science, Lund University, Lund, Sweden LAWRENCE P. KISS, MD, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA JIRI KLIMA, PhD, Institute of Animal Physiology and Genetics, The Academy of Sciences of the Czech Republic, Libechov, Czech Republic; and Center for Cell Therapy and Tissue Repair, Charles University, Praha, Czech Republic JAMES J. KOHLER, PhD, Department of Pathology, Emory University School of Medicine, Atlanta, GA ATSUSHI KOIKE, Division of Molecular Pharmacology and Pharmacogenomics, Department of Genome Sciences, Kobe University Graduate School of Medicine, Kobe, Japan TETSUYA KOMINAMI, DSc, Department of Biology, Faculty of Science, Ehime University, Matsuyama, Japan ANDRÁS KÓNYA, MD, PhD, M.D. Anderson Cancer Center, Division of Diagnostic Imaging, Department of Diagnostic Radiology, Section of Interventional Radiology, University of Texas, Houston, TX

CONTRIBUTORS

PUNITA KOUSTUBHAN, BS, The Forsyth Center for Regenerative and Developmental Biology and Harvard School of Dental Medicine, Boston, MA T. RAJENDRA KUMAR, PhD, Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS TAKAYOSHI KUNO, Division of Molecular Pharmacology and Pharmacogenomics, Department of Genome Sciences, Kobe University Graduate School of Medicine, Kobe, Japan EMMA L. LANE, PhD, Brain Repair Group, School of Biosciences, Cardiff University, Cardiff, UK PAMELA L. LARSEN, PhD, Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX MICHAEL S. LEVIN, PhD, The Forsyth Center for Regenerative and Developmental Biology and Harvard School of Dental Medicine, Boston, MA WILLIAM LEWIS, MD, Department of Pathology, Emory University School of Medicine, Atlanta, GA BRIAN A. LINK, PhD, Departments of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI ELWOOD A. LINNEY, PhD, Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC YOUHUA LIU, PhD, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA DELPHINE LOGEART-AVRAMOGLOU, PhD, Laboratoire de Recherches Orthopediques, Université Denis Diderot, Paris, France M. LYSETSKIY, PhD, Department of Physiology and Interdepartmental Neuroscinece Program, Feinberg School of Medicine, Northwestern University, Chicago, IL DAMODAR K. MAHAJAN, MSc, PhD, Department of Obstetrics and Gynecology, Louisiana State University Health Sciences Center, Shreveport, LA JOHN B. MAILHES, PhD, Department of Obstetrics and Gynecology, Louisiana State University Health Sciences Center, Shreveport, LA PAOLA MAIOLINO, DVM, Associate Professor, Department of Pathology and Animal Health, Faculty of Veterinary Medicine, Naples University Federico II, Naples, Italy WILLY J. MALAISSE, MD, PhD, Laboratory of Experimental Hormonology, Brussels Free University (CP 626), Brussels, Belgium MICHAEL A. MATAR, MD, Ministry of Health Mental Health Center, Anxiety and Stress Research Unite, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel SEAN G. MEGASON, PhD, Biological Imaging Center, Beckman Institute, California Institute of Technology, Pasadena, CA MARILYN MENOTTI-RAYMOND, PhD, Laboratory of Genomic Diversity, National Cancer Institute-Frederick, Frederick, MD ANNA RITA MIGLIACCIO, PhD, Department of Pathology, University of Illinois at Chicago, Chicago, IL; and Department of Hematology, Oncology and Molecular Medicine, Instituto Superiore di Sanita, Rome, Italy YANN S. MINEUR, PhD, Departments of Psychiatry, Neurology and Pharmacology, Yale School of Medicine, New Haven, CT

xiii

SHOUKHRAT MITALIPOV, PhD, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR JAN MOTLIK, DVM, PhD, DSc, Institute of Animal Physiology and Genetics, The Academy of Sciences of the Czech Republic, Libechov, Czech Republic; and Center for Cell Therapy and Tissue Repair, Charles University, Praha, Czech Republic SHAHEEN M. NAKEEB, DVM, PhD, Carmel, IN RICHARD NASS, PhD, Department of Pediatrics and Pharmacology, Vanderbilt University Medical Center, Nashville, TN STEPHEN J. O’BRIEN, PhD, National Cancer Institute-Frederick, Fort Detrick, Frederick, MD RAYMOND R. O’NEILL, PhD, Division of Comparative Medicine, National Center for Research Resources, National Institutes of Health, Bethesda, MD RICARDO OCAÑA-RIOLA, PhD, Department of Statistics, Escuela Andaluza de Salud Publica, Campus Universitario Cartuja, Granada, Spain TOMAS OLSSON, MD, PhD, Department of Clinical Neuroscience, Neuroimmunology Unit L8:04, Center of Molecular Medicine, Karolinska Hospital, Stockholm, Sweden JITKA OUREDNIK, PhD, College of Veterinary Medicine, Iowa State University, Ames, IA VACLAV OUREDNIK, PhD, College of Veterinary Medicine, Iowa State University, Ames, IA MAIOLINO PAOLA, DVM, Department of Pathology and Animal Health, Faculty of Veterinary Medicine, Naples University, Naples, Italy JAMES PARKER, PhD, Retired, Public Information Officer, Oregon National Primate Research Center, Portland, OR HERVÉ PETITE, PhD, Laboratoire de Recherches Orthopediques, Faculte de Medecine Lariboisiere Saint-Louis, Universite Denis Diderot Paris 7, Paris, France RADEK PROCHAZKA, DVM, PhD, College of Veterinary Medicine, Iowa State University, Ames, IA YUREEDA QAZI, MBBS, Department of Biological and Biomedical Sciences, The Aga Khan University, Karachi, Pakistan MICHAEL S. RAND, DVM, DACLAM, Chief, Biotechnology Support Services, Department of Animal Care, University of Arizona, Tucson, AZ CAROLINE A. RICKARDS, PhD, US Army Institute of Surgical Research, Fort Sam Houston, TX JIN Y. RO, PhD, Department of Biomedical Sciences, Baltimore College of Dental Surgery, University of Maryland Baltimore, Baltimore, MD SARA ROCKWELL, PhD, Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT KAREN R. ROCKWELL, PhD, Adj. Assistant Professor, Center for Pharmaceutical Biotechnology and Nanomedicine, School of Pharmacy, Northeastern University, Bouvé College of Health Sciences, Boston, MA UTE CHRISTINE ROGNER, PhD, Departement de Biologie du Développement, Unite de Genetique Moleculaire Murine, Institut Pasteur, Paris Cedex 15, France DILEEP KUMAR ROHRA, MBBS, MPhil, PhD, Department of Biological and Biomedical Sciences, The Aga Khan University, Karachi, Pakistan APRIL E. RONCA, PhD, Departments of Obstetrics and Gynecology and Neurobiology and Anatomy, Wake Forest University School of Medicine, Winston-Salem, NC

xiv

CONTRIBUTORS

JAMES D. ROSE, PhD, Department of Zoology and Physiology, University of Wyoming, Laramie, WY NICOLE MARIE ROY, PhD, Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC ROBERT J. RUSSELL, DVM, Director, Laboratory of Animal Medicine, Harlan Genetic Testing Services, Indianapolis, IN MINORU SAITOE, PhD, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan RICHARD SALVI, PhD, Center for Hearing and Deafness, University at Buffalo, Buffalo, NY STEVEN J. SCHAPIRO, PhD, The Michale E. Keeling Center for Comparative Medicine and Research, Department of Veterinary Sciences, The University of Texas M.D. Anderson Cancer Center, Bastrop, TX ABDULLAH SENER, PhD, Laboratory of Experimental Hormonology, Brussels Free University, Brussels, Belgium AFONSO C. SILVA, PhD, Cerebral Microcirculation Unit, Laboratory of Functional and Molecular Imaging, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD KRISHNA B. SINGH, MD, Department of Obstetrics and Gynecology, LSU Health Sciences Center, Shreveport, LA ALISON C. SMITH, DVM, Department of Comparative Medicine, Medical University of South Carolina, Charleston, SC BART M.G. SMITS, PhD, McArdle Lab for Cancer Research, University of Wisconsin-Madison, Madison, WI DEBRA SOROCCO, ALM, Department of Cytokine Biology, The Forsyth Institute, Boston, MA ROLF SPRENGEL, Dr. rer.nat., Max Planck Institute for Medical Research, Heidelberg, Germany CRAIG W. STEVENS, PhD, Professor of Pharmacology, Director of the Analgesia Research Laboratory, Oklahoma State University-Center for Health Sciences, College of Osteopathic Medicine, Tulsa, OK BOJANA STEFANOVIC, PhD, Cerebral Microcirculation Unit, Laboratory of Functional and Molecular Imaging, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD REIKO SUGIURA, Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University Higashi-Osaka, Japan; and Division of Molecular Pharmacology and Pharmacogenomics, Department of Genome Sciences, Kobe University Graduate School of Medicine, Kobe, Japan M. SUH, PhD, Department of Neurological Surgery, Weill Medical College of Cornell University, New Yorok, NY M. MICHAEL SWINDLE, DVM, Department of Comparative Medicine, Thurmond/Gazes Research, Medical University of South Carolina, Charleston, SC HIROFUMI TAKADA, Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Higashi-Osaka, Japan HIROMI TAKATA, DSc, Department of Biology, Faculty of Science, Ehime University, Matsuyama, Japan VESTEINN THORSSON, PhD, Institute for Systems Biology, Seattle, WA

STANISLAV TOMAREV, PhD, National Eye Institute, National Institutes of Health, Bethesda, MD KARL THOMASETH, PhD, National Research Council, Institute of Biomedical Engineering ISIB-CNR, Padova, Italy JOHN G. VANDENBERGH, PhD, Professor, Zoology Department, North Carolina State University, Raleigh, NC ALESSANDRO M. VANNUCCHI, MD, Division of Hematology, University of Florence, Florence, Italy VÉRONIQUE VIATEAU, DMV, PhD, Unité Pédagogique de Pathologie Chirurgicale, Ecole Nationale Veterinaire d’Alfort, Maisons Alfort, France GEMMA VILAHUR, DVM, PhD, Centro de Investigacion Cardiovascular, CSIC-Institut Catala de Ciences Cardiovasculares, Hospital de la Santa Creu I Sant Pau, Barcelona, Spain JEAN-LUC VILLEVAL, PhD, INSERM, Universite Paris, Institut Gustave Roussy, Villejuif, France PETR VODICKA, MSc, Department of Reproductive and Developmental Biology, Institute of Animal Physiology and Genetics, The Academy of Sciences of The Czech Republic, Libechov, Czech Republic ORIANNE WAGNER-BALLON, MD, INSERM, Universite Paris, Institut Gustave Roussy, Villejuif, France; AP-HP, Laboratoire d’Hematologie, Hopital Henri Mondor, Creteil, France ERIK WALLSTRÖM, Department of Neurology, Karolinska University Hospital, Karolinska Institute, Stockholm, Sweden C. JANE WELSH, PhD, Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX SHERYL J. WILDT, MS, Laboratory of Animal Medicine, Harlan Genetic Testing Services, Indianapolis, IN DON P. WOLF, PhD, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR MARY W. WOOD, MLS, UC Center for Animal Alternatives, Carlson Health Sciences Library, University of California at Davis, Davis, CA RICHARD J. WOOD, PhD, Department of Nutritional Sciences, University of Connecticut, Storrs, CT C. JEFFREY WOODBURY, PhD, Department of Zoology and Physiology and Neuroscience Program, University of Wyoming, Laramie, WY KENNETH C. WRIGHT, PhD, M.D. Anderson Cancer Center, Division of Diagnostic Imaging, Department of Diagnostic Radiology, Section of Interventional Radiology, University of Texas, Houston, TX ANNETTE B. WYSOCKI, PhD, Division of Plastic Surgery, Department of Surgery, The University of Mississippi Medical Center, School of Medicine, Jackson, MS COLIN R. YOUNG, PhD, Departments of Veterinary Integrative Biosciences and Psychology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX JOSEPH ZOHAR, MD, Department of Psychiatry, Chaim Sheba Medical Center, Sackler Medical School, Tel-Aviv University, Tel-Hashomer, Israel

List of Color Plates

Figure 51–3. Changes in the CBV response to amphetamine (A) before and (B) 4 months after cessation of MPTP treatment in a cynomolgus monkey, showing an almost complete loss of amphetamine-induced CBV signal changes in dopaminergic regions. Parkinsonian primates had a prominent loss of response to amphetamine, with relative sparing of the nucleus accumbens and parafascicular thalamus. (Modified from Jenkins et al.183 Copyright 2004 Society for Neurosciences.)

Color plates to follow page 240. Figure 8–1. Example of enrichment items for laboratory mice. The hut is tinted such that the mouse cannot see out, but human caretakers can see in. (Photo by Jill Rawlins.) Figure 13–4. Imaging in live zebrafish embryos. (A) Whole embryos can be quickly imaged on a fluorescent dissecting microscope in their chorions to sort positive transgenics (left) from wild-type siblings (right). These embryos were not treated with PTU so the melanocytes are visible. (B–E) Confocal microscopy permits much higher resolution imaging. (B) A quick method for labeling is to inject RNA encoding fluorescent proteins, in this case a histone2B-EGFP fusion, and a membrane localized mCherry was used to image all the cells of the inner ear. (C) GFP transgenics can be used to image neuronal projections from the trigeminal ganglion as they extend. (D) GFP transgenics can mark specific populations of cells, in this case rhombomeres 3 and 5. (E) GFP fusion proteins can reveal the subcellular localization pattern of proteins, in this case a cytoplasmic protein in the Rohon-Beard and motor neurons of the spinal cord. (Images form S.G. Megason, L.A. Trinh, and S.E. Fraser, unpublished.)

Figure 51–4. (A) Patterns of the fMRI response in the mouse main olfactory bulb (MOB) and accessory olfactory bulb (AOB, pink circle in slice 5) to the pheromone 2-heptanone, one of the urinary chemosignal compounds in mouse. The arrows point to two foci of activation suggestive of a pair of the nearly mirror projections of the receptor neuron subsets to the same MOB. Scale bar = 500 µm. (B) A flattened view of the olfactory bulb indicating the orientation of the odor maps shown in (C) and (D): A, anterior; D, dorsal; L, lateral; M, medial; P, posterior, V, ventral. (C, D) The odor maps of 2-heptanone (Hep) in two different mice show that this pheromone not only activates large regions of the MOB but also generates similar patterns across subjects. Interestingly, the odor map for amyl acetate (AA), a common odorant with an odor quality similar to that of 2-heptanone, was also similar to that of 2-heptanoneb. (Adapted from Xu et al.217 Copyright 2005 Wiley-Liss, Inc.)

Figure 13–5. Creating genetic mosaics in zebrafish. (A) Donor cells are first lineage labeled with a tracer dye at the one-cell stage. Donor embryos can also be injected with morpholinos, RNA, or DNA. At the 1000-cell stage, totipotent blastula cells are transplanted into regions of the host embryo fated to give rise to specific structures. Donor and host embryos can be of either mutant or wild-type genotypes. Resultant chimeras are grown for subsequent analysis. (B) Fate-map of the pregastrula stage embryos. (Modified from Woo and Fraser, 1995.) (C) Mosaic embryo at 24 hpf showing bright-field (left) and fluorescent image (right) of rhodamine-dextran-labeled donor cells targeted to the eye and forebrain. (B.A. Link, unpublished.)

Figure 51–5. Signal changes in CBV-weighted fMRI obtained during stimulation of the cat visual cortex according to predetermined stimulus orientations. (A) Raw gray-scale functional map obtained by subtraction of images during 0º stimulation from prestimulus control. The center of each patch is marked with a green + sign. (B) Four different grating orientations (0º, 45º, 90º, and 135º) were presented in the study of one animal, enabling a composite angle map to be generated through pixel-by-pixel vector addition of the four single-condition maps. In the left hemisphere (marked by a white rectangular box), changes between pixels preferentially activated by a particular stimulus orientation were smoothed by a 3 × 3 Gaussian Kernel. (C) A composite angle map was generated with the region indicated by the white ROI in (B) “Pinwheel” structures indicated by small white dots were observed where domains for all orientations converge. (Adapted from Zhao et al.114 Copyright 2005 Elsevier.)

Figure 25–1. Feline chromosome maps (labeled at top) and homologous synteny blocks (HSBs) in the human (H) and dog (D) genomes. HSBs are shown to the right of each cat chromosome map (only the map scale is shown). The dark cross-marks on each cat chromosome correspond to 100-cR5000 intervals. The inferred centromere positions are shown by dark circles. HSBs are color coded by human or dog chromosome, defined by the key in the bottom right corner. (Reprinted from Murphy et al.7 Copyright 2007, with permission from Elsevier.)

Figure 51–6. Auditory activation in the songbird telencephalon shows (A) statistical maps illustrating the localization of

xv

xvi

LIST OF COLOR PLATES

significant signal intensity changes during auditory stimulation consisting of white noise (wn), a concerto of Bach (music), and a stimulation with song from a male starling (song). (B) The location of the activated areas (top), together with the average BOLD response amplitude for each stimulus (bottom). (Adapted from Van Meir et al.231 Copyright 2005 Elsevier.)

Normal control; (B and D) diabetic mice. Arrows indicate injured glomeruli. (E) Albuminuria develops in diabetic mice in a time-dependent manner. Data are presented as means ± SEM. *p < 0.05. (F) Glomerular collagen deposition score in diabetic and normal mice. *p < 0.05. (Adapted and modified from Dai et al.46)

Figure 61–1. The effect of single PSS exposure versus unexposed control on rat anxiety-like behavior and acoustic startle response and habituation. The representation of the data from both paradigms (EPM and ASR) shows two obvious and rather distinct features. First, it is clear that PSS exposure alters the response of the majority of individuals to at least some degree. Second, the cluster of individuals that forms in the upper left hand corner of the graph (i.e., had the more extreme responses to exposure) is quite distinct from the majority of individuals.36,48

Figure 68–3. Interstitial fibrosis in the mouse model of obstructive nephropathy. (A, B) Representative micrographs show the cross sections and gross morphology of the obstructed kidneys at day 14 after ureteral ligation. (A) Sham control; (B) UUO. (C) Western blot analyses demonstrate a marked induction of αsmooth muscle actin (α-SMA), a molecular marker for myofibroblasts, in the obstructed kidney at day 14 after UUO. (D) Double immunofluorescence staining shows the α-SMA (red) and proximal tubular epithelial cell marker, fluorescein isothiocyanate (FITC)-conjugated lectin from Tetragonolobus purpureas (green). (E) Quantitative determination of total kidney collagen contents in sham and obstructed kidneys. Data are presented as means ± SEM. *p < 0.01. (Adapted and modified from Yang et al.59)

Figure 68–2. Diabetic nephropathy induced by streptozotocin (STZ) in uninephrectomized mice. One week after uninephrectomy, male CD1 mice received an intravenous injection of STZ. (A–D) Representative micrographs demonstrate glomerular enlargement, mesangial expansion, and segmental glomerulosclerosis (periodic acid–Schiff staining; A, B) and glomerular collagen deposition (Masson–Trichrome staining; C, D) (A and C)

Figure 73–4. A pathogenetic model for the cytokine-mediated stromal reaction observed in MMM.

INTRODUCTION

I

1

Animal Models for Human Diseases An Overview JANN HAU

ABSTRACT This chapter provides an introduction to the concept of laboratory animal models, focusing on a general classification of animal models for the study of human diseases. Animal models can be grouped into one of the following five categories: (1) induced (experimental) models, (2) spontaneous (genetic, mutant) models, (3) genetically modified models, (4) negative models, and (5) orphan models. This is followed by a discussion of how knowledge concerning human biology and pathobiology can be extrapolation from results obtained from studies of animals. Finally the chapter discusses how the difference in body size and metabolic rate between small laboratory animals and humans has an impact on the calculation of relevant doses for animals used as models for humans in experimental studies. Key Words: Animal model concept, Induced animal model, Spontaneous animal model, Transgenic animal model, Negative animal model, Orphan animal model, Body size—scaling, Extrapolation, Laboratory animal science—definition.

Laboratory animal science may be defined as the study of the scientific, ethical, and legal use of animals in biomedical research, i.e., a multidisciplinary field encompassing comparative biological and pathobiological specialties for the optimal scientific use of animals as models for human or other species. Basic laboratory animal science is concerned with the quality of animals as sentient tools in biomedical research. It encompasses the comparative biology of laboratory animals, aspects of breeding, housing, and husbandry, anesthesia, euthanasia, and experimental techniques. For animal welfare reasons as well as for scientific reasons it is vital that scientists using animal models in their research are competent and have a good knowledge of basic laboratory animal science. This sourcebook provides a thorough introduction to the use of animal models for human diseases. High quality animals combined with first class animal care ensure the highest possible health and welfare status of the animals and are a prerequisite for good science and public acceptance of the use of animals in research.

INTRODUCTION

THE ANIMAL MODEL CONCEPT

Throughout history ethical and religious considerations as well as social prohibitions have prevented experimental studies of human biology and pathobiology. Even studies of human anatomy were for long periods of time in history a criminal offense and thus not possible. Although impressive anatomical teaching theaters were established in many old European universities, postmortem dissection was often restricted exclusively to criminals executed for their offenses. In the more quiet corners of Europe the teaching theaters remained unused for decades. Consequently, most of our present basic knowledge of human biology, physiology, endocrinology, and pharmacology has been derived from initial studies of mechanisms in animal models.1 Throughout history scientists have performed experiments on animals to obtain knowledge of animal and human biological structure and function.2,3 Often such studies have not been conducted, and are not possible to conduct, in humans. This may not only be due to ethical or religious considerations. Often practical, economic, and scientific reasons make initial studies in animals the best solution for studies of a biological phenomenon.

A laboratory animal model describes a biological phenomenon that the species has in common with the target species. A key word for understanding the concept of animal models is “analogy.”4 A model should not be considered a claim of identity with what is being modeled, but a convergent set of several kinds of analogies between the “target” phenomenon to be understood and the system that is being studied as a substitute for the target phenomenon. A more comprehensive definition has been given by Held on the basis of Wessler’s original definition5: “a living organism in which normative biology or behavior can be studied, or in which a spontaneous or induced pathological process can be investigated, and in which the phenomenon in one or more respects resembles the same phenomenon in humans or other species of animal.” What is generally understood by the term animal model is modeling humans. It is not the image of the used animal that is the focus of research but the analogy of the physiological behavior of this animal to our own (or another) species. It would thus perhaps be more correct to refer to animals as “man models” in this context. Laboratory animal science, comparative medicine, and animal experiments are indeed much more about humans than about any other animal species.6 The practice of studying biological phenomena and diseases in laboratory animals and transferring the findings into solutions

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

3

4

SECTION I / INTRODUCTION

and treatments for improving human health and welfare has a long history and is well established in biomedical sciences. The significance and validity with respect to usefulness in terms of ”extrapolatability” of results generated in an animal model depend on the selection of a suitable animal model. A good knowledge of comparative anatomy and physiology is an obvious advantage when developing an animal model. Animal models may be found throughout the animal kingdom, and knowledge about human physiology has been obtained from species far removed from humans in terms of taxonomy. A good example is the importance of the fruit fly for the original studies of basic genetics. Animal models are used in most fields of biomedical research as reflected in the respective chapters of this book.

CLASSIFICATION OF ANIMAL MODELS A plethora of animal models has been used and is being used and developed for studies of biological structure and function in humans. The models may be “exploratory,” aiming to understand a biological mechanism whether this is a mechanism operative in fundamental normal biology or a mechanism associated with an abnormal biological function. Models may also be developed and applied as so-called “explanatory” models, aiming to understand a more or less complex biological problem. Explanatory models need not necessarily be reliant on the use of animals but may also be physical or mathematical model systems developed to unravel complex mechanisms. A third important group of animal models is “predictive” models. These models are used to discover and quantify the impact of a treatment, whether this is to cure a disease or to assess toxicity of a chemical compound. The anatomy or morphology of the model structure of relevance to the studies may be of importance in all three of these model systems. The extent of resemblance of the biological structure in the animal to the corresponding structure in humans has been termed fidelity. A high fidelity model with close resemblance to humans may seem an obvious advantage when developing certain models. What is often more important, however, is the discriminating ability of the models, in particular the predictive models. When using models to assess the carcinogenicity of a substance it is essential that at least one of the model species chosen responds in a manner that is predictive of the human response to this substance. Thus the similarity between human and model species with respect to relevant biological mechanisms is often more important than the fidelity of the model. Often the two go hand in hand and high fidelity models offer the best opportunity to study a particular biological function. An animal model may be considered homologous if the symptoms shown by the animal and the course of the condition are identical to those of humans.7 Models fulfilling these requirements are relatively few, but an example is well-defined lesion syndromes in, e.g., neuroscience.8 An animal model is considered isomorphic if the animal symptoms are similar, but the cause of the symptoms differs in humans and the model. However, most models are neither homologous nor isomorphic but may rather be termed partial. These models do not mimic the entire human disease, but may be used to study certain aspects or treatments of the human disease.8

CLASSIFICATION OF DISEASE MODELS The majority of laboratory animal models are developed and used to study the cause, nature, and cure of human disorders.

Disease models may conveniently be categorized in one of the following five groups, of which the three first are the numerically most important9: 1. 2. 3. 4. 5.

Induced (experimental) models Spontaneous (genetic, mutant) models Genetically modified models Negative models Orphan models

INDUCED (EXPERIMENTAL) MODELS As the name implies, induced models involve healthy animals in which the condition to be investigated is experimentally induced, e.g., the induction of diabetes mellitus with encephalomyocarditis virus,10 allergy against cow’s milk through immunization with minute doses of protein,11 or partial hepatectomy to study liver regeneration.12 The induced model group is the only category that theoretically allows a free choice of species. Although it might be presumed that extrapolation from an animal species to the human species is better the closer this species resembles humans (high fidelity), phylogenetic closeness, as fulfilled by primate models, is not a guarantee of validity of extrapolation, as the unsuccessful chimpanzee models in AIDS research have demonstrated.13 It is just as decisive that the pathology and outcome of an induced disease or disorder in the model species resemble the respective lesions of the target species. Feline immunodeficiency virus (FIV) infection in cats may therefore for many studies be a better model for human AIDS than HIV infection in simians. Although mice and rats have many biological characteristics in common, they do not necessarily serve equally well as models of human disease. For example, schistosomiasis (mansoni) infection may be studied in experimentally infected mice, but not in rats whose immune system is able to fight the infection effectively.14 Most induced models are partial or isomorphic because the etiology of a disease experimentally induced in an animal is often different from that of the corresponding disease in humans. Few induced models completely mimic the etiology, course, and pathology of the target disease in humans. SPONTANEOUS ANIMAL MODELS These models of human disease utilize naturally occurring genetic variants (mutants). Many hundreds of strains/stocks with inherited disorders modeling similar conditions in humans have been characterized and conserved (see, e.g., www.jax.org). The best known spontaneous models include the athymic nude mouse,15 the use of which represented a turning point in the study of heterotransplanted tumors and enabled the first description of natural killer cells. Some of these mutants were discovered almost a century ago, like the famous spontaneous model Snell’s Dwarf mouse without a functional pituitary16 and the curly tail mouse in which fetuses develop a whole range of neural tube defects.17 Many of the mutants are available in inbred strains with corresponding coisogenic or congenic strains. This is very useful because the influence of just one affected gene or locus may then be studied against a reference strain with a genetic background similar to the mutant. An extensive literature is available on spontaneous models and the majority of these involve mice and rat models, although a wide range of mutants in many different species has been described. A good example of the amount of information available is the publication of Migaki,18 referencing more than 200 diseases in animals exclusively caused by inborn errors of metabolism.

CHAPTER 1 / ANIMAL MODELS FOR HUMAN DISEASES

The spontaneous models are often isomorphic displaying phenotypic similarity between the disease in the animal and the corresponding disease in man, the so-called face validity, e.g., type I diabetes in humans and insulin requiring diabetes in the BB rat. This phenotypic similarity often extends to similar reactions to treatment in the model animal and the human patient, and spontaneous models have been important in the development of treatment regimens for human diseases. However, if the object of a project is to study the genetic causes and etiology of a particular disease then comparable genomic segments involved in the etiology of the disorder—construct validity—is normally a requirement. It should be remembered, however, that an impaired gene or sequence of genes very often results in activation of other genes and mobilization of compensating metabolic processes. These compensatory mechanisms may of course differ between humans and the animal model species. GENETICALLY MODIFIED MODELS The rapid developments in genetic engineering and embryo manipulation technology during the past decade have made transgenic disease models the most important category—in terms of numbers—of animal disease models. The technology and ability to genetically modify mice resulted in a substantial increase in the numbers of laboratory animals used worldwide, which is a trend that seems to continue. A multitude of animal models for important diseases have been developed since this technology became available in the 1980s, and the number of models has increased quickly. Mice are by far the most important animals for transgenic research purposes, but farm animals and fish are also receiving considerable interest. Many physiological functions are polygenic and controlled by more than one gene, and it will require considerable research activities to identify the contribution of multiple genes to normal as well as abnormal biological mechanisms. The insertion of DNA into the genome of animals, or the deletion of specific genes, gives rise to sometimes unpredictable outcomes in terms of scientific results as well as animal well-being in the first generations of animals produced. Thereafter transgenic lines can be selected and bred or cloned to avoid or select for a specific genotype. It is not an accurate science, although the methodology is constantly improving, with the aim of eliminating unwanted effects. The embryo manipulation procedures in themselves do not appear to affect the welfare of offspring in the mouse,19 and the large offspring syndrome common in farm animals has so far not been reported in the literature for rodents, although it has been observed (Johannes Wilbertz, Karolinska Institute, personal communication). Mutations induced by the use of mutagens like ethylnitrosourea is another approach to the generation of new mutants, which may serve as models of human disorders. In many aspects these mutants may be similar to spontaneous mutants and to the ones generated by transgenic embryo manipulation. The maintenance of a line raises issues for chemically induced genetic mutants similar to those for animals genetically modified through embryo manipulation. The recent completion of the maps of the genomes of mouse (and other model animals) and humans will increase the research activities in functional genomics and proteomics, and using high density microarray DNA chip technology in human patients as well as in animals will make it possible to investigate which genes are switched on or off in different diseases.20

5

Having both the human and the mouse genome maps available, this new technology is expected to rapidly increase our knowledge on the genetic background and etiology of important diseases. This paves the way for a range of new homologous animal models with homology between animal and humans (construct validity) for genotype as well as for phenotype. This development may result in a change in animal use from models for the identification of causative genes to models for studying the effects of changes in genetic pathways, gene–gene interactions, and gene–environment interactions.21 The characterization and application of genetically modified mouse models are slowed down by difficulties in phenotyping the animals. There is a need for accurate and reliable behavioral assessment, biotechnology development for physiological assessment, and analysis of complex data as well as training scientists in whole-organism research.22 NEGATIVE MODELS Negative model is the term used for species, strains, or breeds in which a certain disease does not develop, e.g., gonococcal infection in rabbits following an experimental treatment that induces the disease in other animal(s). Models of infectious diseases are often restricted to a limited number of susceptible species and the remaining unresponsive species may be regarded as negative models for this particular human pathogenic organism. Negative models thus include animals demonstrating a lack of reactivity to a particular stimulus. Their main application is studies on the mechanism of resistance to gain insight into its physiological basis. Occasionally negative models give rise to the characterization of new spontaneous (mutant) models. Examples are found in studies of infectious diseases and carcinogenicity where individuals exhibiting resistance to a treatment may be developed into new spontaneous models.23 ORPHAN MODELS An orphan model disease is the term used to describe a functional disorder that occurs naturally in a nonhuman species but has not yet been described in humans, and which is recognized when a similar human disease is later identified. Examples include Marek’s disease, papillomatosis, and bovine spongiform encephalopathy (BSE), Visna virus in sheep, and feline leukemia virus. When discovering that humans may suffer from a disease similar to one that has already been described in animals the literature generated in veterinary medicine may be very useful.

EXTRAPOLATION FROM ANIMALS TO HUMANS When experimental results have been generated in an animal model they have to be validated with respect to their applicability to the target species, which normally is the human. The term extrapolation is often used to describe how data obtained from animal studies reliably can be used to apply to humans. However, extrapolation is generally not performed in its mathematical sense where data fit a certain function that may be described graphically, and the graph extended beyond the highest or lowest sets of data to describe a situation outside the window of observation. Establishing toxicity data in animals and using these to determine safe levels of exposure for people is perhaps what comes closest to mathematical extrapolation in animal studies. However, most studies of animal structure and function are never extrapolated to be applicable to describing the corresponding features in humans; this is not relevant. What laboratory animal experimentation is about is very similar to other types of experiments. The scientists aim to obtain answers to specific questions. Hypotheses are being

6

SECTION I / INTRODUCTION

tested and the answers obtained, analyzed, and published. As an example of this, we might question the possible health hazards of a new synthetic steroid and ask a number of relevant questions to be answered in animal studies before deciding on its potential usefulness as a human hormonal contraceptive: Does it exist in the same form in humans and animals? How does it affect the estrus cycle in rodents? How does it affect endogenous hormone levels in rodents and other species? How soon after withdrawal do the animals revert to normal cyclicity? Does it interfere with pregnancy in rodents and primates? Does it affect fetal development in rodents and primates? Is the frequency of fetal malformations in mice affected? Are puberty, the ovarian cycle, and pregnancy in rodent and dog offspring of mothers treated with the substance affected? Analyzing the data from experiments of this nature would provide information on the potential of the new synthetic steroid as a hormonal contraceptive in humans. A large multinational pharmaceutical company survey analyzed data compiled from 150 compounds for the concordance between adverse findings in human clinical data with data that had been generated in preclinical tests in animals.24 The concordance rate was found to be 71% for rodent and nonrodent species, with nonrodents alone being predictive for 63% of human toxicity (HT) and rodents alone for 43%. High concordance rates were found, e.g., for cardiovascular HTs (80%), hematological HTs (91%), and gastrointestinal HTs (85%). Lower concordance rates were observed, e.g., for the neurological group, because it is difficult to identify symptoms such as headache and dizziness in the animals studied. The only gastrointestinal HT that did not correlate with animal studies was, not surprisingly, nausea. One of the conclusions reached in this study was that the choice of species used might be subject to more thoughtful consideration. By tradition studies are often carried out using rats and dogs, without an open-minded consideration of whether alternative species might be more appropriate for testing a specific compound. Although the predictive value of animal studies may seem high if they are conducted thoroughly and have included several species, uncritical reliance on the results of animal tests can be dangerously misleading and has resulted in damage to human health in several cases, including drugs developed by large pharmaceutical companies. What is noxious or ineffective in nonhuman species can be innocuous or effective in humans and vice versa. For example, penicillin is fatal for guinea pigs but generally well tolerated by humans; aspirin is teratogenic in cats, dogs, guinea pigs, rats, mice, and monkeys but obviously not in pregnant women despite frequent consumption.25 Thalidomide, which crippled 10,000 children, does not cause birth defects in rats26 or many other species,27 but does so in primates. Close phylogenetic relationship or anatomical similarity is not a guarantee of identical biochemical mechanisms and parallel physiological response, although this is the case in many instances. The validity of extrapolation may be further complicated by the question of which humans. As desirable as it often is to obtain results from a genetically defined and uniform animal model, the humans to whom the results are extrapolated are genetically highly variable, with cultural, dietary, and environmental differences. This may be of minor importance for many disease models but can become significant for pharmacological and toxicological models. It is not possible to provide reliable general rules for the validity of extrapolation from one species to another. This has to be

assessed individually for each experiment and can often be verified only after first trials in the target species. An extensive and useful overview on the problem of predictive anthropomorphization, especially in the field of toxicology research, is Principles of Animal Extrapolation by Calabrese.28 The rationale behind extrapolating results to other species is based on the extensive homology and evolutionary similarity between morphological structures and physiological processes among different animal species and between animals and humans.29

MODEL BODY SIZE AND SCALING The use of laboratory animals as models for humans is often based on the premise that animals are more or less similar with respect to many biological characteristics and thus can be compared. However, there is one striking difference between mouse and human, and that is body size. In proportion to their body size mammals generally often have very similar organ sizes expressed as percentage of body weight. Take the heart, for instance, which often constitutes 5 or 6 g/kg body weight, or blood, which is often approximately 7% of total body weight. It is well known that the metabolic rate of small animals is much higher than that of large animals. It has also been demonstrated that capillary density in animals smaller than rabbits increases dramatically with decreasing body weight.30 However, considering that most animals are similar in having heart weights just above 0.5% of their body weight and a blood volume corresponding to 7% of their body weight, it becomes obvious that in order to supply the tissues of small animals with sufficient oxygen for their high metabolic rate it is not sufficient to increase the stroke volume. The stroke volume is limited by the size of the heart and heart frequency is the only parameter to increase, which results in heart rates well over 500 per minute in the smallest mammals. Other physiological variables, like respiration and food intake, are similarly affected by the high metabolic rate of small mammals. This means that scaling must be an object for some consideration when calculating dosages of drugs and other compounds administered to animals in experiments. If the object is to achieve equal concentrations of a substance in the body fluids of animals of different body size then the doses should be calculated in simple proportion to their body weights. If the object is to achieve a given concentration in a particular organ over a certain time period the calculation of dosage becomes more complicated and other factors including the physicochemical properties of the drug become important. Drugs and toxins exert their effect on an organism because of the way they are metabolized, the way they and their metabolites are distributed and bound in the body tissues, and how and when they are finally excreted. However, metabolism or detoxification and excretion of a drug are not directly correlated with body size, but more accurately with the metabolic rate of the animal (see Schmidt-Nielsen30,31 for more details). Kleiber32 in 1932 was the first to demonstrate that in a log–log plot of mammalian body weight to metabolism the graph forms a straight line with a slope of 0.75. The metabolic rate of an animal as expressed by oxygen consumption per gram body weight per hour is related to body weight in the following manner: M = 3.8 × BW−0.25

CHAPTER 1 / ANIMAL MODELS FOR HUMAN DISEASES

where M is the metabolic rate (oxygen consumption in milliliter per gram body weight per hour) and BW is body weight in grams. This equation may be used to calculate dosages for animals of different body weights if the dose for one animal (or man) is known.33 Dose1/Dose2 = BW1−0.25/BW2−0.25

3.

4.

Dose1 = Dose2 × BW1−0.25/BW2−0.25 These equations should be considered as assistance for calculating dosages, but caution should be exerted with respect to too broad a generalization of their use, and the 0.50 power of body weight should be employed when dealing with animals having body weights below 100 g.34 Some species react with particular sensitivity toward certain drugs and marked variations in the reaction of animals within a species occur with respect to strain, pigmentation, nutritional state, time of day, stress level, type of bedding, ambient temperature, etc.33

5.

6.

7.

8.

CONCLUSIONS The selection of an animal model depends on a number of factors relating to the hypothesis to be tested, but often more practical aspects associated with the project and with project staff and experimental facilities play a significant role. The usefulness of a laboratory animal model should be judged on how well it answers the specific questions it is being used to answer, rather than how well it mimics the human disease.35 Often a number of different models may advantageously be used in order to scrutinize a biological phenomenon and for major human diseases such as diabetes, a whole range of well-described induced models are available as are spontaneous models in both mouse and rat strains. Most of the regulating authorities require two species in toxicology screening, one of which has to be nonrodent. This does not imply that excessive numbers of animals will be used because an uncritical use of one-species models may mean that experimental data retrospectively turn out to be invalid for extrapolation, representing a waste of animals. The appropriateness of any laboratory animal model will eventually be judged by its capacity to explain and predict the observed effects in the target species.36 The free choice of species when developing animal models is more or less restricted to the induced models making use of clinically healthy animals, in which a condition deviating from normality is experimentally induced. Although all laboratory animal species are in principle available for model development, it has been a clear trend during the past 30 years that the most popular species, the house mouse and the Norwegian rat, are increasing in popularity at the expense of farm animal species and pet species, while the use of nonhuman primates seems to remain stable.37,38 The completion of the map of the mouse genome and the dominating position of mice in transgenic research seem to indicate that the dominance of the mouse as the most popular model for humans will increase even more in the future.

REFERENCES 1. Coffey DS, Isaacs JT. Requirements for an idealized animal model in prostatic cancer. In: Murphy GP, Ed. Models for Prostate Cancer. New York: Alan R. Liss, 1980:379. 2. Held JR. Muhlbock memorial lecture: Considerations in the provision and characterization of animal models. In: Spiegel A, Erichsen S, Solleveld HA, Eds. Animal Quality and Models in Biomedical

9.

10.

11.

12.

13. 14.

15. 16.

17.

18.

19.

20. 21. 22.

23.

24.

7

Research, 7th ICLAS Symposium Utrecht 1979. Stuttgart, Germany: Gustav Fisher Verlag, 1980. Loew FM. Scholarship and clinical service; comparative and laboratory animal medicine. In: Nevalainen T, Hau J, Sarviharju M, Eds. Frontiers in Laboratory Animal Science. Proceedings of Joint International Conference of ICLAS, Scand-LAS and FinLAS, Helsinki 1995. Scand J Lab Anim Sci 1996;23(Suppl. 1):13. Overmier JB. Animal models on human pathology: A bibliography of a quarter century of behavioral research, 1967–1992. In: Overmier JB, Burke PD, Eds. Bibliographies in Psychology: Number 12. Washington, DC: American Psychological Association, 1992:vii–xiv. Wessler S. Introduction: What is a model? In: Animal Models of Thrombosis and Hemorrhagic Diseases. Bethesda, MD: National Institutes of Health, 1976:xi. Salén J. Animal models, principles and problems. In: Svendsen P, Hau J, Eds. Handbook of Laboratory Animal Science, Vol. II, Animal Models. Boca Raton, FL: CRC Press, 1994:Chapter 1. Kornetsky C. Animal models: Promises and problems. In: Hanin I, Usdin E, Eds. Animal Models in Psychiatry and Neurology. Oxford, UK: Pergamon Press, 1977:1. Mogensen J, Holm S. Basic research and animal models in neuroscience—the necessity of “co-evolution.” Scand J Lab Anim Sci 1989;16(Suppl. 1):51. Hau J. Animal Models. In: Hau J, Van Hoosier G, Eds. Handbook of Laboratory Animal Science, Vol. II, Animal Models. Boca Raton, FL: CRC Press, 2003. Hau J, Buschard K. Effect of encephalomyocarditis (EMC) virus on murine foetal and placental growth monitored by quantification of maternal plasma levels of pregnancy-associated murine protein-2 and alpha-fetoprotein. Acta Pathol Microbiol Immunol Scand Sect B 1986;94:339. Poulsen OM, Hau J, Kollerup J. Effect of homogenization and pasteurization on the allergenicity of bovine milk analysed by a murine anaphylactic shock model. Clin Allergy 1987;17:449. Hau J, Cervinkova Z, O’Brien D, Stodulski G, Simek J. Serum levels of selected liver proteins following partial hepatectomy in the female rat. Lab Anim 1995;29:185. King NW. Simian models of acquired immunodeficiency syndrome (AIDS): A review. Vet Pathol 1986;23:345. Farah IO, Kariuki TM, King CL, Hau J. An overview of animal models in experimental schistosomiasis and refinements in the use of non-human primates. Lab Anim 2001;35:205. Pantelouris EM. Absence of thymus in a mouse mutant. Nature 1968;217:370. Hau J, Poulsen OM, Dagnæs-Hansen NF. Induction of pregnancyassociated murine protein-1 (PAMP-1) in dwarf (dw) mice by growth hormone. Lab Anim 1990;24:183. Jensen HE, Andersen LLI, Hau J. Fetal malformations and maternal alpha-fetoprotein levels in curly tail (ct) mice. Int J Feto-Matern Med 1991;4:205. Migaki G. Compendium of inherited metabolic diseases in animals. In: Animal Models for Inherited Metabolic Diseases. New York: Alan R. Liss, 1982:473. Van der Meer M, Baumans V, Olivier B, et al. Behavioral and physiological effects of biotechnology procedures used for gene targeting in mice. Physiol Behav 2001;73:719. Lander ES. Array of hope. Nat Genet 1999;21(1 Suppl.):3. Van Zutphen LFM. Is there a need for animal models of human genetic disorders in the post-genome era? Comp Med 2000;50:10. Committee on New and Emerging Models in Biomedical and Behavioral Research. Biomedical Models and Resources. Current Needs and Future Opportunities. Washington, DC: National Academy Press, 1998. Cui Z, Willingham MC, Hicks AM, Alexander-Miller MA, Howard TD, Hawkins GA, Miller MS, Weir HM, Du W, DeLong CJ. Spontaneous regression of advanced cancer: Identification of a unique genetically determined, age-dependent trait in mice. Proc Natl Acad Sci USA 2003;100(11):6682. Olson H, Betton G, Robinson D, Thomas K, Monro A, Kolaja G, Lilly P, Sanders J, Sipes G, Bracken W, Dorato M, Van Deun K,

8

25. 26. 27.

28. 29.

30.

SECTION I / INTRODUCTION

Smith P, Berger B, Heller A. Concordance of the toxicity of pharmaceuticals in humans and in animals. Regul Toxicol Pharmacol 2000;32:56. Mann RD. Modern Drug Use. An Enquiry on Historical Principles. Lancaster: MTP, 1984. Koppanyi T, Avery MA. Species differences and the clinical trial of new drugs: A review. Clin Pharmacol Ther 1966;7:250. Lewis P. Animal tests for teratogenicity, their relevance to clinical practice. In: Hawkins, DF, Ed. Drugs and Pregnancy: Human Teratogenesis and Related Problems. Edinburgh: Churchill Livingstone, 1983;17. Calabrese EJ. Principles of Animal Extrapolation. Chelsea, MI: Lewis Publishers Inc., 1991. Beynen AC, Hau J. Animal models. In: Van Zutphen LFM, Baumans V, Beynen AC, Eds. Principles of Laboratory Animal Science. New York: Elsevier, 2001:Chapter 10. Schmidt-Nielsen K. How Animals Work. London: Cambridge University Press, 1972.

31. Schmidt-Nielsen K. Animal Physiology, Adaptation and Environment. London: Cambridge University Press, 1975. 32. Kleiber M. Body size and metabolism. Hilgardia 1932;6:315. 33. Hau J, Poulsen OM. Doses for laboratory animals based on metabolic rate. Scand J Lab Anim Sci 1988;15:81. 34. Bartels H. Metabolic rates of mammals equals the 0.75 power of their body weight. Exp Biol Med 1982;7:1. 35. Snider GL, Lucey ED, Stone PJ. Animal models of emphysema. Am Rev Respir Dis 1986;133:149. 36. Frenkel JK. Choice of animal models for the study of disease processes in man. Fed Proc 1969;28:160. 37. Hagelin J, Hau J, Carlsson H.-E. The refining influence of ethics committees on animal experimentation in Sweden. Lab Anim 2003;37(1):10. 38. Carlsson HE, Schapiro SJ, Farah IO, Hau J. The use of primates in research: a global overview. Am J Primatol 2004;63(4):225.

2

Selection of Biomedical Animal Models MICHAEL S. RAND

ABSTRACT

humans. It was not until the late sixteenth century that this error began to be recognized. Modern research principles can be attributed to three physiologists from the 1860s. In 1865, Claude Bernard, a French physiologist, published An Introduction to the Study of Experimental Medicine.2 This book was intended to provide physicians with guidance in experimental research. It proposed the use of chemical and physical induction of disease in animals, thus becoming the first published book to advocate creating “induced animal models” for biomedical research. His peers of the time were Louis Pasteur in France and Robert Koch in Germany. Louis Pasteur and Robert Koch introduced the concept of specificity into medicine and the “germ theory of disease.” The turning of the century saw the development and use of animal models for infectious diseases and screening and the evaluation of new antibacterial drugs based upon the work of these three researchers. During the first quarter of the nineteenth century, animal studies were crucial for less than one-third of the major advances that occurred. With the contributions of Claude Bernard, Louis Pasteur, and Robert Koch, animal studies contributed to more than half of the significant discoveries made thereafter. Since 1901, two-thirds and 7 of the last 10 Nobel Prizes in medicine have relied at least in part on animal research.3 Today, researchers rely on the identification and development of animal models to explore all avenues of medical science to include assessment of pathogenic mechanisms, diagnostic and therapeutic procedures, nutrition and metabolic diseases, and the efficacy of novel drug development.

Laboratory animals play a crucial role in research discovery and technological advances, and they will continue to take part in improving the lives of people and other animals. It is incumbent upon the researcher to know his subject well in order to provide relevant information to the scientific world. In an effort to assist the biomedical researcher in gaining this knowledge, this chapter provides the following key elements: definition of types of animal models, legislative and legal requirements, criteria for choosing a model, extrapolation validity recommendations, and descriptive features for publication. Key Words: Animal models, Laboratory animal(s), Animal model types, Animal use criteria, Choosing animal model, Animal factors, Extrapolation, Animal description.

INTRODUCTION Over the last one and one-half centuries, almost all medical knowledge, treatment regimes, and medical device development have involved research using animals. The key factor in using animals in research is in its extrapolatability of results to humans. Animals in research have been and still are essential in developing treatments for asthma, HIV/AIDS, cancer, birth defects, bioterrorism medical countermeasures, vaccines, antibiotics, high blood pressure, and much more. Additionally, they have been vital in the development of antibiotics, vaccines, and organ transplantation techniques.1 As the rise in emerging infectious diseases (e.g., West Nile virus and avian influenza) continues, animals will be key and essential in the development of preventive and treatment modalities.

THE CONCEPT OF ANIMAL MODELS

THE HISTORY OF ANIMAL USE IN RESEARCH

WHAT IS AN ANIMAL? Etymologically, the word “animal” derives from the Latin animal meaning soul/spirit, thus describing living organisms that are animated. WHAT IS A MODEL? A model is an object of imitation, something that accurately resembles something else, a person or thing that is the likeness or image of another. The Holy Bible tells us that God said, “Let us make man in our image, in our likeness, so God created man in his own image, in the image of God he created him; male and female he created them.” God created man out of the dust of the ground and then breathed into his nostrils the breath of life to animate him. Thus, humans are “animal models” of God. Consequently, combining the two definitions, an “animal model” is an animated object of imitation, an “image of Man” (or other species), used to investigate a physiological or pathological circumstance in question.4

The use of animals to study human physiology and anatomy can be traced back to the second century AD in which Galen was a Greek physician and philosopher. His research was based almost exclusively on studies using apes and pigs. Unfortunately, this initiated many errors based on his accepted authority and the prohibition by the Church of using human cadavers for research purposes. Galen was later blamed for using incorrect methods in research when in truth it would be more accurate to say that he drew wrong conclusions based on uncritical interspecies extrapolation of data. That is, he assumed that all extracted information derived from his use of animals could be directly applied to

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

9

10

SECTION I / INTRODUCTION

WHAT IS AN ANIMAL MODEL? The U.S. National Research Committee on Animal Models for Research on Aging attempted to define the term “laboratory animal model” as “an animal in which normative biology or behavior can be studied, or in which a spontaneous or induced pathological process can be investigated, and in which the phenomenon in one or more respects resembles the same phenomenon in humans or other species of animal.” Using the term animal model can be confusing because what is often meant by the term “animal model” is actually studying human conditions. In other words, it is not the image of the preferred animal that is the focus of research but the analogy of the physiological behavior of this animal to our own (or another) species. It would, thus, be more correct to speak of “human models” in this context. Indeed, although using animals in research can benefit other animals, it is much more focused on improving the human condition.

TYPES OF ANIMAL MODELS When animals are used in research to study biological and functional systems in humans, they are broken down into the following categories: 1. Exploratory. Animals used in this category are used to gain an understanding of fundamental biological mechanisms, whether normal or abnormal. An example would be the use of a novel animal model of aging, particularly for identifying genes and biochemical pathways regulating longevity. 2. Explanatory. Animals used in this category are used to gain an understanding of complex biological problems. An example would be the use of cognitive and psychosocial animal models to provide an etiology for anorexia nervosa.5 3. Predictive. Animals used in this category are used to discover and quantify the impact of investigative treatments whether for diseases or chemical toxicities. Predictive animal testing models are important in improving the success of a drug or medical device in clinical trials and for generating new data in support of the ongoing marketing of existing products. When animals are used in disease research, they are broken down into the following categories: 1. Induced (or Experimental). Induced models are ones in which normal animals are experimentally created either through surgical modifications, genetic modifications, or chemical injections. An example would be a myocardial infarction induced by coronary artery surgical ligation. 2. Spontaneous. Spontaneous models are genetic variants, which mimic the human condition. The variance occurs naturally through mutation and not by experimental induction. The nu mutation was first reported in 1966 in a closed stock of mice in a laboratory in Glasgow, Scotland. It was not until 1968, however, that it was discovered that the homozygous nude mouse also lacked a functional thymus, i.e., it was athymic. The mutation produces a hairless state, generating the name “nude.” The other, unique defect of nude mice is the failure of the thymus to develop normally to maturity. The thymus remains rudimentary and produces reduced numbers of mature T cells. This means nude homozygotes (animals with identical mutant genes at corresponding chromosome loci) do not reject allografts and often do not reject xenografts (tissue from another species). The discovery that

human neoplasms (tumors) could be grown in nude mice was immediately recognized as an important research tool. Thus, the spontaneous mutation of nu among laboratory mice was a serendipitous development that led to the nude mouse becoming the first animal model of a severe immunodeficiency. In the decades since, the nude mouse has been widely utilized by researchers studying factors regulating transplantable human tumor growth and cancer metastasis.6 3. Transgenic. Transgenic models are induced models in which DNA is inserted into or deleted (knockout) from the genome of the animal. The term “transgenic” was coined in 1981 by Gordon and Ruddle to describe an animal in which an exogenous gene was introduced into its genome. In the late 1980s, the term transgenic was extended to gene-targeting experimentation and the production of chimeric or “knockout” mice in which a gene (or genes) has been selectively removed from the host genome. Today, a transgenic animal can be defined as one having any specific, targeted genetic modification. Transgenic animals are most commonly produced through (1) germline modifications of gametes, (2) microinjection of DNA or gene constructs into zygotes (unicellular embryos), or (3) incorporating modified cells, including embryonic stem (ES) cells, into later stage embryos. After gamete or embryo modifications, the resultant embryos are matured to term in a recipient female.7 4. Negative. Negative models fail to react to a disease or chemical stimulus. Thus, their main use in biomedical research is for studies on the mechanism of disease resistance. A classic example is the failure of gonococcal infection to develop in rabbits after an experimental treatment that induces the disease in other animals. Negative animal models have become increasingly important with the advent of transgenic technology. For example, a novel transgenic mouse was created to study the lack of development of autoimmune thyroiditis with the injection of self-thyroglobulin. This strain of mice lacked certain surface epitopes to account for this negative reaction.8 5. Orphan. Orphan models are the opposite of negative models. Orphan models are animals in which a disease occurs but there is not a corresponding disease in humans. Orphan models may become induced models when a similar disease is recognized in humans later on. Historically, scrapie in sheep was such a model, but now is useful as a model for the human spongiform encephalopathies that are of so much concern (e.g., BSE, “mad cow disease,” and CWD, chronic wasting disease in deer). All categories above may be further subcategorized with the following divisions: 1. Fidelity. The extent a biological structure in an animal resembles that of a human. Thus, a high fidelity animal model gives a highly relevant biological closeness to the human structure. Model fidelity is best conceptualized as a continuous spectrum, ranging from low to high fidelity. Examples of low-fidelity models include bench models made of simple materials that often have little anatomical resemblance to reality. However, these models incorporate some of the key constructs of the simulated tasks. At the other end of the spectrum are high-fidelity models such as human or animal cadavers or the new array of virtual reality simulators. These simulators usually incorporate highly realistic visual and tactile cues in the midst of a highly interactive model. In between these two extremes, almost any kind of intermediate fidelity can exist.

CHAPTER 2 / SELECTION OF BIOMEDICAL ANIMAL MODELS

2. Homologous. The symptoms shown in the animal are identical to those shown in the human. For instance, the recent discoveries of swine hepatitis E virus (HEV) from pigs and avian HEV from chickens afforded an opportunity to develop small homologous animal models for HEV.9 3. Isomorphic. The animal’s symptoms or anatomy are similar to those in the human but the etiology or genetic character is different. For example, there is a set isomorphism between the human and mouse heart at the organ level and also at the organ part level: each species has a heart and a corresponding set of cardiac chambers (right and left atrium, right and left ventricle) and the wall of each chamber has a corresponding set of layers (epicardium, myocardium, endocardium). 4. Partial. These models do not mimic the entire human disease but enough similarities exist to allow their use in studying aspects of the disease or treatments. For instance, animal models of Alzheimer’s disease can be created based on the accumulation of increased levels of amyloid-ß peptide in the brain and have many amyloid plaque deposits; however, they have only subtle behavioral and electrophysiological deficits, thus providing only a partial model of the human condition.10 5. Face validity. The degree to which there is a similar phenotypic display between the disease in the animal and the corresponding disease in the human. For example, it could be argued that the demonstration of drug effects in an animal model for depression after a period of chronic administration is important for establishing its face validity, but is not relevant to the model’s predictiveness and therefore to its ability to serve as a screening test for treatments for the modeled disease.11 6. Construct validity. The degree to which there is a similar genetic display between the disease in the animal and the corresponding disease in the human. As an example of high construct validity, research was performed on three candidate dopaminergic genes (DRD2, DRD4, and DAT-1) that were sequenced in spontaneous hypertensive (SHR) and Wistar Kyoto (WKY) rats. No differences were found in DRD2 or DRD4 genes, but several variations were found in the DAT-1 gene that are of significance because several ADHD families show linkage to DAT-1. It also strengthened the validity of using WKY as a control for SHR, because their behavioral characteristics are similar to those of other rat strains.12

LEGISLATIVE AND LEGAL REQUIREMENTS FOR USING ANIMALS IN RESEARCH Biomedical research is among the most regulated industries in the world. A comprehensive overview of global requirements can be found in the Handbook of Laboratory Animal Science, 2nd edition, Chapter 3.13 Failure to comply with regulatory requirements can result in fines levied against the institution, suspension of authority to operate, permanent revocation of the facility’s license, and withdrawal of public funding. One newly regulated aspect of biomedical research not covered in this chapter occurred after the terrorist attack on September 11, 2001. The attack increased concerns in the United States for the possibility of bioterrorism using agents that would destroy human, animal, and plant life. This concern escalated the need for research that involved the development of therapeutic and preventive measures against such agents. In response, congress passed and President Bush signed into law the “Public Health Security and

11

Bioterrorism Preparedness and Response Act of 2002” (Public Law 107–188) on June 12. The purpose of the act was to improve the capacity of the United States to prevent, prepare for, and respond to bioterrorism and other public health emergencies and to enhance the control of dangerous biological agents and toxins. The Centers for Disease Control and Prevention (CDC) is the agency with the primary responsibility for implementing the provisions of the Act with regard to human pathogens and toxins and the United States Department of Agriculture (USDA) with regard to animal and plant pathogens and toxins. The regulation provides for expanded regulatory oversight of select agents and toxins, and a process for limiting access to persons who have a legitimate need to possess, use, or store these agents. The regulation also establishes a requirement for a security risk assessment performed by the Federal Bureau of Investigation for those persons needing access to select agents and toxins. It also establishes and enforces safety and security procedures, including measures to ensure proper training and appropriate skills to handle agents and toxins; a requirement to designate an institutional Responsible Official to ensure compliance with the regulations; and a requirement to obtain a certificate of registration when there is a need to possess, use, or transfer select agents and toxins. Infectious agents labeled as “select” as determined by the CDC and USDA, registration forms, and other information concerning the Select Agents Program may be found at http://www.selectagents.gov.14

CHOOSING THE RIGHT MODEL To quote the philosopher, Bernard Rollin, “The most brilliant design, the most elegant procedures, the purest reagents, along with investigator talent, public money, and animal life are all wasted if the choice of animal is incorrect.” Once it has been determined that the use of laboratory animals is necessary, the most appropriate species, breed, and strain with the closest homology to humans must be chosen in order to give the research face and/or construct validity. Because new animal models are continually being identified and characterized and the field of biomedical research has become global in nature, the search for the appropriate animal model should start with a thorough literature search and a check of appropriate web sites (see Chapter 7). The Institute for Laboratory Animal Research maintains a very practical and useful search engine for this purpose.15 Selection of a species should not be based solely on availability, familiarity, or cost. Animals that meet these criteria may not provide the genetic, physiological, or psychological facets needed or wanted for the proposed project. It is almost impossible to give specific rules for the choice of the best animal model, because the many considerations that have to be made before an experiment can take place differ with each research project and its objectives. Nevertheless, some general rules can be given. RESEARCH FACTORS • Appropriateness as an analogue. Ensuring that the part or organ being studied has a function similar to the target species is vital in applying research-derived data from the chosen model. • Transferability of information. The usual goal of research using an animal model is to define a process in a system with the hope of transferring the data gained to a more complex system. Traditionally, one-to-one modeling is

12

SECTION I / INTRODUCTION

•

•

•

•

•

sought: modeling in one group of organisms that can be transferred to another group that has several analogous features of interest. This is especially helpful in modeling disease states. However, in modern research, many-tomany models are mainly used. This technique begins by analyzing the component parts of a process or disease, and then finding for each component analogous models in many taxa of living species.16 This is especially helpful when a plurispecies approach is needed to gain approval for new medications or medical devices. Generalizability of the results. The ability to generalize results to the target species is important. Federal regulations prohibiting the unnecessary duplication of previous research highlights the importance of choosing an animal model in which testing results can be easily repeatable and verifiable on which to build new research. In May, 2006, the world was shocked when famed South Korean cloning scientist Dr. Hwang Woo-suk was charged with fraud and embezzlement when scientists could not verify his published data. In addition, if the ultimate target species is human, it is well known that this species is genetically highly variable, with cultural, dietary, and environmental differences. This may be of lesser importance in disease modeling since most diseases do not choose its victim based on genetic variability. However, this is now well known to be of importance in pharmacological and toxicological modeling and has opened up the new field of research in pharmacogenetics. Ethical implications. Certainly research must start with justification for using an animal at all. Federal regulations require the use of alternate methods if feasible. Alternate methods could consist of using cell lines, bacteria, computer models, or even human volunteers. The three Rs of Russell and Burch (replacement of existing experiments with animal-free alternatives, or reduction in the number of animals used, or refined methods to reduce animal suffering) help to meet the ethical concerns.17 Numbers needed. Certainly consultation with a biostatistical analyst prior to submitting a proposal is highly recommended. Numbers needed to provide scientific validity, especially for publication, will impact many other factors such as cost and housing availability. Customary practice within a particular discipline. Caution must be displayed when using this criterion. Customary practices may not always mean that the most appropriate animal model has been used. The “customary” animal may not represent the most accurate genetic, microbiological, physiological, or psychological facets needed for the study. Historical evidence has revealed that using animal models just because others have has led to substandard results. However, customary practices when justified and supported by the other criteria listed can be a satisfactory and faster route of choosing the animal model needed. Existing body of knowledge of the problem under consideration. This criterion again emphasizes the need for a thorough literature search before forming the basis for the research project. The literature search will emphasize what is already known to prevent accidental duplication, but will also reveal what is not known. It will also make

known published authorities in the discipline that may serve as a consultation source to prevent unnecessary and competitive research projects. • Natural versus experimentally produced models. Unavailability of natural models will require the use of experimentally produced models. Depending on the objectives of the study, both may be needed. ANIMAL CARE FACTORS • Cost and availability. Certainly cost and availability are important factors when choosing an animal model, but they can be disastrous if the decision is based solely on cost and not the other listed factors. Cost also includes ongoing care not only for husbandry but also from experimental manipulations. Certainly, the best animal model can be in short supply as illustrated by the CNN news report on August 9, 2003. This report emphasized the increased demands in research due to public health crises such as AIDS and the threat of bioterrorism. The increased demands have led to a national shortage of rhesus macaques. In addition, the shortage has skyrocketed the cost per monkey. • Housing availability. Another practical consideration in choosing the animal model is the accessibility of housing. Research animal housing requirements are stringent and may lessen the availability according to the species chosen. For instance, choosing a nonhuman primate may require the purchase of new caging and the hiring of additional personnel to provide specialized husbandry care, as opposed to choosing mice, which can be placed several to a cage and hundreds in a room. • Husbandry expertise. Some models require not only special housing, but also special care. • Stress factors. Stress sources from many different causes can affect the animal’s physiology, biochemistry, and behavior. Sources of stress can be transportation, handling and manipulations, overcrowding, lack of environmental enrichment, and the research project itself. PHYSICAL AND ENVIRONMENTAL FACTORS • Ecological consequences. While the best animal model may be available only by capturing in the wild, ecological consequences must be considered in its removal. In addition, safety measures must be in place to prevent accidental escape from the research facility. A prime example is the Xenopus spp. frog. If it escapes, it can overrun local ponds and rivers endangering natural amphibian populations. Furthermore, care must be taken not to violate the Endangered Species Act (http://www.fws.gov/endangered/ wildlife.html) or the Convention on International Trade in Endangered Species of Wild Fauna and Flora (http://www. cites.org). • Hazardous components. Many research projects entail the use of chemicals, infectious agents, and radioisotopes. The uses of these components are highly regulated and the appropriate proper authority (Institutional Biosafety Committee, Radiation Safety Committee, Occupational Health and Safety Committee, Environmental Health and Safety Committee, and Institutional Animal Care and Use Committee) within each institution must give approval for

CHAPTER 2 / SELECTION OF BIOMEDICAL ANIMAL MODELS

its use. In addition, all those who will be exposed, whether from the research side or the animal care side, must be notified. • Environmental influences. Environmental aspects may be important to a particular animal species.18 Environmental factors that fit into the broad categories of physical, chemical, biological, and social may impact the physiological and behavioral responses of animals. These factors include humidity, ventilation, light cycle and quality, noise, cage size and bedding materials, diet and water, and room temperature.19 As an example, high temperature and humidity have been proven to impair memory in mice.20 ANIMAL-RELATED FACTORS • Genetic aspects. Uniformity of organisms may be necessary where applicable. “This insidious evolution of the inbred genotype is known as genetic drift. It is capable of subverting the conclusions reached about comparable research results coming from different laboratories when each uses its own subline of the same inbred strain (Bailey DW, 1977).”21 The importance and methods of preventing genetic drift in biomedical research can be found at http:// jaxmice.jax.org/geneticquality/drift.html. In addition, it is important to remember that to be in compliance with the National Institutes of Health’s Guidelines,22 work with transgenic animals requires the approval of the Institutional Biosafety Committee as well as the Institutional Animal Care and Use Committee. • Background knowledge of biological properties. Knowledge of biological properties such as generalized and specialized function of body components is needed in order to validate interspecies transfer of information. Certainly, a rat would not be the best choice in biliary studies due to the absence of a gallbladder. Knowing the biological properties also aids in the decision of whether the animal is a spontaneous model or must be experimentally induced. • Ease of and adaptability to experimental manipulation. This is unquestionably a practical matter. Guinea pigs have highly inaccessible blood vessels and would be impractical in studies requiring repeated blood sampling. Prairie dogs and woodchucks can be vicious to handle; therefore, knowing the response to experimental manipulation may also influence the choice. • Size of the animal. This item is important from several different aspects. The size of the animal impacts housing and husbandry availability. However, size is also important to consider when tissue sampling or blood collection is necessary. For instance, many proposals are rejected because the researcher failed to abide by published guidelines for removal of blood.23 In addition, it is also important to incorporate the size of the animal into the decision-making apparatus when physiological or morphological properties such as joint strain or organ size must be identical to that of a human, especially when developing medical devices. • Life span and age. Studies requiring components at different stages of life can certainly impact the species chosen. The average lifespan of a rat is 2.5–3.5 years, whereas it can be over 30 years for a rhesus monkey.

13

• Sex. The alternating cycle of hormonal production in the female gender and its influence on the data outcome must be considered when planning for the research project. • Progeny needed. Female mice and rats can produce 5–10 progeny per month, whereas the rhesus monkeys only one or two per year. Xenopus sp. frogs produce thousands of ova during their lifetime, whereas mammals produce only dozens. • Diseases or conditions that might complicate results. An excellent historical review on the struggle against pathogens in laboratory rodents can be found in Weisbroth.24 The effects on research can be found in Baker.25 Both publications emphasize the need for disease-free animals in research to prevent adverse effects on resultant data. Just as in human AIDS, the realm of disease-causing organisms changed with the advent of immune deficient models. Special caging and care procedures are fundamental in minimizing such infections. • Special features of the animal such as unique responses or microflora. It is important to be familiar with unique anatomical or physiological features of the species you will be working with. The results could be quite unexpected otherwise. For example, in rabbits, the terminal portion of the ileum empties into an enlarged rounded viscus called the sacculus rotundus and not the colon as in humans. This unique feature of the rabbit is important to know when designing gastrointestinal studies. Forming the above standards into a checklist will help to fulfill the criteria needed to choose the best model for the proposed research project. Model selection is the privilege of individual researchers, but they must be very cautious in their selection because in the end, it is up to them to convince the rest of the scientific community that they made the right choice. Before choosing, consultations should occur with scientists who have already used the animal model. Just as with equipment purchase, communicating with previous users can be very helpful in learning unique features of the selected species, breed, and strain. Not all attributes (especially negative ones) are published, making it even more important to contact those who have experience with the animal model you choose. Preparatory consultation should also occur with those who will be responsible for housing and maintaining the animals, as they will be the most familiar with the care of the animal and its physical and environmental needs. Preparatory consultation with the laboratory animal veterinary practitioner should also occur to discuss the animal-related factors.

EXTRAPOLATION FROM ANIMALS TO HUMANS Extrapolation from animals to humans does not necessarily mean that biomedical research data obtained from using animals are then used to find a corollary in a human. Rather, a hypothesis is formed first based on human relevancy, and then tested on an animal. Answers are obtained, analyzed, and published based on the hypothesis. Although true in many cases, caution must be exerted in assuming that a close phylogenetic relationship or anatomical similarity guarantees an identical biochemical or physiological response in the animal. In addition, it must be realized that humans to whom the results are being extrapolated are genetically highly variable due to cultural, dietary, and environ-

14

SECTION I / INTRODUCTION

mental differences. This is of minor importance when developing disease models but is highly important for pharmacological and toxicological models. So, how can the validity of extrapolation be verified? Complete reliability cannot be guaranteed; however, following the following vital requirements will help to avoid several of the mistakes of the past and overcome problems of the future: • Taking a plurispecies approach. Most of the regulating authorities require two species in toxicology screening, one of which has to be nonrodent. This does not necessarily imply that excessive numbers of animals will be used. The uncritical use of one-species models can mean that experimental data retrospectively turn out to be invalid for extrapolation, representing real and complete waste of animals. Using more than one species is, of course, no guarantee for successful extrapolation either. • Metabolic patterns and speed and body size must match between species. The use of laboratory animals as models for humans is often based on the premise that animals are more or less similar with respect to many biological characteristics and thus can be compared with humans. However, there is one striking difference between mouse and human, and that is body size. In proportion to their body size, mammals generally have very similar organ sizes expressed as percentage of body weight. Take the heart for instance, which often constitutes 5 or 6 g per kilogram of body weight, or blood, which is often approximately 7% of total body weight. It is well known that the metabolic rate of small animals is much higher than that of large animals and, thus, provisions must be made to adjust the study accordingly. Drugs and toxins exert their effect on an organism not per se but because of the way that they are metabolized, the way that they and their metabolites are distributed and bound in the body tissues, and how and when they are finally excreted. Adjusted doses should include the following provisions: If the object is to achieve equal concentrations of a substance in the body fluids of animals of different body size, then the doses should be calculated in simple proportion to the animals’ body weights. If the object is to achieve a given concentration in a particular organ over a certain time period, the calculation of dosage becomes more complicated, and other factors, including the physicochemical properties of the drug, become important. Metabolism or detoxification and excretion of a drug are not directly correlated with body size but, more accurately, to the metabolic rate of the animal. Some species react with particular sensitivity toward certain drugs, and marked variations in the reaction of animals within a species occur with respect to strain, pigmentation, nutritional state, stress level, type of bedding, ambient temperature, age, sex, route or time of administration and sampling, diurnal variation, and season of the year. As much as possible, these items must be controlled.4 • Experimental design and the life situation of the target species must correspond. A model cannot be separated from the experimental design itself. If the design inade䊊

䊊

䊊

䊊

quately represents the “normal” life conditions of the target species, inaccurate conclusions may be drawn, regardless of the value of the model itself.

DESCRIPTION OF ANIMAL MODELS16 Unlike the old days when the researcher could write in the materials and methods section “black mice were used in the study,” modern obligations require an exact description of the model. The description should include the following. • Genetic strain and substrain using correct international nomenclature.26,27 • Special genetic features. • Microbial status of the animal. • Age. • Housing standards. • Maintenance procedures. • Diet. • If used in infectious disease studies, the description should also include Strain of the organism. Method of inoculum preparation. Route of inoculation. 䊊 䊊 䊊

CONCLUSIONS Laboratory animals play a crucial role in research discovery and technological advances, and they will continue to take part in improving the lives of people and other animals. It is incumbent upon the researcher to know the subject well in order to provide relevant information to the scientific world. The final judgment in the choice of the animal model will always be in its ability to elucidate and predict the observed effects in the target species.

REFERENCES 1. Americans for Medical Progress. Everyday wonders. (Accessed August 16, 2006, at http://www.amprogress.org.) 2. Bernard C. Introduction to the Study of Experimental Medicine. Mineola, NY: Dover Publications, 1957. 3. Foundation for Biomedical Research. Nobel prizes: The payoff from animal research. (Accessed on August 30, 2006, at http://www.fbresearch.org/education/nobels.htm.) 4. Hau J. Animal models. In: Hau J, Van Hoosier GL, Eds. Handbook of Laboratory Animal Science, 2nd ed., Vol. II. Boca Raton, FL: CRC Press, 2003:1–9. 5. Connan F, Campbell IC, Katzman M, Lightman SL, Treasure J. A neurodevelopmental model for anorexia nervosa. Physiol Behav 2003;79:13–24. 6. Immunodeficient rodents opening new doors for investigators. Res Anim Rev 1996;1(2):1–8. 7. Pinkert CA. The history and theory of transgenic animals. Lab Anim 1997;26:29–34. 8. Yan Y, Panos JC, McCormick DJ, et al. Characterization of a novel H2A(−)E+ transgenic model susceptible to heterologous but not self thyroglobulin in autoimmune thyroiditis: Thyroiditis transfer with Vbeta8+ T cells. Cell Immunol 2001;212(1):63–70. 9. Meng XJ. Discoveries of Animal Strains of Hepatitis E Virus: Implications for Animal Models and Zoonosis. Ithaca, NY: American College of Veterinary Pathologists and American Society for Veterinary Clinical Pathology, 2004. (Accessed at http://www.ivis.org.) 10. St. George-Hyslop PH, Westaway DA. Antibody clears senile plaque. Nature 1999;400:116–117. 11. Willner P. The validity of animal models of depression. Psychopharmacology 1984;83:1–16.

CHAPTER 2 / SELECTION OF BIOMEDICAL ANIMAL MODELS

12. Sagvolden T, Russel VA, Aase H, Johansen EB, Farshbaf M. Rodent models of attention-deficit/hyperactivity disorder. Biol Psychiatry 2005;57:1239–1247. 13. Bayne K, deGreeve P. An overview of global legislation, regulation, and policies on the use of animals for scientific research, testing, or education. In: Hau J, Van Hoosier GL, Eds. Handbook of Laboratory Animal Science, 2nd ed., Vol. I. Boca Raton, FL: CRC Press, 2003:31–50. 14. Fleischauer AT, Ellis B, Ostorff S, Hemphill MS. The select agents and toxins interim final regulations: Applications to the small animal facility. Contemp Top Lab Anim Sci 2003;42(4):126–140. 15. Animal models and strains search engine. Washington, DC: Institute for Laboratory Animal Research. (Accessed August 16, 2006, at http://dels.nas.edu/ilar_n/ilarhome/search_amsst.shtml.) 16. Davidson MK, Lindsey JR, Davis JK. Requirements and selection of an animal model. Isr J Med Sci 1987;23:551–555. 17. Russell WMS, Burch RL. The Principles of Humane Experimental Technique. London: Methuen, 1959. 18. Environmental control for animals and plants. In: ASHRAE Handbook. Atlanta, GA: American Society of Heating, Refrigerating & Air Conditioning Engineers, Inc., 2005:Chapter 10. 19. Rose, MA. Environmental factors likely to impact on an animal’s well-being-an overview. In: Baker RA, Jenkin G, Mellor DJ, Eds.

20. 21. 22. 23. 24. 25.

26.

27.

15

Improving the Well-Being of Animals in the Research Environment. Adelaide, Australia: ANZCCART, 1994:284–301. Inan SY, Aksu F. Amnesic effects of relative humidity and temperature in mice. Lab Animal 2002;31(2):40–48. Bailey DW. Genetic drift: The problem and its possible solution by frozen-embryo storage. Ciba Found Symp 1977;291–303. NIH guidelines for research involving recombinant DNA molecules. Bethesda, MD: National Institutes of Health, 2002. Morton DB, Abbot D, Barclay R. Removal of blood from laboratory mammals and birds. Lab Anim 1993;27:1–22. Weisbroth SH. Post-indigenous disease: Changing concepts of disease in laboratory rodents. Lab Anim 1996;25:25–33. Baker DG. Natural pathogens of laboratory mice, rats, and rabbits and their effects on research. Clin Microbiol Rev 1998;11(2): 231–266. Mouse Genome Informatics. Rules for nomenclature of mouse and rat strains, 2005. (Accessed August 30, 2006, at http://www.informatics.jax.org/mgihome/nomen/strains.shtml.) Mouse Genome Informatics. Rules for nomenclature of genes, genetic markers, alleles, and mutations in mouse and rat, 2005. (Accessed August 30, 2006, at http://www.informatics.jax.org/ mgihome/nomen/gene.shtml.)

3

Improved Models for Animal Research ROLF SPRENGEL, LEONID ESHKIND, JAN HENGSTLER, AND ERNESTO BOCKAMP

ABSTRACT

expression, are examples that can prevent the generation of a useful animal model. To overcome these limitations and more precisely control gene expression or gene deletion in a tissue- and time-specific fashion, conditional mouse models are used and are becoming increasingly popular. Therefore, these secondgeneration models may significantly improve our ability to examine gene function in vivo. In this chapter, we will discuss different conditional transgenic and gene-targeting techniques, as well as provide a brief overview regarding conventional mouse transgenesis and germ line gene targeting. In addition, we will examine the emerging technology to knock down gene expression in vivo through small interfering RNA (siRNA) methods. While this chapter is not intended to be comprehensive or to provide specific technical details, we encourage the interested reader to explore more focused reviews on this topic.1–6

Experimental animal models are critical to understand gene function and human disease. Many rodent models are presently available providing avenues to elucidate gene function and/or to recapitulate specific pathological conditions. To a large extent, successful translation of clinical evidence or analytical data into appropriate mouse models is possible through progress in transgenic or gene deletion technology. Despite these significant improvements, major limitations still exist in manipulating the mouse genome. For this reason and to maximize success, the design and planning of mouse models need good knowledge concerning the requirements and limitations of commonly used strategies and emerging technologies. The purpose of this chapter is to provide a current overview of strategies for manipulating the mouse genome. Key Words: Transgenic mice, Knockout mice, Conditional mouse models, Cre and Flp recombinase, Tetracycline system, RNAi, Knockdown mice, Functional genomics.

STANDARD MOUSE TRANSGENESIS Since the pioneering work from Gordon and colleagues reporting the successful generation of transgenic mice by microinjection of DNA into the pronucleus of one-cell embryos, the genetic manipulation of the mouse embryo has been extremely useful for creating thousands of murine models for biomedical research.7 Today, two different methods are routinely used for generating genetically modified mice.

INTRODUCTION One of the central issues facing biomedical research is the need to transform in vitro data into knowledge about gene function in mice or humans. In this respect genetically engineered laboratory mice are an excellent tool for modeling genetic disorders, assigning function to genes, evaluating the action of drugs and toxins, and answering fundamental questions in basic science. Animal models account for factors such as age, stress, cell-to-cell communication, pathogen–host interaction, physiology, immune response, brain function, and other key issues, providing an advantage over in vitro assays or computer models. For example, tumor initiation, progression, and spreading cannot be recapitulated in vitro but can be addressed with animal models. Nevertheless, animal models represent only an experimental surrogate and results obtained from mouse experiments do not necessarily recapitulate the human situation. Furthermore, introducing genetic changes to the germ line of the mouse may indeed identify gene function, but also might result in severe developmental consequences, complicating or preventing analysis. Embryonic lethal phenotypes, frequently associated with null alleles or compensatory pleiotropic gene expression, induced through the absence or increased gene

TRANSGENIC MICE First there is microinjection of recombinant DNA into the pronuclei of fertilized mouse eggs or infection of germ cells/early embryos with viral vectors carrying the foreign gene.3,8 With this technique the introduced DNA is more or less randomly inserted in one or multiple copies into the mouse genome. Typically, the recombinant constructs used for pronucleus injection are composed of a selected DNA sequence linked to a promoter or promoter/enhancer combination that determines the expression pattern and expression level of the selected DNA sequence in a given tissue or developmental stage. Using this approach, it has been possible to characterize the function of well-defined transgenic gene products, dominant negative or constitutively active gene mutations, or specifically designed proteins. Furthermore, in vivo suppression of a particular endogenous gene can be achieved by transgenic expression of antisense mRNA, ribozymes, and small hairpin (shRNAs) or micro-RNAs (miRNAs).2,9–15 Besides gene products, transcriptional control elements as enhancers,

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

17

18

SECTION I / INTRODUCTION

silencers, promoters, or complete locus control regions can be studied using transgenic mice.16–18 One recurrent problem observed in transgenic mouse models are variegational position effects often disturbing or masking the specific function of the used transcriptional control elements. Theoretically, it might be assumed that after stable integration into the genome the recombinant DNA should readily manifest its predicted mission. However, many experiments reveal that the genetic surrounding of the inserted transgenic construct is modulating the expression pattern of the transgene itself.19 Obvious reasons for positional variegation are that the injected DNA (1) integrates into or near an endogenous gene locus with strong transcriptional control activity, affecting in cis the promoter of the transgenic construct or (2) integrates in a chromatin structure prone to be inactivated during development, which would lead to silencing of the transgenic construct. To avoid this recurring issue, single-copy integration of transgenes into a selected target locus or the use of so-called insulator elements has been reported.20,21 Alternatively, large recombinant constructs like bacterial artificial chromosomes (BACs) or yeast artificial chromosomes (YACs) can be used for generating position-independent transgenic mouse lines.22,23 As one of the consequences of the genome sequencing projects, a collection of well-characterized BAC and YAC clones that cover nearly the entire human and mouse genomes is now available (for example, http://www.rzpd.de/products/clones; http://bacpac.chori.org; http://www.sanger.ac.uk). In addition to that, convenient methods for site-specific modification of BACs and YACs have been established.24–27 However, it has to be emphasized that by the random chromosomal insertion of BAC or YAC constructs endogenous gene loci may be destroyed and also the expression of neighboring genes might be modified.28 In case these endogenous genes will be indispensable to life, this will become obvious when the transgenic mouse variant can be maintained only as a heterozygous line. For the above reasons, a correlation between phenotype and transgene function is obvious only when at least two independent transgenic mouse lines with identical BAC, YAC, or transgenic constructs show the same phenotype. If only one transgenic line is analyzed, it can never be completely excluded that the observed phenotype is not linked to the transgene itself but reflects the compromised expression of neighboring endogenous genes.

CONVENTIONAL GENE TARGETING The second widely used method for the generation of gene manipulated mice makes use of pluripotent mouse embryonic stem (ES) cells. Targeted gene modification is built on the finding that mammalian cells have the enzymatic machinery for exact homologous recombination between identical (homologous) DNA sequences.29–31 Thus precise predefined modifications of an endogenous gene are possible in cultivated mammalian cells including mouse ES cells. Genetically modified ES cells can then be used to generate gene “knockout” or “knockin” mice that carry the planned DNA modification.32 Normally, pluripotent ES cells are injected in early mouse embryos at the blastula stage or aggregated with morula stage embryos. During embryonic development the ES cells participate in the formation of different tissues including the germ cells and as a result the embryo is composed of two different genetic backgrounds, derived either from the wild-type cells or the genetically modified ES cells. However, in case ES cells have contributed to the germ cells of the animal, offspring

from these chimeric mice will harbor the desired genetic modification in the germline (germline transmission). To generate mice with a defined background, inbreeding with animals of the same genetic background as the original ES cell will produce genetically identical homozygous offspring. However, mice crossed to commonly used ES cell mouse backgrounds often poorly reproduce and are therefore difficult to expand. For this reason, for most experiments chimeric mice are crossed to mouse strains with good breeding efficiencies. Typically, the targeting construct for homologous recombination is composed of a central core region flanked by two regions— the so-called homology arms—that are identical in sequence to the nucleotide sequence of the target region in the genome. The homology arms are required for correct site-specific integration or replacement of the endogenous gene segment by the DNA of the targeting vector. The core region of the targeting construct incorporates the planned genetic modification together with a positive selection cassette, conferring resistance to ES cells containing the targeting construct. Most of the genomic insertions of the targeting vector are random, somewhere in the genome. In a few cases a replacement of the gene segment by the targeting vector takes place. Therefore, enrichment strategies for correctly recombined ES cell clones have been developed. These enrichment strategies make use of negative selection cassettes such as the herpes simplex type 1 thymidine kinase gene or the gene for the diphtheria toxin α-chain. The negative selection cassettes are placed outside the homology arms and are lost during homologous recombination, whereas during random integration the entire targeting vector including the negative selection marker is inserted into the genome of the ES cell. This permits a counterselection against randomly integrated clones and leads to an enrichment of ES cell clones with correct trageting.33–35 For complex multistep genome manipulations including large chromosomal deletions or translocations convenient combinations of different selection markers have been described.34 Most of the published gene targeted mice are summarized in several electronically searchable databases in the internet (see http://www.bioscience.org/knockout/knochome.htm, http://www. nih.gov/science/models/mouse/index.html, and http://www.informatics.jax.org/imsr/index.jsp). In addition to the already existing gene targeted mice, academic institutes and commercial companies have generated gene trapped ES cell libraries that can be used as a source for generating knockout mice. Each individual ES cell clone of such a library harbors a single integration of a viral construct, which in turn serves as a signpost for identification of the trapped gene. Recently, the major gene trapping groups have centralized the access to all publicly available gene trap ES cell lines.36 In this portal (www.genetrap.org) a collection of at the time 45,000 well-characterized ES cell clones is available on a noncollaborative basis. In addition, gene-trap-derived gene-specific knockout ES cell clones are commercially obtainable (http://www.lexicon-genetics.com/ discovery/omnibank_ebiology.htm). Importantly, studies involving gene targeted mice have to consider the genetic background. Depending on the mouse strain that was used the in vivo function or loss of function of a particular gene can be very different.37,38 For example, γ-protein kinase C knockout mice showed a considerable difference in sensitivity to ethanol that was completely dependent on the genetic background.39 This example illustrates that the effect of a single genetic

CHAPTER 3 / IMPROVED MODELS FOR ANIMAL RESEARCH

alteration can be strongly affected by other genes and that the phenotype of a knockout mice is not a strict readout of the missing or destroyed gene but is the concerted action of the remaining genes. Therefore, in traditional genetics a phenotypic impairment of a gene knockout can be taken as just a hint for the function of a particular gene. The definitive proof is a rescue of the impaired phenotype of the knockout by, e.g., transgenic reexpression of the destroyed gene. However, the transgenic rescue of knockout mice is time consuming, expensive, and labor intensive. A simple shortcut strategy is usually applied to minimize the contribution of the individual genetic background. Knockout mice with the genetic origin of the 126SV mouse strain are crossed with C57BL6 or other inbred strains to generate F1 hybrids that contain 50% 129SV and C57BL6 as genetic background. Next, the F1 hybrids are intercrossed to generate knockout, heterozygous, and wild-type littermates. By analyzing knockout and control littermates from the F1 hybrids, the influence of the genetic background is minimized since it will be very different from animal to animal. However, if this approach is used, the number of analyzed mice has to be high. As an alternative, the phenotype of the knockout can be analyzed in two very different genetic backgrounds, e.g., C57BL6 versus DBA. For this, C57BL6 and DBA congenic animals have to be generated. Congenic animals are genetically identical except for the modified region. However, 2–3 years of backcrossing to the defined mouse strain are necessary to achieve a statistically >99% homogeneous genetic background.40 The Jackson Laboratory and the Center for Inherited Disease Research offer possible shortcuts for this time-consuming procedure that will approximately half the number of backcrosses (for more information about marker-assisted breeding see http://jaxmice. jax.org/services/speedcongenic.html and http://www.cidr.jhmi. edu/mouse/mouse_strp.html).

CONDITIONAL MOUSE MODELS In conventional transgenic and gene knockout mice studies the clear-cut gene-function relation is hampered by several factors. Two of those factors were already discussed: the positional variegation and the genetic background. In addition, it has to be considered that the genetic manipulation is set in the early embryo at the one-cell stage. Therefore, the lack or malfunction of the manipulated gene can disturb the development. Consequently, the phenotype in adult mice might be caused by this abnormal development and not by the lack of proper gene function in adults. Likewise, the dysfunction of a particular gene can lead to the upor downregulation of compensatory genes. Those pleiotropic compensatory effects would than mask a phenotype in adults. A gene regulation system that permits the induction or deletion of a gene in a specific tissue or cell type at any given time would erase all the issues mentioned above. With a controlled gene switch it would be possible to study the function of a gene in a single animal before and after the gene is switched on or off. In an “off” and “on” state the genetic background and integration site are the same; the animal can develop normally since the gene switch can be turned after development and the time window for compensatory mechanism is narrow. For this reason, it is obvious that the use of conditional mouse models is a much better choice than relying on conventional transgenic or knockout approaches. To decide which conditional animal model is most suitable, a detailed knowledge of the different established systems is essential. First, it is important to

19

define the criteria that have to be met. The perfect regulatory system (1) should allow a tightly controlled regulation of the target gene without background activity, (2) should be reversible, (3) should be fast in induction kinetics, (4) should be effective in all target cells, and (5) should use a highly specific and nontoxic inducer. Among the growing number of different conditional in vivo systems, the tetracycline (tet) regulated and the Cre-mediated site-specific recombination systems are most commonly used.1,41,42 The scientific community has made a considerable effort to provide public resources comprising conditional mouse mutants (http://nagy.mshri.on.ca/PubLinks/indexmain.html and http:// www.zmg.uni-mainz.de/tetmouse/index.htm). The activity of these initiatives provided the basis for sharing resources, valuable animal strains, and scientific information beyond the scientific scope of individual research projects. Taking a closer look at these resources prior the initiation of a conditional mouse project is therefore good practice and saves time and effort.

THE TET SYSTEM Pioneering work from Gossen and Bujard established a general application of the Escherichia coli Tn10-derived tet resistance operon for regulating transgenic expression in mammalian cells.43 In the TET system the tet repressor (TetR) is fused to the transcriptional transactivation domain VP16 of the herpes simplex virus, giving rise to the tet-controlled transactivator (tTA). In the absence of tetracycline [or other tetracycline derivatives like doxycycline (DOX), anhydrotetracycline, or 4-epidoxycycline], tTA specifically binds to the tTA-dependent promoter (Ptet) to initiate transcription of the Ptet-controlled transgene. Normally, Ptet is composed of seven copies of the tet operator consensus sequence (tetO) and a transcription initiation site (in most cases from the CMV immediate early promoter) that is placed close to the translational start codon of the Ptet transgene. If tetO7 is flanked by two transcription initiation sites (Ptet-bi), two genes can be regulated simultaneously by tTA.44 Adding DOX to the system induces a conformational change of the transcriptional activator tTA, prevents its DNA binding, and at the Ptet promoter transcription is shut down. Thus the TET system requires two building blocks: as an effector the tet-dependant transactivator tTA and as responder a Ptet promoter-regulated gene. The well-established pharmacological properties of tetracycline and its derivatives suggest that these compounds are safe and reliable regulators for tet-controlled gene function in vivo. The properties include predictable pharmacokinetics, good bioavailability and tissue distribution, known half-life times, lack of toxicity at the established working concentrations, and the ability to cross mammalian cell membranes, the blood–brain barrier, and the placenta. Until recently the only downside of using tetracycline or tetracycline derivatives in mice was a possible imbalance of the intestinal flora, which may result in diarrhea and to a less extent in colitis due to the antibiotic action of these compounds. In case unwanted antibiotic action of tetracycline or tetracycline derivatives poses a problem, 4-epidoxycycline, which is equally suitable for controlling tet on/off gene switches but lacks antibiotic activity, can be used instead.45 Almost immediately after the initial publication of the tTA “TET off” system in tissue culture experiments, the first report of a tet-responsive conditional mouse model provided evidence for the enormous potential of this system for reversibly controlling

20

SECTION I / INTRODUCTION

gene expression in vivo.46 Although this first tet-based mouse model sparked much enthusiasm, certain limitations of the TET system became obvious. Major problems included residual background activity of transgene expression in the off state, cellular toxicity of the original transactivator, low sensitivity to DOX in certain tissues, internal cryptic splice acceptors, suboptimal eukaryotic codon usage, and slow in vivo induction kinetics. During the last decade these problems have been addressed and an exquisite toolbox for the generation of tet-based conditional mouse models is available now. The first major improvement of the TET system was the development of the reverse “TET on” transactivator (rtTA), which is activated and induces transcription of Ptet responder genes upon DOX administration.47 Using the rtTA system in transgenic mice increased the speed of transgene induction, reaching in some tissues complete Ptet–gene activation in 1 h as compared to the slow activation kinetics of up to 1 week for tTA.48,49 This marked difference in responsiveness between the tTA and rtTA system in vivo is explained by the metabolic half-life time of DOX in different tissues, i.e., by the fact that in certain tissues it will take some time before DOX is completely eliminated. This difference in induction kinetics can be a major determinant when designing tet-based mouse models. Using the tTA “TET off” system might permit fast shut down and slow induction kinetics contrasting with the rtTA “TET on” system suitable for immediate induction and slow extinction of tet-controlled transgene expression. An additional initial drawback of the TET system was the occasionally observed leakiness leading to unwanted Ptet–gene expression. The Ptet leakiness was addressed by developing antagonistic repressors that silence any residual Ptet–gene expression in the noninduced state. The antagonistic repressors are heterologous fusions of a tet-responsive DNA-binding domain with a strong transcriptional repressor, capable of silencing the residual activity of a Ptet50,50a. This strategy uses an antagonistic combination of tTA and the repressor that—depending on the administration of DOX and either tTA or the repressor—exclusively binds to tetO7. One way to avoid leakiness of Ptet is the generation of a tet repressor mouse line that ubiquitously expresses the tetresponsive repressor and the crossbreeding of this Ptet silencer line to the leaky tet-responder mouse51 (unpublished data from our laboratory). Alternatively, it is also possible to use constructs that express both a transactivator and an antagonizing repressor.51–54 Finally, engineered responder cassettes containing repositioned TetO elements have also been reported to minimize leakiness of the TET regulatory system.55,56 High tTA and rtTA expression can lead to cytotoxicity, most probably due to pleiotropic effects of high levels of the VP16 transactivation domain.57 Therefore, a second generation of tTAs and rtTAs was developed.58–60 Remodeling tTA and rtTA improved several features of the TET system including increased DOX sensitivity, faster activation kinetics, improved codon usage, abrogation of cryptic splice sites, and extended DNA-binding and dimerization properties.56,61,62 These remarkable advances helped to optimize the design of conditional mouse models and to adjust the experimental setup according to the specific needs of the planned experiment. Since the first report of a tet-controlled transgenic mouse model, the TET system has become increasingly popular. Today the huge number of tet-based mouse models represent an invaluable resource for combinations of tissue-specific or generalized

tTA (rtTA) expressing and Ptet responder mice. However, the scope of this chapter does not allow for a detailed description of already generated “tet on/off” mouse models. The reader is encouraged to visit the specialized electronic databases as an additional resource (http://www.zmg.uni-mainz.de and http:// www.tetsystems.com). Besides its fundamental appeal as an established conditional regulatory system in mice, the “TET on/off” system has attracted considerable interest because of its unique potential of reversible gene regulation. Consequently, the TET system will be an excellent choice for the study of gene function relationships, for example, in behavioral neuroscience.

SITE-SPECIFIC RECOMBINASES The first site-specific recombination system that was used in the mouse was the Cre/loxP system.63,64 Initially discovered in the filamentous P1 phage, the Cre/loxP system has become a valuable tool for the induction of site-specific DNA recombination in vivo.65 Based on the enzymatic activity of Cre (for “causes recombination”) and depending upon the position and orientation of loxP sites (for “locus of crossover P1”), mice carrying gene deletions, duplications, inversions, or chromosomal translocations can be generated.66,67 In a similar way the Saccharomyces cerevisiae recombinase Flp can be used for site-specific recombination between FRT (for “Flp recombination target”) sites in mice.68 Both the Cre and Flp recombinases have been applied for the induction of ubiquitous or cell-specific recombination in mice. The success of any recombinase-mediated conditional in vivo experiment depends on the “DELETER” strains that express the Cre recombinase. However, some of Cre expressing mouse lines are “leaky,” with widespread instead of specific Cre-mediated recombination. Consequently, recombination will not be restricted to the anticipated cell type but will also take place elsewhere. In most cases this effect is due to the early developmental activity of the promoter driving Cre expression. Drug-inducible Cre recombinases can bypass unwanted Cre expression during development and can be used as conditional gene switches in vivo. This can be achieved by tTA-controlled Cre expression in triple transgenic mice. These mice harbor a tissue-specific tTA transgene, a PtetO controlled Cre, and the floxed target gene.69,70 Another option is Cre recombinase fusions with mutant estrogen receptor binding domains that selectively bind 17β-estradiol analogues like tamoxifen.41,71 In this scenario Cre–estrogen receptor fusion proteins are retained in the cytoplasm but are translocated to the nucleus upon addition of the synthetic ligand. As in the TET system, protein engineering provided better recombinases including codon improved Cre and Flp enzymes, Flp recombinases with better in vivo activities in mammals, and modified recombinases recognizing novel target sites.68,72–75 Again, before starting experiments, writing grants, and planning the generation of novel transgenic or knockout mice, it will be rewarding to have a closer look at already established resources (http://nagy. mshri.on.ca/PubLinks/indexmain.html). The application of mouse lines with low Cre activity revealed that an incomplete rearrangement of the target sequence can also depend on the accessibility of the loxP sites in the target gene.76 In addition, the observed recombination efficiencies at any given Cre indicator locus can be taken as only a first hint for the useful-

CHAPTER 3 / IMPROVED MODELS FOR ANIMAL RESEARCH

ness of a particular recombination system. Therefore, it is good practice to analyze the efficiency of the Cre-mediated recombination at the target gene for each series of experiments.

RNA INTERFERENCE RNA interference (RNAi) is a sequence-specific eukaryotic surveillance mechanism initiated by double-stranded RNA that ultimately will result in the repression of specific target genes.77,78 Building on the observation that in mammalian cells doublestranded RNAs (dsRNAs) of less than 30 bp will induce specific degradation of a target mRNA but will not trigger a general translational block or interferon response, RNAi knockdown technology has become very popular.79 In 2002 Hasuwa and colleagues reported the generation of knockdown mice and rats, thus providing direct evidence that RNAi technology can be used for studying the functional consequences of gene inactivation in animal models.80 The relative simplicity, speed, and cost-effectiveness of using RNAi for studying gene function in vivo have sparked great expectations and have led to the development of an increasing number of novel tools.2,81–84 However, there are several important issues to be considered. First, in vivo knockdown strategies might significantly deplete the mRNA levels of the targeted gene but might not be sufficient to abolish gene function. Second, in different tissues the level of knockdown might be variable, also being determined by the targeted sequence, copy number of the inserted RNAi transgene, and its particular integration site85 (unpublished observations from our laboratory). Nevertheless, a partial knockdown of a gene function can be of advantage since “mild” phenotypes might in some cases be more suitable for uncovering gene function as compared to a knockout.86 Third, an additional concern is specificity. Indeed, RNAi-mediated knockdown may not be limited to the selected target gene but instead may also affect the function of other genes, an effect known as off-targeting.87 This issue is further compounded by the fact that our present general understanding of the different mechanisms involving short RNAs, like micro-RNAs (miRNAs) or repeat-associated short interfering RNAs (rasiRNAs), remains sketchy. For this reason, the possibility that a particular small RNA might inadvertently bring on unwanted gene regulatory effects other than the sequence-specific knockdown of the target gene cannot be excluded. For this reason, it will be essential to implement workable and standardized controls before interpreting and validating RNAi data.88 Finally, highlevel shRNA or miRNA expression might compete for limiting cellular factors and thus severely interfere with endogenous siRNA regulatory pathways.89 Two alternative strategies have been used for gene selective knockdown in mice: cytoplasmic delivery of synthetic short siRNA oligonucleotides mimicking the active intermediate of an endogenous RNAi mechanism or the stable expression of recombinant shRNAs or miRNAs . The first approach uses small molecules, expression constructs, or viral particles that are injected or infused in a more or less transient fashion.90–96 Possible applications for transient knockdown experiments in mice are the testing of siRNAs as gene therapeutic tools for humans or their use in antiviral treatments.82,97,98 For many applications, however, the use of germ-line transmitting knockdown mice might be the preferred option. In this case the knockdown can be either mediated through ubiquitous expression of shRNAs using RNA polymerase III or tRNA promoters

21

or, alternatively, through miRNA expression from housekeeping or tissue-restricted RNA polymerase II promoters. During the past years several groups have reported the generation of germline-transmitting knockdown mouse lines.99–101 The methodology used for the generation of these mice is similar to the approaches discussed above including standard transgenesis or gene targeting. Likewise, conditional activation of shRNA or miRNA expression in mice has been demonstrated.13,14,102–104 With conditional RNAi systems knockdown-induced lethality can be bypassed and loss of gene function can be studied during any given time window and/or in specific tissues. At present, possible avenues for implementing conditional knockdown in mice either allow the permanent induction/ extinction of the knockdown (Cre/loxP-based systems) or facilitate the reversible induction of a knockdown (tet on/off-based systems). The first strategy reported for inducing conditional knockdown phenotypes in mice relied on Cre-mediated activation or extinction of shRNA expression.14,102,103 In this situation a transcriptional and translational stop element (loxP-STOP-loxP) is placed between the shRNA transcription start site and the upstream regulatory elements, needed for shRNA expression. Upon removal of the loxP-STOP-loxP element by Cre, transcription of the shRNA or miRNA is set off and the knockdown is initiated. Conversely, this strategy also allows for conditionally removing an actively transcribing shRNA expression element and thus permanently extinguishing the knockdown.14 The alternative approach for generating conditional knockdown mice is to use drug-controlled systems. In this respect TET systems were applied for regulated shRNA and miRNA expression in mice. In a recent publication Szulc and colleagues demonstrated reversible silencing of an RNA polymerase IIItranscribed shRNA by virtue of a DOX-dependent KRAB repressor.13 Although RNA polymerase III promoter-based conditional strategies are normally efficient in all cell types and provide good knockdown levels, a limitation of these systems is their lack of tissue specificity. The development of RNA polymerase II promoter-mediated miRNA-based shRNA TET on/off systems overcomes this limitation and initial in vivo experiments demonstrated an application of this approach.105 Similarly, recent documentation of second-generation shRNA- or miRNA-based libraries that cover most if not all predicted mouse genes provides an excellent resource for constructing conditional RNAi mouse models.106,107

CONCLUDING REMARKS Within the last few years, researchers have expanded the toolbox to manipulate the expression of mouse genes by developing several novel techniques, including RNAi knock-down, improved tools for conditional gene regulation in vivo, and the ability to engineer large BAC clones by “recombineering”. In addition, searchable databases, providing detailed information on established mouse lines, together with available collections of pre-characterized ES cell clones and libraries, will facilitate the use of genetically modified mice. Although, this chapter only provides a general overview on the use and limitations of mouse models, we hope the here provided information will serve as a primer illustrating the exciting possibilities and exquisite tools available for in vivo investigating gene function in mice.

22

SECTION I / INTRODUCTION

REFERENCES 1. Bockamp E, Maringer M, Spangenberg C, et al. Of mice and models: Improved animal models for biomedical research. Physiol Genomics 2002;11:115–132. 2. Prawitt D, Brixel L, Spangenberg C, et al. RNAi knock-down mice: An emerging technology for post-genomic functional genetics. Cytogenet Genome Res 2004;105:412–421. 3. Nagy A, Gertsenstein M, Vintersten R, Behringer M. Manipulating the Mouse Embryo—A Laboratory Manual, 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Press, 2002. 4. Sung YH, Song J, Lee HW. Functional genomics approach using mice. J Biochem Mol Biol 2004;37:122–132. 5. Ristevski S. Making better transgenic models: Conditional, temporal, and spatial approaches. Mol Biotechnol 2005;29:153–163. 6. Glaser S, Anastassiadis K, Stewart AF. Current issues in mouse genome engineering. Nat Genet 2005;37:1187–1193. 7. Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci USA 1980;77:7380–7384. 8. Rulicke T. Pronuclear microinjection of mouse zygotes. Methods Mol Biol 2004;254:165–194. 9. Efrat S, Leiser M, Wu YJ, et al. Ribozyme-mediated attenuation of pancreatic beta-cell glucokinase expression in transgenic mice results in impaired glucose-induced insulin secretion. Proc Natl Acad Sci USA 1994;91:2051–2055. 10. Erickson RP. Antisense transgenics in animals. Methods 1999;18:304–310. 11. Kashani-Sabet M, Liu Y, Fong S, et al. Identification of gene function and functional pathways by systemic plasmid-based ribozyme targeting in adult mice. Proc Natl Acad Sci USA 2002;99:3878–3883. 12. Larsson S, Hotchkiss G, Andang M, et al. Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme. Nucleic Acids Res 1994;22:2242–2248. 13. Szulc J, Wiznerowicz M, Sauvain MO, Trono D, Aebischer P. A versatile tool for conditional gene expression and knockdown. Nat Methods 2006;3:109–116. 14. Ventura A, Meissner A, Dillon CP, et al. Cre-lox-regulated conditional RNA interference from transgenes. Proc Natl Acad Sci USA 2004;101:10380–10385. 15. Stegmeier F, Hu G, Rickles RJ, Hannon GJ, Elledge SJ. A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proc Natl Acad Sci USA 2005;102:13212–13217. 16. Sanchez MJ, Bockamp EO, Miller J, Gambardella L, Green AR. Selective rescue of early haematopoietic progenitors in Scl(-/-) mice by expressing Scl under the control of a stem cell enhancer. Development 2001;128:4815–4827. 17. Navas PA, Swank RA, Yu M, Peterson KR, Stamatoyannopoulos G. Mutation of a transcriptional motif of a distant regulatory element reduces the expression of embryonic and fetal globin genes. Hum Mol Genet 2003;12:2941–2948. 18. Sinclair AM, Gottgens B, Barton LM, et al. Distinct 5′ SCL enhancers direct transcription to developing brain, spinal cord, and endothelium: Neural expression is mediated by GATA factor binding sites. Dev Biol 1999;209:128–142. 19. Wilson C, Bellen HJ, Gehring WJ. Position effects on eukaryotic gene expression. Annu Rev Cell Biol 1990;6:679–714. 20. Bronson SK, Plaehn EG, Kluckman KD, Hagaman JR, Maeda N, Smithies O. Single-copy transgenic mice with chosen-site integration. Proc Natl Acad Sci USA 1996;93:9067–9072. 21. Giraldo P, Rival-Gervier S, Houdebine LM, Montoliu L. The potential benefits of insulators on heterologous constructs in transgenic animals. Transgenic Res 2003;12:751–755. 22. Sparwasser T, Gong S, Li JY, Eberl G. General method for the modification of different BAC types and the rapid generation of BAC transgenic mice. Genesis 2004;38:39–50. 23. Giraldo P, Montoliu L. Size matters: Use of YACs, BACs and PACs in transgenic animals. Transgenic Res 2001;10:83–103.

24. Gong S, Yang XW, Li C, Heintz N. Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6Kgamma origin of replication. Genome Res 2002;12:1992–1998. 25. Lee EC, Yu D, Martinez de Velasco J, et al. A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 2001;73:56–65. 26. Wang J, Sarov M, Rientjes J, et al. An improved recombineering approach by adding RecA to lambda Red recombination. Mol Biotechnol 2006;32:43–53. 27. Warming S, Costantino N, Court DL, Jenkins NA, Copeland NG. Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res 2005;33:e36. 28. Rijkers T, Peetz A, Ruther U. Insertional mutagenesis in transgenic mice. Transgenic Res 1994;3:203–215. 29. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154–156. 30. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 1981;78:7634–7638. 31. Smithies O, Gregg RG, Boggs SS, Koralewski MA, Kucherlapati RS. Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature 1985;317: 230–234. 32. Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 1987;51: 503–512. 33. Borrelli E, Heyman R, Hsi M, Evans RM. Targeting of an inducible toxic phenotype in animal cells. Proc Natl Acad Sci USA 1988;85: 7572–7576. 34. Chen YT, Bradley A. A new positive/negative selectable marker, puDeltatk, for use in embryonic stem cells. Genesis 2000;28: 31–35. 35. Yanagawa Y, Kobayashi T, Ohnishi M, et al. Enrichment and efficient screening of ES cells containing a targeted mutation: The use of DT-A gene with the polyadenylation signal as a negative selection maker. Transgenic Res 1999;8:215–221. 36. Nord AS, Chang PJ, Conklin BR, et al. The International Gene Trap Consortium Website: A portal to all publicly available gene trap cell lines in mouse. Nucleic Acids Res 2006;34:D642–648. 37. Phillips TJ, Hen R, Crabbe JC. Complications associated with genetic background effects in research using knockout mice. Psychopharmacology (Berl) 1999;147:5–7. 38. Sanford LP, Kallapur S, Ormsby I, Doetschman T. Influence of genetic background on knockout mouse phenotypes. Methods Mol Biol 2001;158:217–225. 39. Bowers BJ, Owen EH, Collins AC, Abeliovich A, Tonegawa S, Wehner JM. Decreased ethanol sensitivity and tolerance development in gamma-protein kinase C null mutant mice is dependent on genetic background. Alcohol Clin Exp Res 1999;23:387–397. 40. Sigmund CD. Viewpoint: Are studies in genetically altered mice out of control? Arterioscler Thromb Vasc Biol 2000;20:1425–1429. 41. Garcia-Otin AL, Guillou F. Mammalian genome targeting using site-specific recombinases. Front Biosci 2006;11:1108–1136. 42. Mallo M. Controlled gene activation and inactivation in the mouse. Front Biosci 2006;11:313–327. 43. Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 1992;89:5547–5551. 44. Baron U, Freundlieb S, Gossen M, Bujard H. Co-regulation of two gene activities by tetracycline via a bidirectional promoter. Nucleic Acids Res 1995;23:3605–3606. 45. Eger K, Hermes M, Uhlemann K, et al. 4-Epidoxycycline: An alternative to doxycycline to control gene expression in conditional mouse models. Biochem Biophys Res Commun 2004;323:979– 986. 46. Furth PA, St Onge L, Boger H, et al. Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc Natl Acad Sci USA 1994;91:9302–9306.

CHAPTER 3 / IMPROVED MODELS FOR ANIMAL RESEARCH

47. Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H. Transcriptional activation by tetracyclines in mammalian cells. Science 1995;268:1766–1769. 48. Hasan MT, Schonig K, Berger S, Graewe W, Bujard H. Long-term, noninvasive imaging of regulated gene expression in living mice. Genesis 2001;29:116–122. 49. Kistner A, Gossen M, Zimmermann F, et al. Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc Natl Acad Sci USA 1996;93:10933– 10938. 50. Freundlieb S, Schirra-Muller C, Bujard H. A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells. J Gene Med 1999;1:4–12. 50a. Bockamp E, Christel C, Hameyer D, et al. Generation and characterization of tTS-H4: a novel transcriptional repressor that is compatible with the reverse tetracycline-controlled TET-ON system. J Gene Med 2007;9:308–318. 51. Zhu Z, Ma B, Homer RJ, Zheng T, Elias JA. Use of the tetracyclinecontrolled transcriptional silencer (tTS) to eliminate transgene leak in inducible overexpression transgenic mice. J Biol Chem 2001; 276:25222–25229. 52. Hayakawa T, Yusa K, Kouno M, Takeda J, Horie K. Bloom’s syndrome gene-deficient phenotype in mouse primary cells induced by a modified tetracycline-controlled trans-silencer. Gene 2006; 369:80–89. 53. Forster K, Helbl V, Lederer T, Urlinger S, Wittenburg N, Hillen W. Tetracycline-inducible expression systems with reduced basal activity in mammalian cells. Nucleic Acids Res 1999;27:708– 710. 54. Uchida S, Sakai S, Furuichi T, et al. Tight regulation of transgene expression by tetracycline-dependent activator and repressor in brain. Genes Brain Behav 2006;5:96–106. 55. Pluta K, Luce MJ, Bao L, Agha-Mohammadi S, Reiser J. Tight control of transgene expression by lentivirus vectors containing second-generation tetracycline-responsive promoters. J Gene Med 2005;7:803–817. 56. Krueger C, Danke C, Pfleiderer K, et al. A gene regulation system with four distinct expression levels. J Gene Med 2006;8(8): 1037–1047. 57. Izumi M, Gilbert DM. Homogeneous tetracycline-regulatable gene expression in mammalian fibroblasts. J Cell Biochem 1999; 76:280–289. 58. Akagi K, Kanai M, Saya H, Kozu T, Berns A. A novel tetracyclinedependent transactivator with E2F4 transcriptional activation domain. Nucleic Acids Res 2001;29:E23. 59. Baron U, Gossen M, Bujard H. Tetracycline-controlled transcription in eukaryotes: Novel transactivators with graded transactivation potential. Nucleic Acids Res 1997;25:2723–2729. 60. Urlinger S, Helbl V, Guthmann J, Pook E, Grimm S, Hillen W. The p65 domain from NF-kappaB is an efficient human activator in the tetracycline-regulatable gene expression system. Gene 2000;247: 103–110. 61. Krueger C, Berens C, Schmidt A, Schnappinger D, Hillen W. Single-chain Tet transregulators. Nucleic Acids Res 2003;31:3050– 3056. 62. Urlinger S, Baron U, Thellmann M, Hasan MT, Bujard H, Hillen W. Exploring the sequence space for tetracycline-dependent transcriptional activators: Novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci USA 2000;97:7963–7968. 63. Lakso M, Sauer B, Mosinger B Jr, et al. Targeted oncogene activation by site-specific recombination in transgenic mice. Proc Natl Acad Sci USA 1992;89:6232–6236. 64. Orban PC, Chui D, Marth JD. Tissue- and site-specific DNA recombination in transgenic mice. Proc Natl Acad Sci USA 1992;89: 6861–6865. 65. Hamilton DL, Abremski K. Site-specific recombination by the bacteriophage P1 lox-Cre system. Cre-mediated synapsis of two lox sites. J Mol Biol 1984;178:481–486. 66. van der Weyden L, Adams DJ, Bradley A. Tools for targeted manipulation of the mouse genome. Physiol Genomics 2002;11:133–164.

23

67. Branda CS, Dymecki SM. Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. Dev Cell 2004;6:7–28. 68. Rodriguez CI, Buchholz F, Galloway J, et al. High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nat Genet 2000;25:139–140. 69. Shimshek DR, Jensen V, Celikel T, et al. Forebrain-specific glutamate receptor B deletion impairs spatial memory but not hippocampal field long-term potentiation. J Neurosci 2006;26: 8428–8440. 70. Schonig K, Schwenk F, Rajewsky K, Bujard H. Stringent doxycycline dependent control of CRE recombinase in vivo. Nucleic Acids Res 2002;30:e134. 71. Brocard J, Warot X, Wendling O, et al. Spatio-temporally controlled site-specific somatic mutagenesis in the mouse. Proc Natl Acad Sci USA 1997;94:14559–14563. 72. Shimshek DR, Kim J, Hubner MR, et al. Codon-improved Cre recombinase (iCre) expression in the mouse. Genesis 2002;32:19– 26. 73. Buchholz F, Stewart AF. Alteration of Cre recombinase site specificity by substrate-linked protein evolution. Nat Biotechnol 2001;19: 1047–1052. 74. Koresawa Y, Miyagawa S, Ikawa M, et al. A new Cre recombinase gene based on optimal codon usage in mammals: A powerful material for organ-specific gene targeting. Transplant Proc 2000;32: 2516–2517. 75. Santoro SW, Schultz PG. Directed evolution of the site specificity of Cre recombinase. Proc Natl Acad Sci USA 2002;99:4185–4190. 76. Vooijs M, Jonkers J, Berns A. A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent. EMBO Rep 2001;2:292–297. 77. Meister G, Tuschl T. Mechanisms of gene silencing by doublestranded RNA. Nature 2004; 31:343–349. 78. Tomari Y, Zamore PD. Perspective: Machines for RNAi. Genes Dev 2005;19:517–529. 79. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:494–498. 80. Hasuwa H, Kaseda K, Einarsdottir T, Okabe M. Small interfering RNA and gene silencing in transgenic mice and rats. FEBS Lett 2002;532:227–230. 81. Sandy P, Ventura A, Jacks T. Mammalian RNAi: A practical guide. Biotechniques 2005;39:215–224. 82. Lu PY, Xie F, Woodle MC. In vivo application of RNA interference: From functional genomics to therapeutics. Adv Genet 2005;54: 117–142. 83. Coumoul X, Deng CX. RNAi in mice: A promising approach to decipher gene functions in vivo. Biochimie 2006;88:637–643. 84. Wiznerowicz M, Szulc J, Trono D. Tuning silence: Conditional systems for RNA interference. Nat Methods 2006;3:682–688. 85. Hemann MT, Fridman JS, Zilfou JT, et al. An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo. Nat Genet 2003;33:396–400. 86. Rosenbauer F, Wagner K, Kutok JL, et al. Acute myeloid leukemia induced by graded reduction of a lineage-specific transcription factor, PU.1. Nat Genet 2004;36:624–630. 87. Jackson AL, Bartz SR, Schelter J, et al. Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 2003;21:635– 637. 88. Huppi K, Martin SE, Caplen NJ. Defining and assaying RNAi in mammalian cells. Mol Cell 2005;17:1–10. 89. Grimm D, Streetz KL, Jopling CL, et al. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 2006;441:537–541. 90. Matsuda T, Cepko CL. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci USA 2004;101:16–22. 91. Hommel JD, Sears RM, Georgescu D, Simmons DL, DiLeone RJ. Local gene knockdown in the brain using viral-mediated RNA interference. Nat Med 2003;9:1539–1544.

24

SECTION I / INTRODUCTION

92. Xia H, Mao Q, Paulson HL, Davidson BL. siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 2002;20:1006– 1010. 93. Kishida T, Asada H, Gojo S, et al. Sequence-specific gene silencing in murine muscle induced by electroporation-mediated transfer of short interfering RNA. J Gene Med 2004;6:105–110. 94. Song E, Lee SK, Wang J, et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med 2003;9:347–351. 95. McCaffrey AP, Nakai H, Pandey K, et al. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol 2003;21: 639–644. 96. Soutschek J, Akinc A, Bramlage B, et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 2004;432:173–178. 97. de Fougerolles A, Manoharan M, Meyers R, Vornlocher HP. RNA interference in vivo: Toward synthetic small inhibitory RNA-based therapeutics. Methods Enzymol 2005;392:278–296. 98. Leonard JN, Schaffer DV. Antiviral RNAi therapy: Emerging approaches for hitting a moving target. Gene Ther 2006;13: 532–540. 99. Kunath T, Gish G, Lickert H, Jones N, Pawson T, Rossant J. Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype. Nat Biotechnol 2003;21:559–561. 100. Rubinson DA, Dillon CP, Kwiatkowski AV, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells,

101.

102.

103.

104.

105.

106.

107.

stem cells and transgenic mice by RNA interference. Nat Genet 2003;33:401–406. Tiscornia G, Singer O, Ikawa M, Verma IM. A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc Natl Acad Sci USA 2003;100: 1844–1848. Coumoul X, Shukla V, Li C, Wang RH, Deng CX. Conditional knockdown of Fgfr2 in mice using Cre-LoxP induced RNA interference. Nucleic Acids Res 2005;33:e102. Chang HS, Lin CH, Chen YC, Yu WC. Using siRNA technique to generate transgenic animals with spatiotemporal and conditional gene knockdown. Am J Pathol 2004;165:1535–1541. Xia XG, Zhou H, Samper E, Melov S, Xu Z. Pol II-expressed shRNA knocks down Sod2 gene expression and causes phenotypes of the gene knockout in mice. PLoS Genet 2006;2:e10. Dickins RA, Hemann MT, Zilfou JT, et al. Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat Genet 2005;37:1289–1295. Moffat J, Grueneberg DA, Yang X, et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 2006;124:1283–1298. Silva JM, Li MZ, Chang K, et al. Second-generation shRNA libraries covering the mouse and human genomes. Nat Genet 2005;37:1281–1288.

GENERAL CONSIDERATIONS

II

4

The Ethical Basis for Animal Use in Research JAMES PARKER

ABSTRACT

Key Words: Ethics, Consciousness, Animals, Biomedical, Research.

The concern of biomedical researchers for the well-being of laboratory animals reflects a consensus that animals are conscious subjects. Partisans of the animal rights movement believe that researchers must go beyond the acknowledgment of consciousness and consequent attention to animal well-being. They should recognize just how similar human and animal consciousness are, and then address animal interests and animal rights. Consideration of these issues would challenge all uses, even painless uses, of animals. In any effort to identify the interests and rights of animals, even animal rights philosophers admit that the nature of consciousness and cognition—human and animal—matters. A theory of human consciousness and cognition developed by B.F.J. Lonergan is helpful in understanding and comparing human and animal consciousness. According to Lonergan, elemental wonder makes human consciousness and cognition a dynamic, self-assembling process moving from presentations given in experience through successive levels of understanding, judgment, and responsibility to the affirmation of values. Questioning sweeps humans across a divide between elementary knowing, which is shared with animals, and a type of knowing that is exclusively human. None of the animal behaviors catalogued by animal rights partisans reveals wonder and the drive to understand, affirm and decide. That drive is a single, restless activity generating successive levels of consciousness. If, as animal rights philosophers agree, animals are incapable of responsible behavior, it must also be that their cognitional feats are qualitatively different from those of humans. Absent questioning and the drive to understand, animals never emerge on the level of intelligent and rational, much less responsible, consciousness. Their cognitional achievements appear to fall within the province of elementary knowing, a realm in which they are accomplished associative learners— clever, to be sure, but not capable of the beginnings of full human knowing. Human consciousness and cognition differ enough from animal consciousness and cognition that humans claim rights while animals do not. So it is that even if we use animals in research, having vouched for their humane care, we use humans as experimental subjects only when they give informed consent. We safeguard the welfare of animals; we guarantee the rights of persons.

INTRODUCTION If René Descartes (1596–1650) had not existed, animal rights philosophers would have had to invent him. They pillory this famous mathematician/philosopher for theorizing that animal operations are mechanical, not conscious—more like the movements of swans, propelled by water pressure, in the royal gardens of Versailles than like those of humans, directed by rational choices. Such a mechanistic understanding of animals, they assert, has provided justification for using them in biomedical research, even in painful experiments—something that we would never do to humans. All philosophers today, however, are as certain as any pet owner that animals are endowed with consciousness. Except for viruses, bacteria, and, probably, insects and crustaceans, animals are aware of themselves and of the world around them that is made present by internal representations. Their movements and communication are more than the workings of mechanical stimulus and response. Biomedical scientists are no less convinced about the existence of animal consciousness. In their professional organizations, in the laws and regulations they help draft, and in their laboratory deliberations they are keenly aware of issues about the humane treatment of research subjects that animal consciousness raises.1 They follow and aid in advancing current “best practices” for ensuring animal well-being, keenly aware that in research institutions as well as in businesses or schools, there is always room for improving procedures. On the topic of the existence of animal consciousness, Descartes has no contemporary disciples, philosophical or scientific. Instead, controversy swirls around the nature and degree of that consciousness. Several philosophers believe that we must go beyond an acknowledgment of consciousness and a concern for animal well-being and take into account the human-like nature of animal consciousness, address the issue of animal interests and animal rights, and challenge any use of animals, painless though it might be.

ANIMAL INTERESTS The reputed father of the animal rights movement, Peter Singer, follows Jeremy Bentham (1748–1832), in positing that ethical reflection arises from our empathy with others in their pleasure or

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

27

28

SECTION II / GENERAL CONSIDERATIONS

pain.2 When ethics is based on calculations of pleasure and pain it includes responsibilities not just to rational humans, but also to any creatures that experience pleasure and pain. Singer quotes a Bentham proclamation that has become “The Great Sentence” of the animal rights movement: “The question is not, Can they reason? nor Can they talk? but, Can they suffer?”3 Bentham proposed that we align ourselves with a hypothetical observer who is impartial, benevolent, and capable of discerning every consequence of a given action. From such a position we would be able to choose actions that achieve the greatest utility— defined as the maximum pleasure and minimum pain—for the greatest number of individuals. In our calculations, or course, “Each [individual] is to count for one and none for more than one.” Singer extends Bentham’s method to animals. He does not claim that humans and animals are equal or demand that we treat them equally. He alleges that most members of the chromosomal species Homo sapiens are persons—they possess rationality and self-consciousness—but most animals are not. Conversely, some chromosomal humans, such as the mentally disadvantaged and senile, are not persons, while some animals such as chimpanzees clearly are.4 Persons or not, all suffer. Consequently, even though we need not treat each sentient creature equally—it makes no sense to extend the vote to dogs—we must consider the potential pleasures and pains of each equally as we ponder how to treat them. If we give special consideration to certain individuals solely on the basis of their membership in the human species we act as “speciesists.” Singer concludes: If the experimenter is not prepared to use an orphaned human infant, then his readiness to use non-humans is simple discrimination, since adult apes, cats, mice, and other mammals are more aware of what is happening to them, more self-directing and, so far as we can tell, at least as sensitive to pain, as any human infant.5

Is it permissible, then, to experiment on and/or kill anesthetized subjects? To answer this question, Singer expands Bentham’s principle of pleasure and pain. He counsels us to consider not just the pleasures and pains, but also the interests and harms of those affected by any action. We can best determine interests and harms by taking account of what would be individuals’ preferences. No sentient subject would prefer being experimented on and killed, no matter how painless the procedures. Because human consciousness is more complex—human pleasures are fuller and human futures richer—the harm and the wrong involved in overriding preferences are usually greater with humans than with animals. Still, the mental life and interests of some human infants are on par with that of many animals. Consequently, we must, if we intend to experiment on and kill animals, be prepared to do the same to those babies.6 Singer’s arguments have provoked several lines of response.7 One contends that measuring potential pleasures and pains, interests and harms that might derive from certain actions cannot serve as a reliable guide to moral decision-making. There is a limit to how well scientists can peer into the future. Seldom can they see clearly the connection between a given experiment and a new treatment, or forecast with accuracy the number of its potential beneficiaries. Scientists are explorers, not clairvoyants.8 Fellow traveler in the animal rights movement, philosopher Tom Regan, makes a second critique, faulting Singer for the “morally callous” idea that the ends justify the means. When

Singer proposed that some human/animal sexual acts might not be wrong—after all, they could be mutually satisfactory—a piqued Regan responded: “The end does not justify the means” is a moral truth that applies beyond the boundaries of our species . . . As a utilitarian, he [Singer] believes that right and wrong depend on how much satisfaction results from our actions, an outlook that leads him to accept many practices that advocates of animal rights reject.9

Regan also notes that because utilitarian ethics aggregates interests and harms—the total of harms weighed against the sum of interests—it is a rickety defense for small numbers of laboratory animals, whose harms might be outweighed by the benefits achieved for many humans.10 Still another response asks if Singer doesn’t undermine his utilitarian starting point by allowing that the harm of killing humans is usually greater than that of killing animals. He makes it appear, after all, that even for him the nature of consciousness and cognition matters very much in ethical theory.

ANIMAL CONSCIOUSNESS AND RIGHTS Consciousness and cognition matter. Regan and animal rights lawyer Steven Wise base their arguments on this conviction.11 They hold that animals are conscious in a way that requires not only humane treatment but also respect for the right of never being used for food, companionship, entertainment, or research. Wise argues that common law should treat chimpanzees and possibly other animal species not as property but as persons. As persons they would enjoy at least some of the same rights as young children or profoundly retarded adults. Among those rights is that of not being caged and not being subjected to medical research. Rights, Wise asserts, must rest on something objective, and that something is the conscious mind that makes us capable of giving responsible direction to our lives: cognition is a very big deal because the fundamental legal right of animals, the least porous barrier against oppression and abuse that humans have ever devised, depends on it.12

Wise describes several apparent similarities between human and animal consciousness and cognition. Then, counseling us to take three aspirin before we read on, he presents his distillation of the “ten top theories of consciousness.”13 Evidently, he believes that if philosophers and neuroscientists are stymied in headsplitting disagreement about the nature of human consciousness, then there is little reason to deny it—whatever it may be—to animals. There is no need for such abject surrender. Regan, for one, goes to great lengths to define consciousness and cognition. By consciousness he means the awareness that mammals have of their environment, and by self-consciousness he means the awareness of oneself. Regan claims self-consciousness for many species of animals because they act on beliefs and in fulfillment of desires, which requires that they experience themselves as identities in time and know the world in the continuum of time and space rather than as something always new and singular. By beliefs and desires, Regan means certain mental states underlying animal behavior. When my dog Fatigué, for example, sees me standing at the door with his leash, he wags his tail and paws my leg. According to Regan, Fatigué both believes that we are going for a walk and desires to get going. He has internal

CHAPTER 4 / THE ETHICAL BASIS FOR ANIMAL USE IN RESEARCH

representations of the world around him, and can associate immediate representations (me at the door, leash in hand) to representations in his memory (previous walks). Moreover, his tail-wagging indicates that he has a welfare that matters to him. He is responding to more than a mindless physiological drive. Fatigué’s beliefs, Regan allows, are not as sophisticated as our concepts.14 He may believe that a particular bone is good to gnaw on, whereas we can add the belief that the bone is part of a skeleton. Fatigué has few of the beliefs that make up a human concept. Still, he and other animals have beliefs, rudimentary as they may be. Regan’s contention requires that we inquire into animal consciousness and cognition. Is it so like ours that it can be said that animals possess rights and animal research is a moral wrong on par with experimentation on human subjects?

HUMAN CONSCIOUSNESS The well-known philosopher of consciousness Daniel Dennett encourages us to “first devis[e] a theory [of consciousness] that concentrate[s] exclusively on human consciousness . . . and then look and see which features of that account apply to which animals, and why.”15 A good guide in the task of devising a theory of consciousness and cognition is the philosopher and theologian Bernard J.F. Lonergan.16 Lonergan begins by noting that conscious operations involve some sort of awareness that is missing in processes such as the growth of a beard. That awareness is immanent in cognitional and volitional acts: seeing, hearing, touching, feeling, smelling (all acts in the process of experiencing); imagining, thinking, conceiving (all acts in the process of understanding); reflecting, testing ideas, marshaling evidence, affirming and denying propositions (all acts in the process of judging); and taking stock of feelings, considering values, making decisions, committing ourselves (all acts in the process of willing). Unless we are in a dreamless sleep or coma, we are involved in these operations. Each of these operations makes objects present to us. In other words, each is an act of knowing or willing objects, whether those objects are external stimuli or internal feelings. Because they make objects present (involving what Regan calls “consciousness”), Lonergan calls them intentional. Simultaneously and without any reflection or “bending back,” each of these acts makes us present to ourselves. Because they make us present to ourselves (involving what Regan calls “self-consciousness”), Lonergan calls them conscious. It is because we—humans and animals alike—are present to ourselves (conscious) that objects can be present to us, or known.17 Consciousness is the self-awareness that makes the knowledge and willing of objects possible. Conscious and intentional operations (hereafter simply called conscious operations) are activities in a dynamic process that unfolds on four distinct but integrated levels. On the first level, we experience presentations that are given in acts of sensing and feeling—sounds and sights, odors, tastes, and feelings, both external and internal. All of these presentations, because they deliver objects, involve a type of knowing. We know the surroundings of sight, sound, smell, taste, and feel; of here, there, up, down, and over; of before and after; and of association (“A, then B”). While brewing the coffee, weaving in and out of traffic, or raking the leaves we know a taken-for-granted world. It is obvious that animals share with humans this knowing that Lonergan calls elementary knowing. They, too, live in the taken-

29

for-granted world, the world that is spontaneously and expected to be “out there” and real.18 Elementary knowing allows animals to win sustenance, bear offspring, and stave off predators. Due, however, to the spontaneous awakening of wonder, humans emerge from their habitat into the universe through a second type of knowing. What Lonergan terms full human knowing begins with questioning that transforms the presentations given in acts of sensing. Lonergan notes: if at moments I can slip into a lotus land in which mere presentations and representations are juxtaposed or successive, still that is not my normal state. The . . . world of mere impressions comes to me as a puzzle to be pieced together. I want to understand, to grasp intelligible unities and relations, to know what’s up and where I stand.19

By virtue of a drive to find the cause and explain, solid, takenfor-granted surroundings are shot through with questions. We are puzzled by what might be causing that noise under the hood of the car, that ache in the back, that turmoil in the Middle East. Driven by the desire to understand we emerge on a second level of consciousness, the level that Lonergan designates as that of understanding. We continue attending to the objects perceived on the first level of consciousness, but we attend to them as questioners so that our consciousness is the awareness of a self that is puzzled, inquiring, and, at least sometimes, understanding. Figuring out a math problem, solving a crossword puzzle, or discovering clues in a murder mystery, we search for suggestive images and adopt a strategy of supposing (“What if . . . ?”). Then, suddenly—usually in a moment of rest after strenuous mental exertion but still with our suppositions—we generate a set of explanatory relationships, an understanding that is not present in sensations and feelings themselves. We relax in delight, “Ah, that’s it!” Insight, or understanding, has occurred. Notice that we can bend our native wonder back on itself in an effort to understand our own cognitive and emotional states. As we seek to understand the process of sensing, understanding, judging, and deciding, we make objects of these cognitional and volitional activities. All the while, of course, we remain aware of ourselves as inquiring, judging, and responsible subjects. We are introspectively conscious. Insight, thrilling as it might be, is not the end point of human knowledge. The dynamic desire to know and decide—Lonergan calls it the eros of the human spirit—continues to unfold, impelling us next to check if our understanding is correct. On this third level of consciousness, what Lonergan calls rational consciousness, we ask, “Is this really so?” We marshal evidence for the concepts and hypotheses that formulate our insights. Scientists devise ways to test their hypotheses, and all of us draw up mental lists of conditions that have to be fulfilled before we can make ordinary judgments that something is true. Eventually our weighing of an idea leads to another insight when we discover that we have indeed fulfilled the conditions for saying, “This is so, this is true.” Most often we weigh conditions and come to insight about their fulfillment instantaneously—think of how quickly we are sure that we have gotten a joke. The rapidity of judgment can cause us to neglect how personal and significant is the act itself. In the act of judgment, as we satisfy ourselves that we are being neither rash nor indecisive, we take personal responsibility for the truth of an idea or the validity of a hypothesis. We realize that we are about to affirm or deny not only that something is true, but

30

SECTION II / GENERAL CONSIDERATIONS

also and simultaneously that we are authentic knowers, in touch with what is. Full human knowing, which implicitly includes affirmation by the knower that he or she is indeed a knower, comes to term in judgment. But the heart remains restless; persistent questioning now turns into deliberation. It opens a gap between what we have affirmed to be and what might and what ought to be. Again, the capacity for introspection becomes crucial. We take stock of feelings about what is, seek to understand those feelings, and assess how those feelings should guide decisions and actions. We ask, “Whence this passion? What is the nature of this fear? Does this friendship call for truth telling or dictate discreet silence?” Our self-awareness becomes the awareness of a subject in the throes of deliberation. Questioning launches us from the domain of fact into the uneasy realm of values. Consciousness acquires the name of conscience. Values, of course, do not already exist in an “out there” reality. It is we who generate values in the process of judging our insights into the data of inner and outer experience. Whether we can say with confidence that something is really true and then truly valuable depends on whether we have paid good attention to the data, made sense of the data in insight, and stand prepared to take responsibility for a judgment of fact about what is and then for a judgment of value about what ought to be.20 The fact that we generate values and attempt to direct our lives in accord with them is the source of the claims that we make on each other. The inescapable burden of having to choose reveals both my freedom and my responsibility. I must decide what to do, and I must decide that in relationship to others who appear to possess the same consciousness. My choice is both a claim on others and a response to their claim on me. These claims are what we call rights. This account of the dynamic desire to know what is, determine what should be, and commit ourselves to what is truly worthwhile reveals the crucial importance of wonder. In questions that express wonder, humans strive to understand, wrestle with the difference between the real and the apparent, and ponder what is to be done or not done. Questioning makes human consciousness and cognition a dynamic, self-assembling process moving from presentations given in experience through successive levels of understanding, judgment, and responsibility to the affirmation of values. Along the way, questioning sweeps us across a divide between elementary knowing, which we share with animals, and a second type of knowing that Lonergan calls full human knowing. A final comment on Lonergan’s analysis of conscious cognition: in its basic structure, it is not revisable. We cannot deny or revise our account of the process without employing the very same operations that we have outlined—operations of sensing, understanding, judging (affirming/denying), and deciding.

HUMAN AND ANIMAL CONSCIOUSNESS COMPARED Following Dennett’s counsel, my present task is to “look and see which features of [the above] account apply to which animals, and why.” I propose to do that in reverse order, beginning with consciousness on the fourth level, the level of conscience. Neither Singer, nor Regan, nor Wise claim responsible consciousness for animals. Instead, they claim that its absence in animals is no more

significant, morally and legally speaking, than its absence in infants, or in senile or mentally exceptional humans. Singer believes that treating such marginal human beings any differently that animals constitutes “speciesism,” and Regan argues that these exceptional persons—he calls them moral patients in contrast to moral agents—are possessors of rights.21 Even animal rights philosopher Mary Ann Warren objects to the ethical relevance of occasional conditions or exceptional cases.22 Infancy, senility, and mental incompetence are conditions that all of us have experienced or will experience in our lifetimes. And, just as we know in a rough and ready sort of way the difference between being conscious and unconscious, so we also know the difference between a smart animal and an exceptional child. Unlike Fatigué, whose lack of moral agency is something expected, a child with a dog’s mental capacity is received into the world as an unexpected cause for grief and extra care.23 If animals do not function on the level of responsible consciousness, do they operate on the level of rational consciousness and full human knowing? We can surmise that none of our philosophers of animal rights would argue that animals take responsibility for the truth by affirming or denying that something is or is not the case. They might contend, however, that animals operate on the second level of intelligent consciousness and have “aha” moments. In fact, it is here that Regan and Wise, who offer several examples of animal cunning, attempt to blur any distinction between humans and animals. I will return to those examples presently, after drawing attention once again to Lonergan’s distinction between elementary knowing, on the one hand, and full human knowing, on the other. It is certain that animals are conscious. And, to be sure, they know their surroundings and act smartly in their habitat. Still, we must ask if they do so with wonder and its expression in that imperious desire to know and to decide that both initiates full human knowing and grounds human rights. Regan, unfortunately, gives us no account of the characteristic human activity of questioning. Understandable as such an omission is—people today tend to think of knowing in terms of information rather than activity—it skews the picture. Paying no attention to the activity of asking questions, Regan fails to notice how significant the difference is between existing as a sentient subject of a life on the one hand and existing as a sentient, understanding, rational, and responsible subject of a life on the other. It is only with the full human knowing and willing generated by questioning that we enter into the realm of responsibility, morality, and law. What goes on in strictly elementary knowing—that is, what goes on in Fatigué’s mind—can be inferred from behavior. Much behavior, human and animal, appears to be coded in genes that evolution has selected for survival. Weeds mimic plants, viruses trick the immune system, birds build nests, predators stalk their prey, and people react to threats with “fight” or “flight” instincts. Such behavior involves no conscious knowing. Other behavior, although selected by blind evolution, is conscious and based on associative learning. Humans and animals connect behavior “A” with consequence “B,” a fact that makes it possible for humans to train children as well as pets. Fatigué’s behavior of standing ready at the door, wagging his tail, can be explained by his having connected a past walk to a present image of me with his leash. That association suffices to create expectation; he does not question the image of me with the leash, and

CHAPTER 4 / THE ETHICAL BASIS FOR ANIMAL USE IN RESEARCH

thus he will never say to himself, “He looks tired; maybe we ought to skip the walk tonight.” As Stephen Budiansky comments: The intelligence of learned associations of this sort lies in part outside the animal’s brain. It is not what is inside the head, but what the head is inside of—to use William Mace’s felicitous phrase . . . the rationality of the world.24

Finally, some conscious behavior, still based on a capacity selected in evolution, entails not just associative learning, but also wonder and its products—insights, judgments, and values. Humans go beyond the givenness of presentations to develop theories, understand causation, reflect on thoughts and emotions, formulate values, and act to bring about our various visions of utopia. Applying Occam’s razor—one should choose the simplest adequate explanation of any phenomenon—we must ask if any of the many examples and stories of animal cunning require the insight, judgment, and responsible decision-making that are generated by questioning and characterize Lonergan’s full human knowing. Fatigué’s behavior tells us this much: in addition to undergoing vital processes such as the formation and nutrition of skeletal and muscular structures, he knows and desires things around him. Nevertheless, he probably does so in an elementary way. Curious as he may appear as he puts his nose to smells on his walk, he is not motivated by the drive to understand. When he has marked his territory, he goes on his way through a habitat that is anything but a world shot through with questions. Is this supposition supported by observation? We can briefly review the fascinating literature of ethology by asking if any of four types of behavior manifests a type of knowing akin to full human knowing, which is initiated by questioning, or, rather, a type of knowing, which, however sophisticated, resembles elementary human knowing. FEATS OF KNOWLEDGE Marian Dawkins explains that many vertebrates possess mental representations, carry around cognitive maps of food stores, retrieve hidden objects, sort and classify objects, count, and make and use tools.25 Mice, to cite an example of tool making, use their excrement to plug vents carrying toxic cigarette smoke into their cages, probably acting on a learned association that we would express as “This (feces) here, then this (no smoke).” All such feats of knowledge and tool making remain within the scope of elementary knowing, in which animals associate “here” with “there,” “before” to “after,” “this means (a finger extended by a piece of straw) to that end (ants within the hill).” SELF-REFLECTION Gordon Gallup reports that chimpanzees recognize themselves in mirrors and claims that this behavior proves their ability to reflect on themselves.26 Dorothy Cheney and Robert Seyfarth, much more cautious, distinguish selfrecognition—an ability to distinguish oneself from others that does not imply any awareness of doing so—from selfconsciousness—a “meta-awareness” in which a subject can watch not just the mirror but himself looking into the mirror and thus have access to what is going on in his own mind. “There is ample evidence from studies of children . . . that many aspects of selfrecognition do not require self-reflection.”27 Chimps not only know and reach for where they itch or feel pain but also recognize as part of themselves the hand they see in a mirror as it moves to the troubled site. Children rapidly acquire this same ability. In addition, however, and at about the time they begin asking ques-

31

tions, they begin to advance beyond such self-recognition to selfreflection as they turn the attention of inquiring intelligence upon their experience of such mental presentations. The different abilities of chimp and child correspond to the two types of knowledge, elementary and full human knowing. ATTRIBUTION OF MENTAL LIFE The ability to attribute a mental life to others reveals the capacity for reflecting on one’s own mental life. Humans come to such introspection by dint of questioning presentations of sense and feeling. If it were true that animals predict and attempt to change others’ behavior because they understand that minds and intentions underlie that behavior, it would suggest that they can perform the introspection that we have claimed only for full human knowing. Cheney and Seyfarth provide a careful analysis of the anecdotal and experimental evidence that chimpanzees possess a theory of mind. They conclude that even the most compelling examples of chimps acting as if they recognize that other individuals have beliefs—examples of apparent teaching and deceiving—can be explained in terms of animals’ keen observation of behavior and their ability to follow associative (“A” then “B”) rules to change that behavior.28 LANGUAGE To use a language is to employ signs or pictures or even words to fulfill a demand, obtain a reward, or achieve a desired end; to understand language is to employ the same instruments not just as means to ends, but also as conveyances of meaning. Computers can be programmed to use words. What they cannot do is understand themselves or understand the events of another’s mind to which words point.29 Wise, who concedes that chimpanzees mostly employ signs as means of obtaining food or fulfilling human commands, is impressed that some chimps have demonstrated the use of more than 3000 words. But children at about the age of three years and without any specific training begin to go beyond the mere use of words to an understanding of thousands of words and the rules for joining them in an infinite number of novel combinations that express meaning in their minds and the minds of others.30 For them, a word is a sign, or one thing standing for another, and also a symbol into which their minds and the minds of others can read significance.31 In summary, none of the animal behaviors catalogued by Wise reveals an underlying activity of asking questions or a basic capacity for wonder. All appear to fall within the province of elementary but not full human knowing. As chimpanzee researcher Daniel Povinelli notes, chimpanzees do not have an ambition to explain the world. Speculating about what would happen if he asked both a 3-year-old child and a chimpanzee why the chicken crossed the road, he concludes the following: For the three-year-old, the question is an opportunity to attribute feelings to the chicken and to make up a story—for example, the chicken must have missed its mommy and spotted her on the other side of the road. The chimpanzee has a much simpler answer: ‘Yes.’32

It was Darwin who observed that the difference between human and animal consciousness is one of degree, not kind. It was also Darwin who noted that there is an enormous practical difference between humans and animals. Humans, he said, are the only “utopian animals.”33 It is the performance of asking questions that sweeps us into the realm of conscience and makes us “utopian animals.” But that performance is a single, restless activity generating successive levels of consciousness. Just as it makes human

32

SECTION II / GENERAL CONSIDERATIONS

behavior responsible, it also makes some acts of knowing acts of understanding. If nonhuman animals are incapable of moral behavior, then, most likely, their cognitional feats are qualitatively different from ours. In the absence of questioning, they probably never emerge on the levels of intelligent, rational, and responsible consciousness. They are clever associative learners in elementary knowing, but probably not capable of the beginnings of full human knowing.

CONCLUSION: THE USE OF ANIMALS IN RESEARCH Two overarching issues frame ethical reflections on the use of animals in research. The first is the issue of pain and suffering. The ground of the contemporary concern for animal pain—ground on which philosophers, scientists, and the general public stand together—is an unchallenged consensus that most vertebrate species are conscious. Scientists are morally and, ever since the Animal Welfare Act of 1966, legally bound to ensure the wellbeing of laboratory animals.34 The second issue is that of animal use. If animal consciousness is no different than that of humans, then any use of animals—as pets, as a source of food and clothing, as subjects of research, even when painless and certainly when it involves their death—is as morally reprehensible as the use of humans for the same purposes. The argument of this chapter is that human consciousness and cognition are evidently different enough from animal consciousness and cognition that humans can claim rights while animals cannot. So even if we use animals in research, having vouched for their humane care, we use each other as experimental subjects only when we give informed consent. We safeguard the welfare of animals; we guarantee the rights of persons.

REFERENCES AND NOTES 1. Cf. American Association for the Advancement of Science: http:// archives.aaas.org/docs/resolutions.php?doc_id=381; Federation of American Societies for Experimental Biology: http://opa.faseb.org/ pages/PolicyIssues/animalresearch_restatement.htm; Society for Neuroscience: http://www.sfn.org/index.cfm?pagename=guidelinesP olicies_UseOfAnimalsandHumans; American Psychological Association: http://www.apa.org/science/anguide.html. Professional societies helped animal protectionists craft and then update the 1966 Animal Welfare Act that sets standards of animal care for research institutions. Cf. The National Research Council–Institute of Laboratory Animal Resources. Guide for the Care and Use of Laboratory Animals. Washington, DC: National Academy Press, 1996. Moreover, they endorse regulations that implement the Act—regulations that stipulate conditions for the housing, feeding, cleanliness, exercise, and medical needs of laboratory animals; prescribe anesthesia for potentially painful procedures and analgesic drugs during postoperative care; and require participation on Institutional Animal Care and Use Committees (IACUCs) to ensure that research animals are treated responsibly and compassionately. 2. Singer P. Animal Liberation: A New Ethic for Our Treatment of Animals, 2nd ed. New York: New York Review of Books, 1990 and Singer P. Practical Ethics. New York: Cambridge University Press, 1993. 3. Hearne V. What’s wrong with animal rights. Harpers 1991;283: 1696:59–64. The citation is from Bentham J. Introduction to the Principles of Morals and Legislation. New York: Hafner Publishing Company, 1946:310–311. 4. See Singer (1990, 18–19),2 and Singer (1993:117)2. 5. See Singer (1990, 81–82).2 6. See Singer (1990, 21).2

7. Cf. Berkowitz P. Other people’s mothers. New Repub 2000:4:434: 27–37; Russell SM, Nicoll CS. A dissection of the chapter “Tools for Research” in Peter Singer’s Animal Liberation. Proc Soc Exp Biol Med 1996;211:109–139; Carruthers P. The Animals Issue. Cambridge, UK: Cambridge University Press, 1992. 8. Singer allows the use of animals when it is clear that small harm to few animals is outweighed by great benefits for many humans. Such cases, however, are very few. Biomedical research—especially basic research—does not proceed in a straight line from a handful of animals to certain specific persons. Ten years after declaring that the routine use of hundreds of thousands of laboratory animals in research “without the remotest prospect of significant benefits for human beings or any other animals” is ethically unacceptable, Singer heralded experiments in which experimentally damaged nervous systems of rats were repaired with stem cells derived from embryos. (Cf. Singer P. Sense and sentience: We might not need pig hearts if the ban on human embryo experiments were lifted. Guardian August 21, 1999:24.) Singer touted these experiments because they could lead to the practice of growing tissues for the repair or replacement of damaged organs, thus eliminating the need of temporary animal transplants for patients on human organ waiting lists. This research, however, depended on decades of basic research, much of it conducted with little understanding of its role in stem cell therapy and all of it involving the routine use of thousands of laboratory rats. None of it would have been approved if Singer’s moral calculus had been used prospectively. 9. Regan T. Editorial. News Observer, Raleigh, NC. April 3, 2001: A11. 10. Regan T. The case for animal rights. In: Singer P, Ed. In Defense of Animals. New York: Harper & Row, 1985:20–21. 11. Regan T. The Case for Animal Rights. Berkeley, CA: University of California Press, 1983. Wise SM. Rattling the Cage: Toward Legal Rights for Animals. Cambridge, MA: Perseus Books, 2000. 12. See Wise SM (2000, 236).11 13. See Wise SM (2000, 125–131).11 14. See Regan T (1983, 56).11 15. Dennett DC. Animal consciousness: What matters and why. Soc Res 1995;62(3):700. 16. Lonergan BJF. Insight. New York: Longmans, Green and Todd, 1953. For readable summaries of Lonergan on consciousness and cognitional activity, cf. Lonergan BJF. Cognitional structure. Continuum 1964;2:230–242; and Novak M. Lonergan’s starting place: The performance of asking questions. Continuum 1964;2:89–101. 17. People are inclined to say, “I am conscious of a bird flying by the window.” Lonergan would say, “I have knowledge of the bird while being conscious or present to myself.” 18. See Lonergan BJF (1953, 251).16 19. See Lonergan BJF (1953, 324).16 20. See Lonergan BJF. Method in Theology. New York: Herder and Herder, 1972:27–41. 21. See Regan T (1983, 279–281).11 22. Warren MA. The case for weak animal rights. In: Leone D, Leone B, Eds. Animal Rights: Opposing Viewpoints. San Diego, CA: Greenhaven Press, Inc., 1996:47. 23. Nelson JL. Animals, handicapped children and the tragedy of marginal cases. J Med Ethics1988;14:191–193. As Nelson reminds us, “The distinction is this: the birth of a ‘marginal’ human, or the reduction of a normal human to a marginal state, is a tragedy; the birth of say, a healthy collie pup, whose potentials are roughly on a par with the human’s is not.” 24. Budiansky S. If a Lion Could Talk. New York: The Free Press, 1998:27–28. 25. Dawkins MS. Through Our Eyes Only? The Search for Animal Consciousness. Oxford: W.H. Freeman Spektrum, 1993:24. 26. Gallup GG Jr. Chimpanzees: Self-recognition. Science 1970;167: 86–87. 27. Cheney D, Seyfarth RM. How Monkeys See the World. Chicago, IL: University of Chicago Press, 1990:240. 28. See Cheney D, Seyfarth RM.27 Ever tentative, the two scientists leave open the possibility that chimpanzees possess a very limited theory

CHAPTER 4 / THE ETHICAL BASIS FOR ANIMAL USE IN RESEARCH

of mind. They may share another’s feelings just as we spontaneously flinch at another’s pain or grin at another’s laugh, and they may be aware that feeling states such as pain underlie actions such as grimaces. Wise identifies such fellow-feeling as an “implicit theory of mind,” while Lonergan names it the intersubjective and “potential meaning” of experience. Whatever it is called, it seems qualitatively different from an explicit theory of mind, which requires that one hold and compare two different states of mind, one’s own and that of another. The ability to do that requires the introspection and active questioning of full human knowing. 29. So it is that the closest any language translation software has come to a meaningful alternative for “The spirit is willing, but the flesh is weak” is “The wine is good, but the meat is spoiled.” 30. See Wise SM (2000, 221).11 Cf. Trefil J. Are We Unique?: A Scientist Explores the Unparalleled Intelligence of the Human Mind. New York: John Wiley & Sons, Inc., 1997:55. 31. Wolfe A. The Human Difference: Animals, Computers and the Necessity of Social Science. Berkeley, CA: University of California Press, 1993:79.

33

32. Wheeler D. With ingenious experiments, a psychologist studies the mental lives of chimps. Chron Higher Ed June 25, 1999:B2. 33. Cited in Budiansky 1998, xv. Compare contemporary philosopher Charles Hartshorne, cited in Derr TS. Animal rights, human rights. First Things 1992;20:23–30, who, although sympathetic to animal rights issues, notes that the human/animal difference is “so vast that for many purposes one can safely forget that it is a matter of degree.” 34. What constitutes animal well-being, we should note, is not immediately obvious. Counterintuitive as it seems, rats, for example, may be put on edge by being placed in larger cages. Researchers have an ethical obligation to advance knowledge about animal well-being, asking ever-new questions that lead to new and sometimes revised judgments and gradually round out the picture of humane animal care.

5

Bibliographic Searching Tools on Disease Models to Locate Alternatives for Animals in Research A Website Companion MARY W. WOOD

AND

LYNETTE A. HART

ABSTRACT

SEARCHING FOR A RANGE OF POTENTIAL MODELS Anyone can quickly become frustrated trying to monitor and locate the relevant information on published research concerning disease models. Following the current literature, which is so widely dispersed, can seem an overwhelming task. The pertinent information is published in numerous journals and books and then organized and indexed among a variety of databases, some of which are freely available and others that are proprietary. New searching tools are now available in a website specifically developed as a companion to this book. The searching tools can be freely accessed at a designated website, where user-friendly searches can be executed at any time according to stored instructions,3,4 in a variety of databases pertaining to the disease models described in this volume. The databases include those freely available and linked on the website, such as PubMed, TOXNET, AGRICOLA, TOXLINE, and NCBI, as well as specialized resources such as FishBase and the JAX Mice Database. Proprietary databases available by subscription are also listed, such as ISI Web of Science, PsycInfo, and Aquatic Sciences and Fisheries Abstracts (ASFA). We have previously developed comprehensive search grids to facilitate bibliographic searching on topics related to alternatives for veterinary medical education.5–7 WEBSITE ON MODELS AND DISEASES As described below and covered extensively in the interactive website, a working list of topics addressed by searching tools (using a variety of databases and resources) has been selected from the following, including nonvertebrate and vertebrate animal models by species: sea urchin embryo; budding yeast; fungus; social amoebae; round worm; Drosophila; bees; Xenopus, fish, and other aquatic animals; mice; rats, guinea pigs, hamsters, rabbits, and ferrets; large and farm animals; exotics; cats and dogs; and nonhuman primates. Searching tools are also organized by disease, including aging, AIDS, alcoholism, Alzheimer’s disease, atherosclerosis, diabetes, eye diseases, hearing disorders, lung cancer, multiple sclerosis, neurodegenerative diseases, obesity, acute and chronic pain, Parkinson’s disease, posttraumatic stress, renal disease, sepsis, thrombosis, and wound healing. The website was developed as a companion to the Sourcebook of Models for Biomedical Research and can be accessed: http://www.vetmed.ucdavis.edu/ Animal_Alternatives/sourcebook.html.

Nonvertebrate and vertebrate models are commonly used in research to study diseases that affect humans and animals and to clarify disease etiology and methods for disease prevention and treatment. The relevant information on models is published in a variety of resources and then indexed among free and proprietary databases. New searching tools specifically developed as a companion to this book can now be freely accessed at a designated website created as a companion for the Sourcebook of Models for Biomedical Research, where user-friendly searches can be executed at any time according to stored instructions, in a variety of databases pertaining to the relevant disease models. Some databases are freely available, such as PubMed, AGRICOLA, TOXLINE, and NCBI, as well as specialized resources such as FishBase and the JAX Mice Database. Other useful databases are proprietary, such as PsycInfo and Aquatic Sciences and Fisheries Abstracts. The website can be accessed: http://www.vetmed. ucdavis.edu/Animal_Alternatives/sourcebook.html. Key Words: Animal models, Laboratory animal(s), Animal model types, Choosing animal model, Diseases, Alternatives, Disease models, Nonvertebrate, Vertebrate, Databases, Searching, Websites.

INTRODUCTION Both nonvertebrate and vertebrate models are commonly used in research to study diseases that affect humans and animals and to clarify disease etiology and methods for disease prevention and treatment. A wide range of databases, both free and proprietary, are available to draw from when searching research literature on these topics.1 Existing tools that use search filters in the PubMed database simplify identifying strains of mice as appropriate disease models.2 In this chapter we describe other improved methods for effective literature searching for disease models, and present some new efficient strategies and tools for searching that are available in a new website companion for the Sourcebook of Models for Biomedical Research.

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

35

36

SECTION II / GENERAL CONSIDERATIONS

The website offers direct links to particularly relevant databases and other resources, and indicates which ones are likely to be especially useful for particular models or diseases. In addition, the website includes some stored searches in complimentary databases, termed embedded search templates, that can be launched at will. With these templates, users can use point-and-click technology to avail themselves of the currently available literature on a particular topic, and then make the search instruction more specific to their needs. The search templates generally draw on the high-speed, comprehensive PubMed database, or TOXLINE or NCBI; all are freely available to anyone. RELEVANT LEGISLATION AND REGULATIONS REGARDING ALTERNATIVES For each use of animals, scientists are required by the USDA’s Animal Welfare Act8 and the related USDA Policy 119 and Policy 1210 to complete animal use protocols that include questions on possible alternatives to procedures causing more than momentary pain or distress, whether in teaching, research, or testing. While researchers seeking improved methods or completing animal-use protocols often find it difficult to locate the relevant information, searching the literature regularly could result in identifying an improved animal (or even yeast or fungus) model for a particular disease. The regulations require that principal investigators provide a written narrative in the protocol concerning the availability of alternatives, including the methods and sources used to determine the availability of refinements, reductions, and replacements. USDA animal care Policy 11 provides a detailed description of painful and distressful procedures. Policy 12 offers detailed guidance on the investigator’s description of the database search for alternatives, including the names of the databases searched, the date the search was performed, the period covered by the search, and the key words and/or the search strategy used. Considering the ongoing creation of new animal models and the development of new technologies, frequent searching and consideration of alternatives are appropriate so as to identify and consider new methods and models. This chapter addresses a gap facing scientists by presenting userfriendly searching tools that are targeted toward (1) locating animal models and (2) conducting effective bibliographic searches on animal models and diseases, as required for animal use protocols. These tools simplify searching by providing streamlined access to the resources being sought. Facilitating efficient and effective searching by users can improve the research and simplify compliance with USDA requirements.

REVIEWING DATABASES BY ANIMAL MODEL OR SPECIES Browsing among the databases pertinent to animal models can be a useful place to begin, to gain familiarity with the array of resources. We have sorted the databases, including 20 complimentary databases, 10 other resources, and 10 proprietary databases listed here, so that users can quickly explore the topics of greatest interest, whether they be animals or diseases in general, or particular animal models or diseases. POTENTIAL ANIMAL MODELS As shown in Figure 5–1, a large number of complimentary databases are available that pertain to animal models. Some of them have more specialized purposes. For example, ECOTOX, INVITTOX, and TOXNET pertain specifically to toxicology testing, indicating substances that may play an adverse role in certain diseases. FishBase and oneFISH deal with literature on fish. AGRICOLA covers the

general literature on veterinary medicine, and PubMed covers the general literature on human medicine. DTIC and DoD Biomedical Research are sites developed in response to military needs, but have general applications for psychological stress and wound healing. A variety of other complimentary resources are available that are useful in finding animal models. Of particular note are the resources dealing with mice, including the International Mouse Strain Resource, the Mouse Models of Human Cancers Consortium, and the NCI Mouse Models. Ensembl Genome Browser taps into the expanding genomic developments. Other databases are available as proprietary products. Many institutions subscribe to some of these, so users may have ready access to them at their worksites. One of the most useful of these, ISI’s Web of Science, offers web access to citation information, allowing a search for all papers that have cited an earlier work. This feature, previously known as Citation Index, offers a feedforward in tracking papers that have cited a classic work to follow significant lines of research. PsycInfo has good coverage of the human psychological literature, and sometimes addresses pain and stress topics in more detail than PubMed, with an added value of going back to very early literature. CAB and Biosis offer more comprehensive coverage of the European literature and the biology literature than either PubMed or AGRICOLA. Zoological Record covers a broad base of literature on animal behavior of many species and goes back to the earliest references. Fish & Fisheries Worldwide is another database that is essential for people working with fish. ERIC is a database specialized for accessing the literature pertaining to education. NONVERTEBRATE MODELS Identifying useful models for a study not involving mammals, or perhaps not even vertebrates, offers a potential for reducing the pain or distress that may result from interventive procedures. A growing number of such models are proving useful. Studies using these can be easily accessed, as indicated in Figure 5–2. Sea Urchin Embryo Search templates offer point-and-click instantaneous searches of some topics where sea urchin embryos have been useful, including cellular activity, the role of metals in early development, and transcription. Other search templates launch TOXLINE or HSDB, two resources on toxicity that are less well known, retrieving studies pertaining to cellular mechanisms and substances with adverse impacts on cellular activity. Yeast Yeast has proven useful in studies of drug design, glucose, and DNA, topics for which search templates are provided. The DNA search retrieves studies on DNA damage from exposure to toxic substances. Fungus The use of nonanimal models, such as fungi, can be reviewed in search templates that reveal studies of circadian oscillations in fungi, as compared with other organisms. Social Amoebas Specialized databases for the social amoebas include DictyBase and Dicty Workbench. Embedded searches focus on cytokinesis, signal transduction, and development. Round Worm The round worm has become a preferred model for studies of aging that relates to studies of human mitochondria. The database, WormBase, is an important special resource for studies of Caenorhabditis elegans. Fruit Fly Drosophila has longstanding importance as an animal model for genetic studies, as reflected in the special database, Homophila. Search templates here pertain to using the fruit

Figure 5–1. Opening page of the website, showing selected resources suitable for browsing to identify potential disease models. Resources shown in shaded boxes are free to all users and are avail-

able via links on the website. The column on the right side lists useful proprietary databases available by subscription. http://www.vetmed. ucdavis.edu/Animal_Alternatives/sourcebook.html.

Figure 5–2. Nonvertebrate models featured on the website, highlighting free and proprietary databases in the middle two columns. The column on the right side indicates the use of the free database

PubMed for stored embedded searches that are available for launching via links on the website. http://www.vetmed.ucdavis.edu/Animal_ Alternatives/sourcebook.html.

38

SECTION II / GENERAL CONSIDERATIONS

Figure 5–3. Vertebrate models that are typically used in laboratories, featured on the website, highlighting free and proprietary databases in the middle two columns. The column on the right side indicates the use of the free database PubMed for stored embedded

searches that are available for launching via links on the website. http://www.vetmed.ucdavis.edu/Animal_Alternatives/sourcebook. html.

fly for studies of hearing, balance, neurodegenerative models, and aging. Bees Studies of alcohol effects are conducted on nonvertebrates, bees. These studies are easily accessed in an embedded search. VERTEBRATE MODELS A number of vertebrate species are familiar in laboratory studies, primarily the aquatic vertebrates and the small mammals. The fish, frogs, and other aquatic animals,

and the mice, rats, and other small mammals are readily accessed as shown in Figure 5–3. Less commonly studied, but also used as models, are large and other farm animals, exotic animals, dogs and cats, and nonhuman primates; searching strategies for these species are presented in Figure 5–4. Fish, Frog, and Other Aquatic Animals The use of fish has expanded in recent years, in many cases as a vertebrate replacement for mammals. Specialized resources are most appropriate

Figure 5–4. Vertebrate models that are less typically used in laboratories, featured on the website, highlighting free and proprietary databases in the middle two columns. The column on the right side indicates the use of the free database PubMed for stored embedded

searches that are available for launching via links on the website. http://www.vetmed.ucdavis.edu/Animal_Alternatives/sourcebook. html.

CHAPTER 5 / BIBLIOGRAPHIC SEARCHING TOOLS ON DISEASE MODELS TO LOCATE ALTERNATIVES FOR ANIMALS

39

specifically toward biomedical research and misses some important work utilizing exotic animals. Dogs and Cats AGRICOLA is worthwhile for searching literature pertaining to dogs and cats, as is PubMed. Nonhuman Primates Specialized databases are useful in locating literature on nonhuman primates, especially the complimentary PrimateLit, with research dating back to 1940.

for searching on these species, including the complimentary FishBase, oneFISH, Xenopus Resources, Zebrafish Gene Collection, and ZFIN, and the proprietary ASFA and Fish and Fisheries Worldwide. Mice Despite the growing number of nonvertebrate and fish models, mammals are essential for some studies. In mainstay models for studies in which a mammal is essential, the number of mice used far exceeds any other mammal. As examples, the search templates use mice as models for various diseases, for studies of Alzheimer’s disease, and for studies of obesity. Specialized databases such as the JAX Mice Database and the Mouse Biology Program are important when searching for appropriate strains of mice. Specialized strategies for searching with search filters also can be employed.2 Rats, Guinea Pigs, Hamsters, Rabbits, and Ferrets Rats are frequently used as models in a wide range of studies where an animal larger than a mouse is needed. Other small mammals are used in specific research, such as ferrets in studies of Helicobacter and guinea pigs in hyperlipidemia. With these species, AGRICOLA provides coverage of some research that complements PubMed. The complimentary ANZCCART Fact Sheets provide practical information on basic parameters for working with small mammals. Large and Other Farm Animals For agricultural animals, AGRICOLA invariably provides more comprehensive coverage of the research than PubMed. The FASS Guide is available at modest cost, providing another valuable resource on agricultural animals. Exotic Animals Specialized databases are more effective when searching on exotic animals than PubMed, which is targeted

Researchers face a dual challenge, to keep updated not only with regard to new animal models, but also concerning new work on the particular disease of interest. DISEASES IN GENERAL As with animal models, a wide range of complimentary databases and resources is available for browsing, as indicated in Figure 5–5. SPECIFIC DISEASES To provide useful examples of search strategies, four embedded search strategies are indicated in Figure 5–6 for each listed disease. For the embedded search templates stored concerning each of the specific conditions and diseases, two searches for each disease draw from PubMed, one focusing on a specific research strategy on a relevant topic for that disease and another addressing models for that disease. A third search for each disease draws from TOXLINE, the National Library of Medicine’s bibliographic database for toxicology, covering the full range of effects of drugs and other chemicals, including biochemical, pharmacological, physiological, and toxicological effects. The fourth search for each disease uses NCBI (National Center for Biotechnology Information), offering an extended

Figure 5–5. Selected resources on the website suitable for browsing by disease, to identify potential disease models. Resources shown in shaded boxes are free to all users and are available via links on the

website. The column on the right side lists useful proprietary databases available by subscription. http://www.vetmed.ucdavis.edu/ Animal_Alternatives/sourcebook.html.

REVIEWING DATABASES FOR ANIMAL MODELS BY DISEASE

40

SECTION II / GENERAL CONSIDERATIONS

Figure 5–6. Search strategies on the website are presented as stored embedded searches by disease, drawing from the free databases PubMed, TOXLINE, NCBI, and DTIC. The stored searches are avail-

searchable library of the life sciences literature, including PubMed, PubMed Central, books, Coffee Break (short reports on recent discoveries), Genes and Disease, and OMIM (catalog of human genes and genetic disorders). Thus, NCBI reflects the emerging emphasis on research pertaining to genetics interacting with other disciplines. We provide embedded searches for the 19 diseases and conditions listed below. Users can use the search templates as a starting point to adapt searches more specifically to their needs. Posttraumatic stress and wound healing are two topics that have broad application and benefit from the comprehensive military emphasis on databases, as found in DTIC and DoD Biomedical Research. • Aging • AIDS • Alcoholism • Alzheimer’s disease • Atherosclerosis • Diabetes • Eye diseases • Hearing disorders

able for launching via the website. http://www.vetmed.ucdavis.edu/ Animal_Alternatives/sourcebook.html.

• Lung cancer • Multiple sclerosis • Neurodegenerative disorders • Obesity • Pain, acute and chronic • Parkinson’s disease • Posttraumatic stress • Renal disease • Sepsis • Thrombosis • Wound healing

CONCLUSIONS A website companion for the Sourcebook of Models for Biomedical Research developed by the UC Center for Animal Alternatives offers streamlined access to information concerning animal models for disease. The site offers expeditious links to relevant databases and provides embedded stored search examples for effective searching for models. The site can be used as an instructional tool to demonstrate how to search in these

CHAPTER 5 / BIBLIOGRAPHIC SEARCHING TOOLS ON DISEASE MODELS TO LOCATE ALTERNATIVES FOR ANIMALS

subject areas. The examples offer a starting point that a user can, with growing experience, use as a springboard for further searching.

ACKNOWLEDGMENTS The extensive electronic library holdings of the University of California make it possible to explore, identify, and distinguish the unique features of the resources presented in these search grids. Hsin-Yi Weng creatively contributed to much of the developing technology offered in this chapter.

REFERENCES 1. Hart LA, Wood MW, Weng H-Y. Effective searching of the scientific literature for alternatives: Search grids for appropriate databases. Anim Welf 2005;14:287–289. 2. Wood MW, Hart LA. Search filters, a new tool in the search for alternatives: Locating mouse strains as disease models. ATLA 2004;32(Suppl. 1):599–602. 3. Wood MW, Hart LA. Considering animal alternatives and welfare via a comprehensive search of the scientific literature. ALTEX 2006;23(Spec. Issue):197–199. 4. Wood MW, Weng H-Y, Hart LA. Guide to bibliographic databases for alternatives searching: Database approach to a search. UC Center for Animal Alternatives, University of California, Davis, School of Veterinary Medicine. Accessed on September 7, 2006, at http://www. vetmed.ucdavis.edu/Animal_Alternatives/databaseapproach.html.

41

5. Wood MW, Hart LA, Weng H-Y. Effective bibliographic searching for animal alternatives in veterinary medical education: The UC Davis web site. J Vet Med Ed 2005;32:468–472. 6. Weng H-Y, Wood MW, Hart LA. Alternatives resources in education: Educational search grids. UC Center for Animal Alternatives, University of California, Davis, School of Veterinary Medicine. Accessed on September 7, 2006, at http://www.vetmed.ucdavis.edu/Animal_ Alternatives/altsearch.htm. 7. Wood MW, Hart LA, Weng H-Y. Search grids for teaching and research protocols on veterinary medicine teaching alternatives. UC Center for Animal Alternatives, University of California, Davis, School of Veterinary Medicine. Accessed on September 7, 2006, at http://www.vetmed.ucdavis.edu/Animal_Alternatives/protocol.htm. 8. United States Animal Welfare Act 7USC 2131–2159, adopted 1966, amended 2002. Accessed September 7, 2006, at http://www.nal.usda. gov/awic/legislat/awa.htm. 9. United States Department of Agriculture, Animal and Plant Health Inspection Service Animal Care. USDA Animal and Plant Health Inspection Animal Care Policy Manual Policy #11—Painful Procedures—April 14, 1997. Accessed September 7, 2006, at http://www. aphis.usda.gov/ac/policy/policy11.pdf. 10. United States Department of Agriculture, Animal and Plant Health Inspection Service Animal Care. USDA Animal and Plant Health Inspection Animal Care Policy Manual Policy #12—Consideration of Alternatives to Painful/Distressful Procedures—June 21, 2000. Accessed September 7, 2006, at http://www.aphis.usda.gov/ac/ policy/policy12.pdf.

6

NIH Policies on Sharing of Model Organisms and Related Research Resources J.P. KIM, CHARLENE CHO,

AND

NORKA RUIZ BRAVO

ABSTRACT

EVOLUTION OF NIH SHARING POLICIES

The National Institutes of Health (NIH) has long recognized that the sharing of research materials and data is critically important to the progress of biomedical research. Effective sharing of research resources benefits the public by facilitating and promoting discovery across the full spectrum of the research community. In this spirit, the NIH Model Organism Sharing Policy builds upon extant NIH sharing policies to allow the NIH to continue to support and promote broad and enabling research effectively and efficiently. This chapter provides guidance, sample plans, approaches, and other resources for investigators considering how best to share model organisms and related research resources with the research community. Key Words: Model, Organisms, Data, Federal, Funding, Grants, Sharing, Research, NIH, Policy.

FROM PUBLICATION TO SHARING OF RESEARCH TOOLS TO DATA AND ORGANISMS The NIH is a part of the U.S. Department of Health and Human Services and is the primary federal agency responsible for conducting and supporting biomedical research in the United States. With a history dating back to 1887, the NIH has played a prominent role not only in biomedical research, but also in the dissemination of research results and materials to the research community. As a steward of federal funds and in keeping with its mission, the NIH has formulated policies regarding the sharing of research resources made possible as a result of public funding (see Table 6–1). The concept of sharing research results has remained a constant element of NIH grants policies, including those related to unique research resources. An early Public Health Service (PHS) Grants Policy Statement set the stage by stating a seminal principle for federally funded research: “Project directors and principal investigators are encouraged to make the results and accomplishments of their activities available to the public.”5 A May 1980 Special Announcement was published in the NIH Guide for Grants and Contracts regarding the “NIH Policy Relating to Reporting and Distribution of Hybridomas Produced Under Grants and Contracts.”6 This was followed by additional announcements highlighting the importance of sharing.7 The Bayh-Dole Act, P.L. 96-517, passed into law on December 1980 to stimulate research and development of government-funded research, prompted additional reminders about the importance and value to scientific research of sharing research resources.8

INTRODUCTION Model organisms have played a critical role in the history of biomedical research.1 As tools of discovery and a means to demonstrate proof of concept, they have been pivotal in a broad range of seminal events, including among others, developing a vaccine against smallpox, testing the artificial heart, and discovering the genetic basis for lactose intolerance.2 The sharing of research materials, including model organisms and associated data, has also been critically important in the progress of biomedical research.3 Common use of model organisms by research laboratories has provided scientists with the opportunity to verify research results, explore previously unsuspected mechanisms of disease, and study complex biological questions using an integrative, holistic approach.4 As a public sponsor of biomedical research, the National Institutes of Health (NIH) is committed to supporting national and international efforts that encourage the sharing and dissemination of important research resources. This chapter will discuss the policies of the NIH on the sharing of model organisms and related research resources and its significance to biomedical research, as well as approaches and considerations of how such resources might most effectively be shared, consistent with NIH funding policies.

THE NIH POLICY ON SHARING OF MODEL ORGANISMS FOR BIOMEDICAL RESEARCH After consultation with the research community, the NIH announced its Policy on Sharing of Model Organisms for Biomedical Research (NOT-OD-04-042) on May 7 2004.9 Under this policy, NIH applications that result or may result in new, genetically modified variants of model organisms are expected to include a plan for sharing those organisms and related research resources. The policy is based on existing NIH Grants Policy [NIH Grants Policy Statements (http://grants.nih.gov/grants/policy/#gps)], including the NIH Research Tools Policy, and essentially serves as implementation guidelines for earlier policies.10,11 As such, this

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

43

44

SECTION II / GENERAL CONSIDERATIONS

Table 6–1 Highlights in the evolution of NIH sharing policies Year

Policy

Publication

Pre-1980s

Policy encouraging making the results and accomplishments of NIH funded activities available to the public Policy on reporting and distribution of hybridomas produced under grants and contracts Policy on reporting and distribution of unique biological materials produced with NIH funding Biological materials policy: “Handling Non-Election of Title to Patentable Biological Materials” NIH research tools policy: “Sharing of Research Resources: Principles and Guidelines for Recipients of NIH Research Grants and Contracts on Obtaining and Disseminating Biomedical Research Resources” 2003 NIH data sharing policy: “Final NIH Statement on Sharing Research Data” NIH model organism sharing policy: “NIH Policy on Sharing of Model Organisms for Biomedical Research”

PHS Grants Policy Statement, October 1976

1980

1984

1996

1999

2003

2004

NIH Guide Supplement, May 1980 NIH Guide Notice, March 1984

NIH Guide Notice, May 1996

Federal Register Notice, December 1999

NIH Guide Notice, February 2003

NIH Guide Notice, May 2004

policy applies to extramural investigators and institutions that are funded by a variety of mechanisms (see below). Although this policy does not apply to NIH intramural investigators, the NIH intramural research program has guidelines that are consistent with those for the extramural community (for example, see http:// www1.od.nih.gov/oir/sourcebook/ethic-conduct/resources.htm). Accordingly, the NIH advocates that investigators share such resources across the entire research community. The policy assumes that sharing will take place in a manner consistent with accepted practice. For example, new, genetically modified model organisms developed with NIH funding may be shared as mature organisms, sperm, eggs, embryos, or even the vectors used to generate transgenic or knockout organisms. RATIONALE FOR THE SHARING POLICY The NIH Model Organism Sharing Policy contributes to scientific progress by making new resources available more quickly to a large number of people and obviates the need to use limited resources and investigator time to reproduce previously developed model organisms. With the Model Organism Sharing Policy, the NIH expects

that new, genetically modified model organisms and related resources generated with NIH funding will be distributed and shared with the scientific community in a timely manner (i.e., at least upon publication of the primary results announcing the development of the genetically modified model organisms). This expectation serves to fulfill the mission of the NIH: “Science in pursuit of fundamental knowledge about the nature and behavior of living systems and the application of that knowledge to extend healthy life and to reduce the burdens of illness and disability.” Sharing of model organisms and related research resources allows the biomedical community, including students, postdoctoral fellows, and investigators, to engage in new scientific research and training opportunities, which can extend the scope of inquiry beyond that envisioned by the original creator of the animal strain. As a result, sharing research resources is likely to result in more rapid breakthroughs for the diagnosis, prevention, and treatment of disease than a single investigator could achieve alone. Broad access and use of research resources also contribute to the many goals of the NIH, including “promoting the highest level of scientific integrity, public accountability, and social responsibility in the conduct of science.” Sharing of model organisms allows investigators a chance to verify research results, reduces needless and costly duplication of effort, and serves as a form of insurance against unforeseeable loss of research resources (e.g., disasters such as floods, fires, and power outages that have resulted in the loss of valuable mouse strains and other resources). Thus, everyone benefits from the sharing of research resources, including investigators, the scientific community, and the public. Indeed, sharing is good practice.12 Journals such as Proceedings of the National Academy of Sciences USA, Cell, Nature, Neuron, Immunity, The Journal of Immunology, The Journal of Neuroscience, and Science require investigators to make unique resources available so that research results can be verified and additional research promoted. Both the Society for Neuroscience, with a membership of 29,000 scientists, and the Federation of American Societies for Experimental Biology (FASEB), consisting of 19 societies and a membership of 66,000 scientists, have taken positions that are consistent with the NIH policy. For example, the policy of the Society for Neuroscience is that “unique and propagatable research materials used in studies being reported must be made available to qualified scientists for bona fide research purposes”(see http://www.sfn.org/index.cfm?pagename =responsibleConduct_authorsOfResearchManuscripts and http:// www.sfn.org/skins/main/pdf/Guidelines/ResponsibleConduct. pdf) under section 1.8 of “Guidelines: Responsible Conduct Regarding Scientific Communication,” Society for Neuroscience, 1998.13 MODEL ORGANISMS DEFINED For purposes of the NIH Model Organisms Sharing Policy, the NIH uses the term “model organism” to describe both mammalian (e.g., mouse and rat) and nonmammalian models (e.g., budding yeast, social amoebas, roundworm, Arabidopsis, and frog). Genetically modified model organisms are those in which spontaneous mutations have occurred and/or those in which mutations have been induced by chemicals, irradiation, transposons, or transgenesis (e.g., knockouts and injection of DNA into blastocysts). Congenic or consomic strains are also considered genetically modified model organisms. Detailed information about model organisms can be found on the NIH Model Organism for Biomedical Research website (http://

CHAPTER 6 / NIH POLICIES ON SHARING OF MODEL ORGANISMS AND RELATED RESEARCH RESOURCES

www.nih.gov/science/models/). Publications and electronic databases as well as websites of stock centers and repositories that list available strains also make the identification of model organisms relatively easy. SCOPE OF THE POLICY The policy covers both model organisms and related resources. Related resources include materials and data necessary for the production and understanding of model organisms, such as vectors, nonhuman embryonic stem cells, established cell lines, protocols for genetic and phenotypic screens, mutagenesis protocols, and genetic and phenotypic data for all mutant strains. As a matter of practice, genetic variants of viruses, bacteria, and other prokaryotic organisms should be made widely available. At this time, however, the NIH is not expecting the submission of a sharing plan from investigators who intend to develop noneukaryotic organisms. It should be noted that this NIH sharing policy applies only to nonhuman model organisms. For example, human cells that are not commercially available would not be subject to this policy. For information on regulations, policies, and guidelines for human subject specimens, see http://grants2.nih.gov/grants/policy/hs/ specimens.htm. Unlike the related, but distinct, 2003 NIH Data Sharing Policy (http://grants2.nih.gov/grants/guide/notice-files/NOT-OD-03032.html), which applies to projects requesting $500,000 or more in direct costs in any given year,14 the NIH Model Organism Sharing Policy applies to all projects that produce or may produce model organisms or related resources. In other words, the NIH expects that all investigators pursuing projects that would or could result in model organisms and related resources will include a sharing plan in their applications, regardless of the requested budget amount or final amount awarded. FUNDING MECHANISMS AFFECTED With respect to NIH grant mechanisms, this policy applies to all research project grants (R mechanisms) and Centers (P mechanisms) as well as to Small Business Innovation Research (SBIR) and Small Business Technology Transfer (STTR) awards. The policy also applies to individual Career Development awards (Ks) if the scope and aims of the project include the development of model organisms. By contrast, institutional training grants (Ts) or individual fellowships (Fs) are not subject to the policy, but sharing would be applicable to such grantees as the production of model organisms presumably would be covered under a mentor’s research grant. This policy also applies to cooperative agreements and to contracts from the NIH that will generate model organisms and related resources as described above. It should also be noted that this policy applies to all tiers of a funding agreement, including international collaborations and foreign grants. For example, if the U.S. institution is the primary grantee, then it would be the responsible for its subgrantee or subcontract arrangements and would be expected to ensure that this policy is adequately addressed in the application. In some cases, different subprojects will have different sharing plans, or only one or a few of the subprojects will need sharing plans. Regardless of how many parties are involved or who is designated as the primary grantee, it is important to state clearly in the application which party [e.g., Principal Investigator (PI) or head of the subproject] will be responsible for the implementation of the proposed sharing plan(s). DEVELOPING SHARING PLANS When submitting sharing plans to the NIH under this policy, such plans should provide

45

information to ensure that sharing will be meaningful for other investigators in the community. For example, the following information should be addressed. How novel strains will be made available to the scientific community, including the following: • The form in which the investigator/institution will provide the organisms (e.g., adults, embryos, sperm). • Related research resources and data that the investigator/ institution will provide. • A reasonable time frame for periodic deposition of material and associated data. • Whether the investigator/institution will share under its own auspices or use a repository, and, if a repository, which one. • For vertebrate animals and for other species for which pathogens or contaminants are potentially serious problems, how the investigator/institution will maintain the strains to minimize the risks of infection or contamination. How technology transfer and intellectual property issues will be addressed, including the following: • How the institution plans to make such organisms and resources widely available to the research community. • How the institution plans to make certain any rights or obligations to third parties are consistent with the terms and conditions of the NIH award to ensure appropriate dissemination of model organisms or related research resources under the NIH award. • A description of the mechanisms that will be used to distribute organisms and related research resources [e.g., material transfer agreements (MTA)]. Sharing plans may vary, depending on the organism, the nature of the resources that will be shared, the extent of intellectual property issues involved, and plans for distributing the resource(s), as well as the particular programmatic goals. The actual content and level of detail included in a sharing plan will be affected by several factors, such as the status of the development of a model organism, the potential impact of intellectual property rights on the availability of the model organism, and the method of sharing. Therefore, the NIH recommends that the investigator contact the funding Institute or Center about the level of detail expected. Samples of sharing plans for model organisms and related resources may be found at http://grants.nih.gov/grants/policy/ modelorganism. These three sample plans have been provided below to assist the applicant community in responding to this policy when submitting a funding application to the NIH: • A simple plan that may be appropriate for a project, which has the goal of producing a model organism but has not produced one (Organism Sharing Simple) (http://grants. nih.gov/grants/policy/model_organism/model_organ_ simple_plan.doc). • A plan for sharing mice (Organism Sharing Mouse) (http://grants.nih.gov/grants/policy/model_organism/ model_organ_mouse_plan.doc). • An example of a complex plan for sharing mice with IP held by various parties (Organism Sharing Complex) (http://grants.nih.gov/grants/policy/model_organism/ model_organ_complex_plan.doc).

46

SECTION II / GENERAL CONSIDERATIONS

Sharing plans may also take into account practical limitations. For example, if a large genetic screen is conducted and thousands of lines are generated, only a few of them may display a phenotype that is of interest to laboratories. Generally, the investigator in such a case would not be required to maintain and distribute all the lines. Similarly, if knowledge of their existence is limited to a publication in which they were described as being used in a cross and do not demonstrate a specific phenotype, there would be no need to maintain or distribute those lines. However, if the collection of lines was generated with NIH funding and with the purpose of serving as a community resource, then there would be an obligation to share the lines. There are certain instances in which sharing of a resource may not be appropriate. For example, an investigator would not be expected simply to redistribute a resource acquired from another source. However, if that resource is used to breed, construct, or generate another resource using NIH funds, there would be an obligation to share the derived resource. In any case, if no sharing plan is included in the NIH application, the applicant would be obligated to state why sharing would be restricted or not possible. Failure to provide an adequate sharing plan may affect the NIH’s decision to make an award. Equally important, failure to comply with the submitted and accepted sharing plan may be considered in future funding decisions for the investigator and the investigator’s institution. GENERAL RESOURCES AND CONSIDERATIONS FOR THE DISTRIBUTION AND SHARING OF MODEL ORGANISMS AND RELATED RESOURCES Sharing of model organisms and related resources is not intended to be challenging for the research community, and yet depending on circumstances (e.g., demand or supply), responding to requests for model organisms can be costly and labor intensive. Also, there could be other considerations that may need to be taken into account when transferring materials. To minimize administrative burdens and to help streamline the processing of such requests, the NIH recommends that developers deposit their model organisms with existing repositories, which would provide for ready, long-term access by the biomedical research community. Repositories such as the Mouse Models for Human Cancer Consortium (http://emice.nci.nih.gov/), the Mutant Mouse Regional Resource Centers (http://www.mmrrc.org/), the Rat Resource and Research Center (http://www.nrrrc.missouri. edu/), and the Zebrafish Information Network (http://zfin.org/) are among some of the examples of repositories funded by the NIH and should be viewed as valuable resources for the sharing of model organisms. If submitting the model organisms with a repository is not an option, the NIH also offers funds for research resource sharing. Applicants may request funds in their applications for expenses associated with sharing model organisms and related research resources, including administrative supplements for unanticipated expenses associated with sharing. Whether it is through a repository or through the auspices of the developer, distributing model organisms and related research resources developed with NIH funding should occur in a manner consistent with the NIH Research Tools Policy. The majority of unpatented transfers to not-for-profit entities should occur under terms no more restrictive than the Uniform Biological Materials Transfer Agreement (UBMTA) (http://www.autm.net/aboutTT/

aboutTT_umbta.cfm) or the Simple Letter Agreement (SLA) (http://www.nhlbi.nih.gov/tt/docs/sla_mta.pdf). The provider may specify whether the model organism can be distributed to other parties. If the materials are patented or licensed to an exclusive provider, other arrangements may be used, but terms providing for commercialization option rights, royalty reach-through, or product reach-through rights back to the provider are inconsistent with the NIH Research Tools Policy. When NIH-funded materials are being transferred to for-profit entities that intend to restrict their use internally, recipients should ensure that the materials are transferred with the fewest encumbrances possible. The SLA may be expanded for use in this instance, and internal-use license agreements with execution or annual use fees may be appropriate, depending on circumstances. Ascertaining which type of agreement to use in transferring materials can present quite a challenge. It is important to keep in mind that if no agreement is used in obtaining a material, there may be no applicable restrictions. Consequently, it is best to confer with the technology transfer office of the investigator’s institution for guidance on such matters. CONSIDERATIONS FOR DEVELOPERS AND PROVIDERS OF MODEL ORGANISMS The NIH Model Organism Sharing Policy states that the NIH expects the submission of sharing plans from investigators whose projects will or could yield new, genetically modified model organisms or related resources. Further, if a given project unexpectedly results in new, genetically modified model organisms or research resources, it would be appropriate to share the model organisms, consistent with NIH grants policy. Consulting with the NIH program official and including a sharing plan in the next noncompeting application or in a separate letter would also be considered appropriate in such a situation. Since sharing plans can be highly complex and can involve issues pertinent to intellectual property, these plans should be consistent with NIH guidelines and in consultation with individuals who have expertise in this area. These experts could be staff from an institution’s sponsored research office, technology transfer office, or office of general counsel. Such consultation often will clarify a given institution’s policies regarding sharing and distribution of research resources such as mutant mice as well as its policies regarding intellectual property. Parties developing and/ or providing research resources should ensure that research use rights are retained for noncommercial research purposes, which are consistent with practices supported by the Association of University Technology Managers (AUTM) and the Council on Governmental Relations (COGR). If sharing plans for NIH awards are in conflict with obligations (i.e., title to the invention) to third parties (e.g., company X), NIH-funded parties will need to revise any such agreements so that they are consistent with the terms and conditions of the NIH funding award. For guidance regarding this issue, see the 2003 NIH Grants Policy Statement,15 as well as “Developing Sponsored Research Agreements: Considerations for Recipients of NIH Research Grants and Contracts”16 and “Intellectual Property Reporting for NIH Grantees That Also Have Involvement with the Veterans Administration.”17 Nonprofit institutions that wish to waive or assign title of inventions developed under an NIH funding award to another party must obtain approval from the NIH, except where such an assignment is made to an organiza-

CHAPTER 6 / NIH POLICIES ON SHARING OF MODEL ORGANISMS AND RELATED RESEARCH RESOURCES

tion that has as one of its primary functions the management of inventions and are subject to the same provisions as the funding recipient. As a term of award, it is the responsibility of the NIH-funded party to ensure that rights to inventions, such as model organisms and related research resources, arising out of NIH funding agreements are properly assigned to the institution. Failure to comply with NIH research resource sharing policies and the accepted plan may be considered by the NIH program staff in future funding decisions for the investigator and the investigator’s institution. An example of the Personal Benefits of Reserving Research Use Rights for the Research Community If the developer of a model organism reserves research use rights for at least noncommercial research purposes across the research community, this not only protects the nonprofit institutions, but it can also protect the developer of the mouse later on in his or her career. For example, in one scenario, a scientist is both an investigator at nonprofit institution A and the Chief Scientific Officer of for-profit X. While at nonprofit A, the investigator develops a model organism. The investigator does not want any competitors to have access to the resource, and therefore asks nonprofit A to license exclusively all rights for this resource to the investigator’s for-profit X, while retaining a research use license only for nonprofit A. The investigator is terminated from both nonprofit A and for-profit X. The investigator moves to nonprofit B, hoping to continue the research, but can no longer access the resource because for-profit X refuses to provide access to the investigator and nonprofit A is not permitted to provide the resource to any third party. If a research use license had been retained for all nonprofit research institutions, the investigator would have been able to continue the research at nonprofit B unhindered. CONSIDERATIONS FOR REQUESTORS AND RECIPIENTS OF MODEL ORGANISMS NIH sharing policies seek to ensure that research tools and results are made available to the biomedical community in a timely manner for furthering the research enterprise. Publications are the major medium by which the biomedical community learns of research results. When model organisms are reported in publications, users of the shared resource should acknowledge the source (repository and/or investigator) and reference the original citation describing the strains in their publications. This citation helps identify the specific material used by identifying the source. Standard genetic nomenclature should be used to designate the strains. Scientific custom is to name providers of materials as authors if providers collaborate with recipients of organisms and contribute intellectually to the manuscript. Otherwise, it is appropriate for providers to be acknowledged as the source of research resources upon which the manuscript is based. This acknowledgment most appropriately appears in the Materials and Methods section of the publication. As noted previously for developers and providers of model organisms, requestors and recipients of model organisms or related research resources produced with NIH funding should be mindful of the policies regarding inventions arising out of NIH funding agreements, including issues related to intellectual property. For example, if the recipient is employed by a not-for-profit institution (e.g., university) and conducts research with a model organism that results in a patentable invention, title to the invention must be properly assigned to the not-for-profit institution.

47

REVIEW AND EVALUATION OF SHARING PLANS When a sharing plan is submitted as part of the funding application, the Scientific Review Group, or study section, will comment on the adequacy of the sharing plan. Reviewers will be asked to describe their assessment of the sharing plan in an administrative note and, except in defined circumstances, will not include their assessment in the overall priority score. For some special initiatives, such as Funding Opportunity Announcements (FOAs) specifically directed to the development of model organisms, reviewers may be asked to integrate their evaluation of the sharing plan with other review criteria and factor their assessment into the overall evaluation of scientific merit. Any concerns must be resolved before an award can be made. Program staff, who will review sharing plans before making funding recommendations, should be the first point of contact for resolving sharing plan deficiencies. Whether a sharing plan is reasonable can be determined by the reviewers on a case-by-case basis, taking into consideration the organism or related resource involved, the timeline, the applicant’s decision to distribute the resource or deposit it in a repository, and other relevant considerations. For mechanisms other than special FOAs, the presence or adequacy of a plan should not enter into the scoring of the application. The sharing plan itself should be discussed after the application is scored and reviewer comments recorded in an administrative note. For special initiatives and research mechanisms, where the text of the announcement specifically includes the adequacy of a sharing plan as a review criterion, the adequacy of the plan will be discussed by the reviewers and will figure into the score. Finally, it should be noted that budget review would be part of the overall review consistent with usual NIH practice. PROGRESS REPORTING AND MONITORING OF SHARING Grants management staff will ensure that an acceptable sharing plan is referenced in the Notice of Grant Award (NGA) either by the standard reference to the funded application or by specific reference to a revised plan. The NGA is a binding legal document. The terms and conditions incorporate, and the grantee accepts, the terms once funds are drawn to conduct the project. The Grants Management Officer works with the NIH program staff to complete this process and address enforcement if the plan is not followed. Recognizing that circumstances can change, grants management and program staff will work with grantees to renegotiate sharing plans as necessary. Each Institute or Center will determine an appropriate response, consistent with the NIH guidelines, if the terms and conditions of the award are violated. Annual progress reports should report the number of sharing requests received and the number fulfilled when submitting a noncompeting renewal progress report, as well as the Final Progress Report. As the expectations and tools available to facilitate model organism sharing continue to evolve, maximum flexibility is encouraged to allow for renegotiations during the project period at the request of either the Institute/Center or the funded institution in response to materially new and/or unforeseen information or developments. Sharing plans should be discussed with the appropriate NIH program contact who can work with the developer to modify the sharing plan accordingly. It should also be noted that the sharing plan holds for the life of the grant. Accordingly, it should address how the institution

48

SECTION II / GENERAL CONSIDERATIONS

plans to make the model organism and related research resources available after the grant has expired, such as through costreimbursable charges or donation to a repository. As noted earlier, repository use is highly encouraged to provide for easy long-term access.

CONCLUSIONS The sharing of research materials and data is critically important in the progress of biomedical research. Along these lines, the NIH Model Organism Sharing Policy builds upon extant NIH sharing policies to continue to fund and promote broad and enabling research effectively and efficiently. This chapter provides sample plans, approaches, guidance, and resources for investigators to consider in how to share model organisms and related research resources with the research community. When done effectively, such sharing of research resources moves research forward and enables additional research to benefit public health.

APPENDIX: EXAMPLES OF REPOSITORIES AND ASSOCIATED RESOURCES FOR SHARING OF MODEL ORGANISMS AND RELATED RESOURCES AND DATA NIH Model Organism for Biomedical Research website: http:// www.nih.gov/science/models/. Examples of rodent resources: NCRR Rodent Resources website: http://www.ncrr.nih.gov/ncrrprog/cmpdir/RODENT.asp. Examples of mouse resources: Trans-NIH Mouse Initiatives: Major resources including sequencing and mapping resources, mutant mouse resources, and databases and repositories: http://www. nih.gov/science/models/mouse/resources/index.html. Mouse Models for Human Cancer Consortium: http:// emice.nci.nih.gov. Mutant Mouse Regional Resource Centers: http://www. mmrrc.org. NIH Knockout Mouse Project (KOMP): http://www.nih. gov/science/models/mouse/knockout/index.html. Partial listing of mouse repositories and databases: http://www. nih.gov/science/models/mouse/sharing/4.html. Examples of rat resources: Rat genomics and genetics: Major resources including rat databases and rat genomic tools: http://www.nih.gov/ science/models/rat/resources/index.html. Rat Resource and Research Center: http://www.nrrrc. missouri.edu. NIH Autoimmune Rat Model Repository and Development Center: http://dvrnet.ors.od.nih.gov/ratcenter/ index.html. Examples of fish resources: NCRR Fish Resources website: http://www.ncrr.nih.gov/ ncrrprog/cmpdir/FISH.asp. Zebrafish Information Network: http://zfin.org.

REFERENCES 1. Grieder FB, Strandberg JD. The contribution of laboratory animals to medical progress–past, present, and future. In: Hau J, Van Hoosier GL Jr, Eds. Handbook of Laboratory Animal Science: Essential Principles and Practices, 2nd ed., Vol. 1. Boca Raton, FL: CRC Press, 2002:1–11. 2. Hendrikson C. A short history of the use of animals in vaccine development and quality control. Dev Biol Stand 1996;86:3–10. 3. Malakoff D. The rise of the mouse, biomedicine’s model mammal. Science 2000;288:248–253. 4. Rogers JR, Bradley A. The mouse genome sequence: Status and prospects. Genomics 2001;77:117–118. 5. Public Health Service Grants Policy Statement. Washington, DC: U.S. Department of Health, Education, and Welfare, 1976. [DHEW Publication No. (OS) 77-50,000.] 6. Special Announcement: NIH Policy Relating to Reporting and Distribution of Hybridomas Produced Under Grants and Contracts. The NIH Guide Supplement for Grants and Contracts, May 1, 1980. Bethesda, MD: National Institutes of Health (NIH), 1980. 7. Notice: NIH Policy Relating to Reporting and Distribution of Unique Biological Materials Produced with NIH Funding. The NIH Guide for Grants and Contracts. No. 13(4):1. Bethesda, MD: National Institutes of Health (NIH), 1984. 8. NIH Procedures for Handling Non-Election of Title to Patentable Biological Materials. The NIH Guide for Grants and Contracts. No. 25(16); PT 36:96–131. Bethesda, MD: National Institutes of Health (NIH), 1996. 9. NIH Policy on Sharing of Model Organisms for Biomedical Research. The NIH Guide for Grants and Contracts. NOT-OD-04-042. Bethesda, MD: National Institutes of Health (NIH), 2004. (Accessed September 1, 2006, at http://grants2.nih.gov/grants/guide/notice-files/NOT-OD04-042.html.) 10. NIH Grants Policy Statements. Bethesda, MD: National Institutes of Health (NIH), 2003. (Accessed September 1, 2006, at http://grants. nih.gov/grants/policy/#gps.) 11. Principles and Guidelines for Recipients of NIH Research Grants and Contracts on Obtaining and Disseminating Biomedical Research Resources: Final Notice. U.S. Federal Register. No. 64(246):72090– 72096. Bethesda, MD: National Institutes of Health (NIH), 1999. 12. National Research Council of the National Academies. Sharing Publication-Related Data and Materials: Responsibilities of Authorship in the Life Sciences. Washington, DC: The National Academies Press, 2003. 13. Society for Neuroscience. Responsible Conduct Regarding Scientific Communication, 1st ed. Washington, DC: Society for Neuroscience, 1998. 14. Final NIH Statement on Sharing Research Data. The NIH Guide for Grants and Contracts. NOT-OD-03-032. Bethesda, MD: National Institutes of Health (NIH), 2003. (Accessed September 1, 2006, at http://grants2.nih.gov/grants/guide/notice-files/NOT-OD-03-032. html.) 15. NIH Grants Policy Statement 12/03, 2003. Bethesda, MD: National Institutes of Health (NIH). (Accessed September 1, 2006, at http:// grants.nih.gov/grants/policy/nihgps_2003/index.htm.) 16. Developing Sponsored Research Agreements: Considerations for Recipients of NIH Research Grants and Contracts. The NIH Guide for Grants and Contracts. No. 23(25); PT 34:94–213. Bethesda, MD: National Institutes of Health (NIH), 1994. (Accessed September 1, 2006, at http://grants2.nih.gov/grants/guide/notice-files/not94-213. html.) 17. Intellectual Property Reporting for NIH Grantees That Also Have Involvement with the Veterans Administration. The NIH Guide for Grants and Contracts. NOT-OD-01-033. Bethesda, MD: National Institutes of Health (NIH), 2001. (Accessed September 1, 2006, at http://grants.nih.gov/grants/guide/notice-files/NOT-OD-01-033. html.)

7

Databases for Biomedical Animal Resources RICHARD L. CRAWFORD AND TIM ALLEN

ABSTRACT

DATABASES

Database searching for research involving warm-blooded animals is required by law in the United States and is also a requirement for performing animal research in many other countries. It is, therefore, necessary for the investigator/researcher to perform a reasonable database search before developing the research protocol. Key Words: Databases, Searching, Websites, Animal models, Resources, Strategy, Protocol, Animal, Area of Study, Sources of Information.

While there are many papers discussing the utility of various bibliographic and specialty databases and information centers in the development of animal research protocols,4–18 it is still useful to point out major providers/suppliers/repositories of animal models for biomedical research. In the United States, the National Institutes of Health (NIH) is the primary resource for the development and funding of biomedical animal models. Their website—Model Organisms for Biomedical Research (http://www.nih.gov/science/models)—is a good starting point for information on various projects under development by the NIH, its grantees, and other national and international laboratories. Among the organisms listed are mammalian models, especially rat and transgenic mouse models, yeast, fungi, round worm, fruit fly, and frogs. Another excellent resource is the NIH, National Center for Research Resources, Division for Comparative Medicine. (http://www.ncrr.nih.gov/comparative_ med.asp). The Division for Comparative Medicine provides access to laboratory-bred nonhuman primates including macaques, baboons, chimpanzees, owl monkeys, squirrel monkeys, and others. The Division also maintains an aquatic animal models program (zebrafish, Xiphophorus, cephalopods, and Aplysia) and has initiatives for invertebrates (Drosophila, Caenorhabditis, Aplysia, Tetrahymena, and cephalopods). The Institute for Laboratory Animal Research at the U.S. National Academies of Science has a unique search engine available that searches the websites of suppliers and repositories that sell or distribute experimental animals. It is available at http://dels.nas.edu/ilar_n/ilarhome/ models.shtml. The European Union (EU) maintains the European Mouse Mutant Archive (EMMA) (http://www.emma.rm.cnr.it/), which preserves and distributes relevant strains collected from repositories and laboratories throughout the EU. The EMMA Strain Database is accessible through an easy to use search utility and can be found at http://andy.emma.cnr.it/jEmma/list.utf8.html. In Japan, the Riken Bioresource Center was established by the government to serve as a global resource for biomaterials. The Center serves as the Japanese repository for an extensive collection of mouse strains. The searchable catalog provides a detailed description of the model, strain information, the developer, and pertinent journal references. The English version is available at http://www.brc.riken.jp/lab/animal/en/. Another excellent inter-

INTRODUCTION The United States (U.S.) Animal Welfare Act,1 U.S. Department of Agriculture (USDA) animal welfare regulations,2 and USDA-Animal Care Policy 123 require that the principal investigator, using warm-blooded animals in research, must ensure that pain and distress are minimized, that alternatives to the proposed procedure or protocol are not available, or if alternatives are available why they were not used, and that the research does not unnecessarily duplicate previous experiments. Also, many countries now have regulations that require researchers to document that they have conducted a search for possible alternatives when applying for permission to conduct animal research. Database searching is the best method to find animal resources and to comply with the requirements of the Animal Welfare Act or similar regulations. It is important to become familiar with the database platform, the subjects covered in each database, and the sources of information for each database to perform an effective and reasonable search effort. An institution’s librarian or information specialist can be of considerable help with database searching and should be consulted prior to developing the research protocol. Training of researchers in how to perform a proper database search is also recommended as the search strategy determines the type and quality of the information obtained.

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

49

50

SECTION II / GENERAL CONSIDERATIONS

Table 7–1 National and international sources of animals Name

Web address

NIH Model Organisms for Biomedical Research NIH Division for Comparative Medicine Institute for Laboratory Animal Research Models search engine European Mouse Mutant Archive (EMMA) EMMA Strain Database Riken Bioresource Center Federation of International Mouse Resources The Jackson Laboratory International Mouse Strain Resource Charles River Laboratories Taconic Farms/Lexicon Genetics Harlan Sprague Dawley Harlan Europe Lab Animal Suppliers Guide Laboratory Animals Breeders and Suppliers

www.nih.gov/science/models www.ncrr.nih.gov/comparative_med.asp dels.nas.edu/ilar_n/ilarhome/models.shtml

national source of information on animal models and their availability is through the Federation of International Mouse Resources (FIMRe). This 11 nation collaboration provides researchers with access to a searchable mutant mouse catalog, searchable database of mouse genome informatics, nomenclature guidelines, and links to repository sites for contributing new strains. It is available at http://www.fimre.org. These websites also provide numerous links to university collections, national laboratories, and commercial vendors. Not surprisingly, commercial vendors also maintain extensive databases of useful animal models. The Jackson Laboratory (http://www.jax.org), which operates as a nonprofit institution, is one of the world’s foremost repositories for unique mouse models and maintains a searchable database of available strains. This database, the International Mouse Strain Resource, contains information on global stocks of inbred, mutant, and engineered mice. It can be found at http://www.informatics.jax.org/imsr/index.jsp. Charles River Laboratories (http://www.criver.com) is a commercial vendor of rodents, rabbits, chickens, and chicken eggs. It maintains a database of disease models and transgenic models and provides access to the Deltagen repository of 900 knockout mouse lines. Taconic Farms (http://www.taconic.com), with its partner Lexicon Genetics, maintains a searchable database of genetically

Table 7–2 Bibliographic databases, biomedical, veterinary, and biological AGRICOLA—agricola.nal.usda.gov CAB Abstracts—www.cabi.org MEDLINE—www.ncbi.nlm.nih.gov/pubmed/ EMBASE—www.embase.com BIOSIS—www.biosis.org Zoological Record—www.biosis.org PsycINFO—www.apa.org/psycinfo/ Scopus—www.scopus.com

www.emma.rm.cnr.it Andy.emma.cnr.it/jEmma/list.utf8.html www.brc.riken.jp/lab/animal/en www.fimre.org www.jax.org www.informatics.jax.org/imsr/index.jsp www.criver.com www.taconic.com www.harlan.com www.harlaneurope.com guide.labanimal.com www.lal.org.uk/breedersandsupp1.html

modified mice and has numerous other rodent models listed with detailed descriptions of their utility. Harlan (http://www.harlan. com) and Harlan Europe (http://www.harlaneurope.com), as the world’s largest suppliers of laboratory animals, provide information resources on a variety of animal models and maintain an extensive catalog of rats, mice, hamsters, guinea pigs, cotton rats, gerbils, and rabbits. More vendor information can be found at http://guide.labanimal.com and http://www.lal.org.uk/breedersandsupp1.html. Table 7–1 summarizes the information listed above. Large bibliographic databases such as the National Library of Medicine’s Pubmed (http://www.pubmed.gov), Elsevier’s EmBase (http://www.embase.com), and Thomson Scientific’s BIOSIS (http://www.biosis.org) are catalogs of the world’s peer-reviewed scientific publishing. As such they contain a wealth of information on different animal models and provide background information on these models. It is important to remember that while there is some overlap in coverage, each database contains unique information not found in the others. Not only does subject coverage vary but the sources of information are varied as well. For example, a search of Medline should also include EmBase, which includes monographs, reports, and other useful sources not included in Medline, which covers only journals. Several core databases, such as Medline, EmBase, and Biosis, should be searched in order to conduct a comprehensive literature search. Elsevier has made this somewhat easier by combining their EmBase with unique records from Medline to create EmBase.com containing more than 17 million biological and pharmacological records. Choosing databases pertinent to the protocol will provide the researcher with better information and more accurate resources for the study and will better comply with the laws and regulations pertaining to animal research. The following tables list selected bibliographic databases for animal resources (Table 7–2), the subjects covered (Table 7–3), and the sources of information for the selected databases (Table 7–4). Table 7–5 lists web addresses for database vendors or portals to a variety of online databases.

CHAPTER 7 / DATABASES FOR BIOMEDICAL ANIMAL RESOURCES

51

Table 7–3 Subject coverage of selected databases AGRICOLA

BIOSIS

CAB

EMBASE

MEDLINE

Zoological Record

General agriculture Animal science

General agriculture

General agriculture

Clinical medicine

Clinical medicine

Zoological information

Aerospace biology

Animal science and production

Experimental medicine

Experimental medicine

Behavior and communication

Chemistry and biochemistry

Biochemistry and anatomy

Crop science

Pharmacology, drugs, potential drugs

Pharmacology

Physiology, immunology

Microbiology

Bacteriology (microbiology)

Forestry

Biochemistry

Microbiology

Biochemistry

Cytology

Cell biology

Pest control

Developmental biology

Administration

Diseases

Human and animal nutrition

Botany

Human nutrition

Forensic medicine

Nutrition

Evolution

Biotechnology

Anatomy

Biotechnology

Health economics

Nutrition

Genetics

Physiology

Physiology

Pesticides

Occupational health

Anatomy and physiology

Histology

Veterinary medicine

Clinical medicine

Veterinary medicine

Toxicology

Veterinary medicine

Taxonomy

Wildlife

Pathology

Machinery and buildings

Occupational medicine

Life cycles/ development

Zoology

Biophysics

Economics

Toxicology

Nomenclature

Entomology

Toxicology

Other medical topics

Paleontology

Source: United States Department of Agriculture, National Agricultural Library, Animal Welfare Information Center.

Table 7–4 Sources of information for selected databases AGRICOLA 1970–present

BIOSIS 1969–present

CAB 1972–present

EMBASE 1974–present

MEDLINE 1966–present

Zoological Record 1978–present

>1000 journals

Approximately 9000 journals

>14,000 journals/ translations

Approximately 3700 journals

4300 journals

Approximately 6000 journals

Books, monographs

Books, monographs

Books, monographs

Books

Books, monographs

Proceedings

Proceedings/abstracts

Symposia

Conference proceedings, symposia, meetings

Conference proceedings, symposia, meetings

Research reports

Technical reports

Technical reports

Special reports

Theses

Nomenclature, rules

Theses, dissertations

Theses

Translations

Annual reviews

Review journals

Bibliographies

Bibliographies

Bibliographies

Electric documents

Patents 86–89

Patents

Audio visuals

Letters, notes

Annual reports

USDA publications

Guides

Guides

Government documents

Research comments

Conferences

Selected newsletters

Conferences

Meetings

Manuals, SOPs

Symposia

Toxicology protocols

Meetings

Source: United States Department of Agriculture, National Agricultural Library, Animal Welfare Information Center.

52

SECTION II / GENERAL CONSIDERATIONS

Table 7–5 Databases, vendors, and portals

Table 7–6 Additional databases available on the web

DIALOG—www.dialog.com National Library of Medicine Databases—www.nlm.nih.gov/databases OVID—www.ovid.com Primate Information Network—pin.primate.wisc.edu AWIC—awic.nal.usda.gov Databases—awic.nal.usda.gov/databases

Scientific and Technical Information Network—stinet.dtic.mil (provides public access to unclassified military biomedical research) Scirus—Scientific Information Search Engine developed by Elsevier Science—www.scirus.com/ PrimateLit—primatelit.library.wisc.edu/

Source: United States Department of Agriculture, National Agricultural Library, Animal Welfare Information Center.

resources listed above, Table 7–6 lists other free resources that may provide useful information. Because many countries require scientists using animals to consider alternative methods or to minimize pain and distress to those animals that are used, a listing of useful websites is provided below. Listed below are a few samples of websites for alternatives to animal use in research: ALTERNATIVES Alternatives to Skin Irritation & Corrosion Testing in Animals—www.invitroderm.com Altweb—altweb.jhsph.edu Animal Welfare Information Center—http://awic.nal. usda.gov AVAR Alternatives in Education Database—www.avar.org Center for Alternatives to Animal Testing (CAAT)—caat. jhsph.edu/ ECVAM: The European Centre for the Validation of Alternative Methods—ecvam.jrc.it/index.htm Fund for the Replacement of Animals in Medical Experiments (FRAME) www.frame.org.uk/index.htm Humane Society of the United States (HSUS)—www. hsus.org Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM)—iccvam.niehs.nih.gov

WEB SEARCHING AND OTHER GENERAL DATABASES AVAILABLE ON THE WEB Web searching is an information source that should not be ignored. There are many websites available and using the terminology “Animal Model” or “Animal Models” in any web search engine will bring up many websites concerning animal models. However, these will also include sites for animal replicas, sporting goods, top dog model, and other similar sites, and a lot of scrolling is required to locate pertinent and specific animal model sites. Consequently, it is necessary to choose your words wisely. Being specific will improve your results, i.e., use more than two or three words in your strategy. Remember, it is the World Wide Web, so consider international spelling, such as behavior, behaviour, tumor, tumour, estrogen, oestrogen, anesthetic, anasthetic, anaesthetic, and other words that may have several spellings. Enclosing a multiword phrase in quotation marks tells the search engine to list only sites that contain those words in that exact order. A plus (+) sign directly in front of a word indicates that the word or phrase must appear in the search results. A minus (−) sign indicates that the word or phrase should not be included in the search results. By using proper searching strategies the results will be more accurate and meaningful to your protocol. In addition to the

Table 7–7 Comparison of alternative websites

Articles Abstracts Bibliographies Conference proceedings Dbase links Document links Grant information Grant program How to search Legislation List of alternative methods Organization links Search engine

AWIC

CAAT

FRAME

HSUS

ICC-VAM

Norwegian Reference Center

X

X

X

X

X

X

X X X X

X X X

X X X

X X

X X X

Altweb

AVAR

X X

X

X X

X

X X

NORINA

X

X X X X

X X X

X X X X

X X

X X X

X X

UC Center for Animal Alternative X

X X

Skin irritation

X X X X

X

X X X X

X X

X

X

X X

X

X

X

X X

X

X X

Source: United States Department of Agriculture, National Agricultural Library, Animal Welfare Information Center.

CHAPTER 7 / DATABASES FOR BIOMEDICAL ANIMAL RESOURCES

The Norwegian Reference Centre for Laboratory Animal Science & Alternatives— oslovet.veths.no NORINA (A Norwegian Inventory of Alternatives)— oslovet.veths.no/NORINA University of California Center for Animal Alternatives— www.vetmed.usdavis.edu/Animal_Alternatives/main. html Table 7–7 indicates the content and subject matter for the various web sites on alternatives to animal use.

SPECIFIC ANIMAL MODEL DATABASES As previously indicated, the Internet has a lot of useful information available through general searches using broad terms such as “animal models of disease.” There are animal model suppliers and vendors, books on animal models, journal articles on animal models and their use, lists of animal models available, and links to useful information on animal models. A good web search combined with a good multiple database search should provide useful information to the researcher that is pertinent to the research protocol. Listed below is a sample of databases to be found from various institutions. • University of Michigan Transgenic Animal Model Core http://www.med.umich.edu/tamc • Plant, Animal & Bacteria Cell Models http://www. cellsalive.com/cells/3dcell.htm • Surgical Animal Models for Cardiovascular Diseases http://www.health.ufl.edu/anires/Models/index.htm • Models for studying AIDS and AIDS associated cancers www.ihv.org/research/animal.html • Animal Model of inherited human disorder www.vet.utk. edu/research/models.shtml • Animal Models of Neuropsychiatric Diseases www. worldscibooks.com/medsci/p421.html • Animal Models Center, Beth Israel Deaconess Medical Center http://cardiogenomics.med.harvard.edu/ component-detail?project_id=233 • Animal Models of Diabetic Complications Consortium www.amdcc.org • XENOGEN—Light Producing Animal Models www. xenogen.com/wt/page/animalmodel_list • Zivic Laboratories Inc. www.zivic-miller.com/technical% 20services%20department.htm • Transgenic Animal Web www.med.umich.edu/tamc/links. html • Canadian Council on Animal Care. Animal Models in Biomedical Research www.ccac.ca/en/CCAC_Programs/ ETCC/Module05/04.html • Zebrafi sh Animal Models for Human Hereditary Ocular Disease www.research-projects.unizh.ch/p5672.htm • University of Arizona—Research Models & Disease Links www.ahsc.arizona.edu/uac/disease.shtml • Animal Models Available from Baylor College of Medicine www.bcm.edu/blg/Animal/Mods.pdf

DATABASE SEARCH STRATEGY Databases and web sites contain a great deal of useful research information, however, little pertinent information will be obtained without a proper search strategy to find the information desired.

53

Listed below are pointers to keep in mind when preparing to do a database search. 1. What is the general area of study (drug testing, cardiology, toxicology, lipid metabolism, etc.)? 2. What is the type of protocol (research, teaching, testing, etc.)? 3. What is the proposed animal species? 4. Identify the systems or anatomy involved in the study. 5. List any drugs or compounds used in the procedure. 6. What are the objectives and end points of the protocol? 7. Use both American and European spelling. 8. List key words using terminology from your responses to the questions above. 9. If searching on a system that allows development of complex search strategies, keep your concepts separate and then combine them to obtain specific and pertinent information. 10. Choose databases that are appropriate to the area of study.

CONCLUSIONS There is a tremendous amount of information available from databases and websites; however, it can be difficult to obtain the specific information desired if the proper databases are not searched and the terminology used is too broad or inappropriate to the protocol. Some databases, such as Agricola and Medline (PubMed), are available to search and download references for free. Other databases require subscriptions and the downloading references for a fee. If a facility is involved in a great deal of research, which necessitates database searching, a subscription to a database vendor would be helpful as multiple databases would be available for searching and those pertinent to the protocol or area of study could be utilized. Two, or more, databases should be searched and these databases should be pertinent to the protocol in both the subjects covered and the sources of information (not just journals). The search strategy, including databases used, key words used, and years searched, should be saved for use again when required or if changes are required in the protocol. Should the investigator not be familiar with database searching strategy, he or she should contact an institutional librarian or information specialist for assistance. Training in proper searching techniques is also very helpful to those who do not have librarians or information specialists available to them and are required to perform periodic database searches. Should neither of these options be available to the investigator, the Animal Welfare Information Center (U.S. Department of Agriculture, National Agricultural Library, Beltsville, MD; Email, [email protected]) is available for suggestions and assistance if desired.

ACKNOWLEDGMENTS The use of trade, firm, or corporation names in this article is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable.

54

SECTION II / GENERAL CONSIDERATIONS

REFERENCES 1. Animal Welfare Act. United States Code, Title 7, Chapter 54, Sections 2143(a)(3)(A)(B)(C). 2. Animal Welfare Regulations. Code of Federal Regulations, Title 9, Chapter 1, Subchapter A—Animal Welfare, Part 2, Subpart C, Sections 2.31 (d)(i)(ii)(iii). 3. Animal Care Policy 12, Written Narrative for Alternatives to Painful Procedures: June 21, 2000. www.aphis.usda.gov/ac/publications/ policy/policy12.pdf. 4. Mattingly CJ, Rosenstein MC, Colby GT, Forrest JN, Boyer JL. The Comparative Toxicogenomics Database (CTD): A resource for comparative toxicological studies. J Exp Zool A Comp Exp Biol 2006;305(9):689–692. 5. Wiley JC, Prattipati M, Lin C-P, Ladiges W. Comparative Mouse Genomics Centers Consortium: The Mouse Genotype Database. Mutat Res 2006;595(1–2):137–144. 6. Sprague J, Bayraktaroglu L, Clements D, Conlin T, Fashena D, Frazer K, Haendel M, Howe DG, Mani P, Ramachandran S, Schaper K, Segerdell E, Song P, Sprunger B, Taylor S, Van Slyke CE, Westerfield M. The Zebrafish Information Network: The zebrafish model organism database. Nucleic Acids Res 2006;34(Database issue): D581–585. 7. Blake JA, Eppig JT, Bult CJ, Kadin JA, Richardson JE. The Mouse Genome Database (MGD): Updates and enhancements. Nucleic Acids Res 2006;34(Database issue):D562–567. 8. Smith AJ, Allen T. The use of databases, information centres, and guidelines when planning research that may involve animals. Anim Welfare 2005;14(4):347–359. 9. Krupke D, Naf D, Vincent M, Allio T, Mikaelian I, Sundberg J, Bult C, Eppig J. The Mouse Tumor Biology Database: Integrated access to mouse cancer. Exp Lung Res 2005;31(2):259–270. http://www. informatics.jax.org.

10. de la Cruz N, Bromberg S, Pasko D, Shimoyama M, Twigger S, et al. The Rat Genome Database (RGD): Developments towards a phenome database. Nucleic Acids Res 2005;33(Database issue): D485–491. 11. Weeks J, Hart RP. SCI-Base: An open-source spinal cord injury animal experimentation database. Lab Anim (UK) 2004;33(3):35–41. 12. Habermann B, Bebin A-G, Volkmer M, Herklotz S, Eckelt K, Pehlke K, Epperlein HH, Schackert HK, Wiebe G, Tanaka E. An Axolotl EST database: Gene information for studying regeneration. Dev Biol 2003;259(2):537. 13. FlyBase Consortium. The FlyBase database of the Drosophila genome projects and community literature. Nucleic Acids Res 2003;31(1):172– 175. http://flybase.bio.indiana.edu. 14. Smith CM, Begley DA, Eppig JT, Finger JH, Hayamizu TF, Hill DP, Kadin JA, McCright IJ, Richardson JE, Ringwald M. The mouse Gene Expression Database (GXD): A resource for developmental biologists. Dev Biol 2003;259(2):492–493. 15. Santi PA, Beattie JW, Lukkes J, Nietfeld J. The Mouse Cochlea Database: Bibliographic Database Development. Society for Neuroscience Abstract Viewer and Itinerary Planner 2002, Abstract No. 162.3. 16. Anagnostopoulos AV (Reprint), Mobraaten LE, Sharp JJ, Davisson MT. Transgenic and knockout databases: Behavioral profiles of mouse mutants. Physiol Behav 2001;73(5):675–689. 17. Aleman CL, Mas RM, Rodeiro I, Noa M, Hernandez C, Menendez R, Gamez R. Reference database of the main physiological parameters in Sprague-Dawley rats from 6 to 32 months. Lab Anim (UK) 1998;32(4):457–466. 18. Allen T. On-line databases and the World Wide Web: What is available? What is missing? In: van Zutphen LFM, Balls M, Eds. Animal Alternatives, Welfare, and Ethics. Developments in Animal and Veterinary Science, Vol. 27. Amsterdam: Elsevier, 1997.

8

Psychological Enrichment for Animals in Captivity KRISTINE COLEMAN

ABSTRACT

WHAT IS PSYCHOLOGICAL ENRICHMENT? Environmental enrichment is “an animal husbandry principle that seeks to enhance the quality of captive animal care by identifying and providing the environmental stimuli necessary for optimal psychological and physiological well-being” (Shepherdson,6 p. 1). In other words, it is a way to functionally simulate the natural environment of research animals, in an effort to increase the opportunity to express species-specific behaviors and decrease the occurrence of abnormal behaviors. The goals of providing psychological enrichment are to have animals that are in good physical condition, display a variety of species-typical behaviors, are resilient to stress, and easily recover (behaviorally and physiologically) from aversive stimuli.7

Animal care in biomedical facilities has undergone a dramatic transformation in the area of psychological well-being and enrichment over the past two decades. Today, attending to the behavioral needs of research animals is considered an integral part of animal care. Enrichment is defined as environmental stimuli provided to research animals in an effort to improve well-being by increasing species-specific and decreasing abnormal behaviors. Such environmental enhancement can help alleviate some of the stress associated with living in captivity, and can thus produce a better research model. This chapter discusses some of the issues surrounding the provision of enrichment and details some basic enrichment strategies, with emphasis on rodents, dogs, and nonhuman primates. Key Words: Environmental enrichment, Psychological wellbeing, Animal welfare, Laboratory animals, Animal behavior, Animal husbandry.

WHY PROVIDE ENRICHMENT? ETHICAL CONCERNS The research community’s views on the care of research animals reflect the significant change in attitude that has occurred in the general public over the past few decades. Much of this shift can be attributed to increases in scientific knowledge regarding the complex nature of animal behavior. Today, scientists and others using animals in research recognize that they have a responsibility to attend to the psychological wellbeing of their subjects.8 In short, providing psychological enrichment, within experimental constraints, is generally considered the “right thing to do.” LEGAL OBLIGATIONS In most countries, including the United States, providing research animals with enrichment is not an option, but rather is required by various regulatory agencies. The Animal Welfare Act (AWA9) is the statute that safeguards laboratory animals in the United States. Nonhuman primates are afforded the greatest protection under the AWA, which mandates that research institutions housing nonhuman primates have in place a plan to ensure their psychological enrichment.9 The AWA also calls for exercise for dogs used in research.9 While the AWA does not contain provisions for mice, rats, or birds, scientists receiving federal funding from most U.S. funding agencies must adhere to the guidelines in the Public Health Service Policy On Humane Care And Use Of Laboratory Animals [“PHS Policy”; National Institutes of Health, Office of Laboratory Animal Welfare (OLAW)]. This policy covers all vertebrates and requires compliance with the AWA and the Guide for the Care and Use of Laboratory Animals (“The Guide”8). The Guide specifies that enrichment be provided for laboratory animals, including social housing when appropriate for the species and the use of objects and cage

INTRODUCTION Animal care in biomedical facilities has undergone a dramatic transformation in the area of psychological well-being and enrichment over the past two decades. In the early part of the twentieth century, the emotional welfare of laboratory animals was largely disregarded. This situation changed as people realized that like humans, animals have certain basic behavioral needs, which, if not met, can adversely affect the behavior and physical health of the individual. Today, providing for the psychological requirements of research animals is accepted as an integral part of animal care.1 Most institutions devote substantial resources to ensure that animals receive enrichment (items provided to animals beyond their basic food and housing needs) of various sorts. Many facilities, particularly those housing nonhuman primates, have entire units dedicated to providing psychological enrichment for subjects.2 Furthermore, enrichment is now provided not only to mammals, but also to birds,3 snakes,4 and fish.5 This chapter discusses the psychological enrichment of laboratory animals. While it focuses on rodents, dogs, and nonhuman primates, most of the concepts are universal, and can be applied to other laboratory species as well.

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

55

56

SECTION II / GENERAL CONSIDERATIONS

complexities that “increase the opportunities for the expression of species-specific postures and activities and enhance the animals’ well-being” (Council,8 p. 37). It also sets the standards for voluntary accreditation through the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), an organization that promotes the humane treatment of animals used in science. ENRICHMENT CAN IMPROVE THE ANIMAL MODEL One of the most compelling reasons to provide enrichment is to reduce stress and improve well-being for laboratory subjects, thereby improving the animal model. Stress can adversely affect many physiological parameters including the reproductive axis,10 immunological parameters,11,12 and brain function.13 Stressed animals are at a higher risk for becoming ill14 or developing maladaptive behaviors such as self-directed aggressive behavior,15 which can affect a variety of research outcomes. Reducing this kind of stress can result in more reliable research subjects, and improve the interpretation of research results. Providing social housing to rhesus macaques is an example of how enrichment can improve the animal model. Macaques and other nonhuman primates are social animals, and have evolved to live in complex societies. Socially housing monkeys affords them the opportunity to engage in species-specific behaviors and to develop social and cognitive skills necessary for group living.16 It also reduces potential confounding factors, which can directly affect research results. For example, singly and group-housed monkeys differ with respect to various immunological parameters, such as natural killer cell activity, cytokine production, and proliferation responses to mitogens.17–19 These differences can affect the results obtained in a variety of studies. Thus, it can be argued that housing monkeys in a more natural condition produces a better model for human conditions.

EFFECTS OF ENRICHMENT Environmental enrichment in rodents has been associated with a variety of behavioral, neural, and physiological parameters, including increased exploratory behavior,20–22 decreased anxiety on the elevated plus maze,23 improved learning ability,24,25 increased brain size and weight,26 increased number of synapses per neuron,27 increased neurogenesis,28 and decreased cell death.26,29–32 Mice in an enriched environment also have higher natural killer (NK) cell activity than nonenriched mice, suggesting better immune function.23 On the other hand, enriched rodents tend to consume more food33 and ethanol22,34 compared to nonenriched rats, an outcome that needs to be carefully considered in the study design. While enrichment has been shown to decrease stress (as measured by serum levels of corticosteroid) in nonhuman primates,35 several studies have shown that it can increase basal corticosteroid (CORT) levels and cause larger adrenal weights in male rats.23,36,37 However, these studies also reported that the CORT levels of the enriched rats were still within normal limits and were not associated with pathology.23 Furthermore, enriched animals also showed less fear and exhibited less response to other stressors, such as predators or experimental stressors, suggesting that enrichment can attenuate the stress response to aversive events.23,25,36,38 This mitigating effect of enrichment on stress response has been found in other species as well, including birds39 and snakes.4 This ability to recover from stressful experiences is considered an important aspect of well-being.7,40

ENRICHMENT AND EXPERIMENTAL VARIABILITY The findings described in the previous section underscore the importance of considering enrichment as a potential variable in studies. Even small changes in enrichment practices can have dramatic effects on many experimental outcomes, leading to the potential for variability among laboratories.41 It is important to carefully document the type of enrichment provided, as well as when and how it is implemented, and to report this information in scientific publications. However, the absence of enrichment can also increase variability between laboratories.42 Lack of enrichment may increase an animal’s vulnerability to the stressors that are being investigated.43 Furthermore, there can be vast differences in how individuals respond to stress.44–46 Using enrichment to decrease stress associated with experimental procedures may reduce variability and the development of stress-related problems, thereby increasing the validity of experimental results.

IMPLEMENTING AN ENRICHMENT PLAN There are many resources available for implementing an enrichment plan for various species,1,47–49 including a special issue of the Institute for Laboratory Animal Research (ILAR) Journal (2005, Volume 46). Below are some general guidelines. ENRICHMENT SHOULD BE SPECIES-SPECIFIC Each species has evolved under different environmental conditions, resulting in unique behavioral patterns. Successful enrichment programs must take these specific behaviors into account in an effort to increase the biological functioning of the captive environment.50 For instance, while most rodents are social, golden hamsters are not. Even within a species, enrichment needs may also vary by strain51 or gender.43 The Animal Welfare Information Center, part of the United States Department of Agriculture, maintains a website (http://www.nal.usda.gov/) containing useful information regarding the behavior of various laboratory animal species. CONSISTENCY IS IMPORTANT Because enrichment can affect many research outcomes, care must be taken to ensure enrichment is given uniformly, and not haphazardly. Enrichment should be considered an integral part of animal care, and not as something extra to be done “when there is time.”1 It is also important for caretakers and researchers to avoid providing extra enrichment to “favorite” subjects. Not only can this be an experimental confound, but it can also affect the well-being of the animals. We have found that if only one or two monkeys in a room get an enrichment device, they are sometimes threatened by other, presumably more dominant, individuals (personal observation), which can lead to self-biting or other behavioral problems. INTEGRATE BEHAVIORAL NEEDS WITH EXPERIMENTAL NEEDS Successful enrichment programs integrate the needs of the animal with experimental constraints. Studies on feeding behavior may prohibit the intake of extra calories, but other enrichment options can be used in lieu of food enrichment. Macaques can be given foraging devices containing toys that can be manipulated (e.g., small cat toys) instead of food.52 New enrichment options should be tested prior to their implementation, to see if they will affect experimental outcomes. ENRICHMENT SHOULD BE RESULTS-BASED An important, but difficult, part of a successful enrichment program is assessment. Enrichment offerings should be periodically evaluated, and provisions that do not have a demonstrable effect upon

CHAPTER 8 / PSYCHOLOGICAL ENRICHMENT FOR ANIMALS IN CAPTIVITY

well-being of the animal should be reconsidered.50 It is not always easy to decide whether or not a particular enrichment offering is effective. Parameters to be assessed should include behavior (enrichment items should increase species-typical and/or decrease abnormal behaviors) as well as physiological variables that indicate well-being (i.e., animals should be healthy, with few signs of chronic stress). It is also important to consider the cost and safety of enrichment items. Items that are difficult to clean may be fomites, increasing the risk of illness.17 Optimally, enrichment items directly affect the well-being of laboratory animals. However, there may be value in enrichment options that have only indirect effects on the animals for which they are designed. Televisions, for example, are often provided to nonhuman primate species, even though it is not part of the normal behavioral repertoire of wild monkeys. Still, because people like to watch television, they assume (rightly or wrongly) that animals would as well. In practice, most animals do not pay a lot of attention to TV enrichment, although some do. However, beyond the direct effects of TV enrichment on the animals (which may be small), it allows the caretakers to feel that they are doing something positive for the animals. This can increase their job satisfaction and improve the relationship between caretakers and the animals, which can, in turn, improve well-being for the primates.53 TYPES OF ENRICHMENT Enrichment can be broken down into five basic categories,47,48 including social (housing gregarious species in groups or pairs), physical (perches, platforms, houses or platforms, toys, and other objects that can be explored or manipulated), food (novel food items, or objects that increase foraging time), sensory (televisions, radio), and occupational (physical and mental activity, including training). These enrichment options are not equally attractive to all species. Ideally, laboratory animals would receive multiple forms of enrichment.

ENRICHMENT FOR COMMONLY USED LABORATORY ANIMALS RODENTS AND RABBITS Rodents and rabbits are among the most commonly used animals in biomedical research. Strikingly, although they have been bred in laboratories for centuries, they still maintain behaviors similar to wild counterparts,54,55 including nest-building, burrowing, hiding, and foraging. Even knockout and genetically modified strains of mice and rats retain many behaviors of wild animals. A successful enrichment program should take species-typical behaviors into account. Most rodents and rabbits are gregarious (although golden hamsters are not), and live in family groups.56 Thus, social housing (in groups or pairs) should be one of the first considerations in implementing an enrichment program for these species. Such housing allows individuals to engage in many species-typical behaviors, including play and grooming.55 Furthermore, having social companionship can buffer individuals against external stresses,57 which, in turn, improves well-being. However, social housing can lead to aggression in some species. Mice tend to be more aggressive than rats. Furthermore, both strain58 and sex can affect aggression in mice. For example, female mice tend to fight less than males, particularly during the mating season. Providing additional enrichment can help ameliorate some of the negative consequences of social housing. Van Loo et al.59 found that adding nesting material can reduce aggression in socially housed male mice.

57

In general, rodents and rabbits are prey species, which shapes much of their behavior. When confronted with an aversive or frightening stimulus, their first reaction is to hide or burrow. Providing the animals with shelter (e.g., tunnels or houses) or substrate in which to burrow affords them the ability to withdraw from aversive situations, which can alleviate fear and stress.55 It also allows animals to exercise some control over their environment,60–62 which itself reduces stress-related behaviors such as stereotypical behavior (defined as repetitive, invariant behavior patterns with no obvious goal or function).37,57,63,64 These items, along with other forms of physical enrichment (e.g., platforms, nesting material, toys, and gnawing sticks), also increase exploration and manipulation and decrease abnormal behaviors in rodents and rabbits.33,37,57,65–67 Many of these items (e.g., tunnels) can easily be made with commonly available materials, such as PVC tubing. Commercially available polycarbonate rodent huts provide shelter, but are tinted to allow observation of the animal by care staff (Figure 8–1). In the wild, rodents and rabbits spend a great deal of their time foraging for food, and thus food enrichment is an important part of an enrichment program.68 Indeed, rodents in the laboratory will work to obtain food, even when it is freely available.69 Feeding enrichment can be as simple as hiding food in the substrate, which promotes foraging. Exercise can also benefit rodents and should be included in an enrichment plan when possible, particularly if social housing is not an option. Exercise on a running wheel can mitigate some of the anxiety that occurs as a result of social isolation in mice.70 In addition to improving animal welfare, enrichment can also increase safety for humans. Providing enrichment can help to decrease the risk of bites and scratches, increasing safety for animal caretakers and researchers.71 Van de Weerd et al.33 found that mice from an enriched environment were easier to handle (e.g., showed more relaxed behavior) compared to mice in a standard environment.33 Providing enrichment is not without problems, however. The addition of objects can increase aggression among socially housed animals,37 due to territoriality over particular items. Making more than one “favored item” available can help to reduce this aggression. Items must be carefully cleaned, to avoid causing illness.

Figure 8–1. Example of enrichment items for laboratory mice. The hut is tinted such that the mouse cannot see out, but human caretakers can see in. (See color insert.) (Photo by Jill Rawlins.)

58

SECTION II / GENERAL CONSIDERATIONS

And, care must be taken in deciding upon things to use. While nesting material is used without problem in many species, it can cause conjunctivitis in some genetically modified mice.72 Therefore, enrichment options should be carefully researched prior to implementation, to ensure that they are appropriate for the species and strain being utilized. DOGS Dogs are unique among laboratory animals in that they have evolved a well-developed bond with humans. Therefore, one of the best forms of enrichment for dogs is positive interactions with human handlers.40 Socializing dogs with humans, particularly when they are puppies, allows them to learn appropriate social cues and behaviors.73 Early socialization may also reduce stress for the dog and help to prevent behavior problems from starting.74 Odendaal and Meintjes75 demonstrated that simply petting a dog can increase affiliative neurotransmitters, including β-endorphins, for both the dog and the human. Working with socialized dogs is also safer for handlers. Dogs reared with human contact are more outgoing, easier to work with, and less likely to bite handlers compared to those without.73,76 Since the dog is a highly gregarious species, social housing, in pairs or groups, is important for their welfare. Social housing helps to increase species-specific behaviors, including play and exploration, and reduce abnormal behaviors such as stereotypies.77 If social housing is precluded due to experimental constraints, dogs should be allowed to see conspecifics.78 Adding physical enrichment items such as beds, resting platforms, and toys can enhance the environment and improve the welfare of laboratory dogs.73,77,79 It can also help increase learning in elderly dogs.80 Providing dogs with a variety of objects from which to choose, including chew toys, can reduce boredom.81 Exercise is another important part of an enrichment plan for laboratory dogs. While exercise has not been shown to substantially improve welfare,82 it is mandated by the Animal Welfare Act. Singly housed dogs must be given space to exercise.9 However, simply giving the dog an area in which to play may not be enough to promote exercise. Dogs generally do not exercise unless they have stimulation from humans or other animals.83 One way to ensure that dogs are getting exercise is to provide them with daily “play time” during which they can interact with other dogs. Another option is for handlers to take dogs for walks. An effective way to improve welfare and refine common husbandry practices with dogs is to train them for various tasks using positive reinforcement training (PRT).73 PRT techniques are a form of operant conditioning84 in which the subject is presented with a stimulus (e.g., a verbal command), responds by performing a specific (i.e., target) behavior (e.g., present a body part for injection), and is provided with reinforcement (e.g., food treat). Training increases mental stimulation for the dogs.73 It also makes dogs more obedient and easier to handle.73 Bookstores are filled with resources on how to train dogs, including “Don’t Shoot the Dog,” 85 which provides an excellent introduction to PRT. NONHUMAN PRIMATES Many species of nonhuman primates are used in biomedical research, although rhesus and other macaque species are among the most common. Unlike rodents or dogs, primates used in research have not been domesticated, and therefore have maintained even more of the behavioral repertoire of their wild counterparts than these other groups. Macaques and other nonhuman primates are social animals and live in complex societies, often with a strict dominance hierarchy. They form intricate social relationships in the wild, where they spend a great

deal of time engaged in social behaviors such as grooming and huddling with other members of the troop. Thus, social housing is considered one of the best forms of enrichment for nonhuman primates.16,86 Options for social housing can vary from large outdoor enclosures to indoor cages, depending upon the constraints of the facility and the experimental design. For animals in cages, pair housing (i.e., housing two monkeys in a double cage with a removable slide) allows social contact between the partners, and as such is considered an acceptable form of social housing for several species of primates.87,88 When full pair housing is not appropriate due to scientific reasons, the use of grooming contact slides89 between adjacent cages should be considered. These slides are designed so that part of the slide is a solid panel while the other part consists of bars wide enough to permit grooming (Figure 8–2). They afford individuals privacy as well as increased social contact with the cage mate.89 Social housing increases the opportunity for animals to engage in many species-typical behaviors including play, feeding, and grooming.90,91 It can also reduce abnormal behaviors such as stereotypical behavior and self-aggressive behavior,92 although this is not always the case (personal observation). Social enrichment is, however, an important tool in preventing abnormal behaviors from occurring.93 One of the main risk factors for the development of self-injurious behavior in macaques is having been reared without close social contact with conspecifics.94 In addition, the presence of known companions can lessen the effects of various stressors,95–97 and alter immune function,18,19 which can improve well-being. Social housing is not a panacea, however. It can result in injury if individuals are not compatible. Factors such as sex and age may affect how well individuals get along with each other. For macaques, males are generally harder to successfully pair house than females.98 Temperament may also play a role in compatibility in pair-housed animals. In a study on female rhesus macaques (Macaca mulatta), we found that animals with similar temperaments (as measured by reaction to a novel object) were more likely to form successful pairs than those of different temperaments.99 Forming groups of monkeys is particularly challenging (see references 100–102 for strategies on group formation). Once formed, groups should remain stable. Introducing a new adult into an established group can result in injury or death to the newcomer. Even in the absence of overt aggression, social housing can be stressful, particularly in species with dominance hierarchies in which some animals have more access to resources than others. In these societies, individuals at the bottom of the hierarchy are more prone to stress and stress-related diseases than dominant individuals.103,104 The provision of physical enrichment is another important part of an enrichment plan for nonhuman primates. This type of enrichment, which includes toys, mirrors, wood, and other manipulatable objects, affords individuals the opportunity to express species-normative behaviors such as play and exploration.35,105 Decisions regarding enrichment items should be based on the behavior of the species. For example, unlike macaques, owl monkeys (Aotus sp.) utilize nests in the wild, and should be provided with nest boxes in captivity. Macaques, on the other hand, utilize perches and other cage furniture.86 Items such as toys should be rotated regularly to keep them novel and thus of interest

CHAPTER 8 / PSYCHOLOGICAL ENRICHMENT FOR ANIMALS IN CAPTIVITY

A

B Figure 8–2. Grooming contact slide. The slide (A) is placed between two adjacent primate cages. The bars allow the partners to groom each other, while the solid part prevents full access to the other cage. (B) A slide within a cage (i.e., from the rhesus macaque’s perspective). (Photos by Kevin Mueller.)

to the monkeys.106–108 Specific enrichment devices, such as paint rollers or foraging boards covered with trail mix or other food items, are often provided to nonhuman primates in an effort to reduce the occurrence of abnormal behaviors such as stereotypy or overgrooming. There is debate as to the therapeutic value of such devices. While enrichment of this kind has been shown to decrease undesired behaviors in some studies,105,109,110 others have found no discernable effect of the devices on abnormal behavior111,112 or that the benefit was limited to the time the apparatus was present.93

59

Physical enrichment can increase the incidence of disease and injury for subjects. It is important to choose enrichment items that can be easily cleaned, to avoid spreading disease or illness.17,113 Animals can injure themselves on certain types of hooks used to keep devices on the outside of cages (e.g., “S” hooks). Further, ropes and wire used to hang devices or swings should be covered with PVC tubing, to reduce the potential for strangulation. Like rodents and rabbits, nonhuman primates spend much of their day foraging for food. This time is greatly reduced in a laboratory setting, where food is typically provided one or two times a day. Providing food enrichment can help functionally simulate the environment of nonhuman primate species.50 Further, the USDA Draft Policy on Environmental Enhancement for Nonhuman Primates suggests that primates be provided with daily opportunities to forage. Devices have been developed to promote foraging and manipulation for the monkeys. These devices, which are typically put on the outside of cages and filled with trail mix and produce (depending on the species and dietary restrictions), provide monkeys with the challenge of obtaining food.114 They have small holes through which the monkeys can forage for the treats. While these devices can be purchased commercially, they can also be made using materials such as PVC tubing (Figure 8– 3). Handmade devices should be sturdy, so that they do not easily break. It is also important that devices can be thoroughly cleaned. Such enrichment devices have been found to decrease the occurrence of some undesirable behaviors, including stereotypies112 and overgrooming,105 when kept on the monkeys’ cages continuously. However, these behaviors tend to return after time, likely due to increased boredom with devices that are kept on cages indefinitely. Rotating devices to keep them novel is recommended.115 Adding a small toy to the device can also increase the amount of time they are utilized by the monkeys52 (Figure 8–3). Many facilities provide research primates with various forms of auditory and visual enrichment, including access to television, radio, brightly colored mobiles, or a window.116 Computer screen savers can also be presented to the animals. Monkeys seem to watch the computer screens about the same amount as a television (personal observation). Television can be aversive if stressors, such as predators, appear on the screen. There is some evidence to suggest that sensory enrichment is beneficial to primates,117 although there are contradictory results as well.118 More work is needed to determine the benefits of this kind of enrichment. Using positive reinforcement techniques to train monkeys to voluntarily cooperate with husbandry and/or research procedures is becoming recognized as an important tool in an effective enrichment program. PRT increases mental stimulation for subjects119 and therefore is an effective form of psychological enrichment. Primates have been successfully trained to perform various tasks, such as presenting a body part for injection120 or veterinary procedures,121 taking oral medications,122 and remaining stationary for blood samples.120,123 PRT can reduce the stress associated with common management procedures.119,121,124 By allowing individuals to cooperate with the procedures, PRT may give primates an increased sense of control over their environment.119 In addition, training can reduce abnormal behaviors in nonhuman primates. In a preliminary study,125 we found that PRT reduced the occurrence of stereotypical behavior (e.g., pacing, rocking) in some, but not all, rhesus macaques. Furthermore, training primates to cooperate with research procedures can reduce the inherent risk of the

60

SECTION II / GENERAL CONSIDERATIONS

they relieve boredom and help the animal learn to cope with environmental circumstances.50 However, some stressors may not be adaptive, particularly if the animal has not evolved appropriate responses to them. These are the stressors that a successful enrichment program should address.

ACKNOWLEDGMENTS I am grateful to Diana Gordon, Adriane Maier, Nicola DeBolt Robertson, Jill Rawlins, Greg Johnson, Kevin Mueller, and Victor Lucas for helpful comments on the manuscript and for their continued help and patience. I also thank Steve Schapiro for valuable conversations that have helped shape my thoughts on enrichment.

REFERENCES A

B Figure 8–3. Foraging devices for nonhuman primates. (A) A device made by drilling extra holes into a wiffle ball. (B) A commercially available device (Crumble Disc Holder, Bio-Serv, Frenchtown, NJ), with a small cat toy added. (Photos by Jill Rawlins.)

procedures. It is clear that working with a cooperative animal is safer for caretakers and handlers than working with a highly stressed, and potentially reactive, animal.126 While training is beneficial for the animal as well as the research, it can also involve a great deal of time. However, the time invested will likely be made up for by the decrease in time it takes to conduct procedures.127

CONCLUSIONS In designing an enrichment program for an animal model it is essential to balance two equally important goals: the reduction of stress associated with the captive environment and the development of a model that (as closely as possible given the overriding need to care for the animal’s physical and mental well-being) approximates the human condition. Life is as full of stressors for humans as it is for other animals. Thus, it is not a realistic goal of any enrichment program to eliminate all stress. Not only is that goal unattainable, but it also is not necessarily in the animal’s best interest. Stressors are not intrinsically detrimental,7 and are a natural part of the behavioral repertoire of many animals.16 Indeed, some can be adaptive, as

1. Stewart K. Development of an environmental enrichment program utilizing simple strategies. Anim Welf Inform Center Newsl 2004;12: 1–7. 2. Baker KC, Weed JL, Crockett CM, Bloomsmith MA. Survey of environmental enhancement programs for laboratory primates. Am J Primatol 2007;69(4):377–394. 3. Meehan CL, Garner JP, Mench JA. Environmental enrichment and development of cage stereotypy in Orange-winged Amazon parrots (Amazona amazonica). Dev Psychobiol 2004;44(4):209–218. 4. Almli LM, Burghardt GM. Environmental enrichment alters the behavioral profile of ratsnakes (elaphe). J Appl Anim Welf Sci 2006;9(2):85–109. 5. Kihslinger RL, Nevitt GA. Early rearing environment impacts cerebellar growth in juvenile salmon. J Exp Biol 2006;209 (Pt. 3):504–509. 6. Shepherdson DJ. Introduction: Tracing the path of environmental enrichment in zoos. In: Shepherdson D, Mellen J, Hutchins M, Eds. Second Nature: Environmental Enrichment for Captive Animals. Washington, DC: Smithsonian Institution Press, 1998:1–12. 7. Novak MA, Suomi SJ. Psychological well-being of primates in captivity. Am Psychol 1988;43(10):765–773. 8. Council NR. Guide for the Care and Use of Laboratory Animals. Washington, DC: National Academic Press, 1996. 9. Regulations AW. Animal Welfare Act. In: Federal Regulations. United States, 1991. 10. von Holst D. The concept of stress and its relevance for animal behavior. In: Moller AP, Milinski M, Slater PJB, Eds. Stress and Behavior. San Diego, CA: Academic Press, 1998:1–109. 11. Hickey MC, Drennan M, Earley B. The effect of abrupt weaning of suckler calves on the plasma concentrations of cortisol, catecholamines, leukocytes, acute-phase proteins and in vitro interferongamma production. J Anim Sci 2003;81(11):2847–2855. 12. Engler H, Dawils L, Hoves S, et al. Effects of social stress on blood leukocyte distribution: The role of alpha- and beta-adrenergic mechanisms. J Neuroimmunol 2004;156(1–2):153–162. 13. Conrad CD, McEwen BS. Acute stress increases neuropeptide Y mRNA within the arcuate nucleus and hilus of the dentate gyrus. Brain Res Mol Brain Res 2000;79(1–2):102–109. 14. Rogers CJ, Brissette-Storkus CS, Hayes LA, Cameron JL, Chambers WH. Selective reduction in CD2 expression on CD2bright/ CD8+ lymphocytes from cynomolgus monkeys (Macaca fascicularis) in response to acute stress. J Neuroimmunol 1998;86(1): 63–73. 15. Novak MA. Self-injurious behavior in rhesus monkeys: New insights into its etiology, physiology, and treatment. Am J Primatol 2003;59:3–19. 16. de Waal FBM. The social nature of primates. In: Novak MA, Petto AJ, Eds. Through the Looking Glass. Washington, DC: American Psychological Association, 1991:69–77. 17. Bayne K. Potential for unintended consequences of environmental enrichment for laboratory animals and research results. ILAR J 2005;46(2):129–139.

CHAPTER 8 / PSYCHOLOGICAL ENRICHMENT FOR ANIMALS IN CAPTIVITY

18. Lilly AA, Mehlman PT, Higley JD. Trait-like immunological and hematological measures in female rhesus across varied environmental conditions. Am J Primatol 1999;48(3):197–223. 19. Schapiro SJ, Nehete PN, Perlman JE, Sastry KJ. A comparison of cell-mediated immune responses in rhesus macaques housed singly, in pairs, or in groups. Appl Anim Behav Sci 2000;68(1):67– 84. 20. Manosevitz M. Early environmental enrichment and mouse behavior. J Comp Physiol Psychol 1970;71(3):459–466. 21. Renner MJ, Rosenzweig MR. Social interactions among rats housed in grouped and enriched conditions. Dev Psychobiol 1986;19(4):303– 314. 22. Fernandez-Teruel A, Driscoll P, Gil L, Aguilar R, Tobena A, Escorihuela RM. Enduring effects of environmental enrichment on novelty seeking, saccharin and ethanol intake in two rat lines (RHA/Verh and RLA/Verh) differing in incentive-seeking behavior. Pharmacol Biochem Behav 2002;73(1):225–231. 23. Benaroya-Milshtein N, Hollander N, Apter A, et al. Environmental enrichment in mice decreases anxiety, attenuates stress responses and enhances natural killer cell activity. Eur J Neurosci 2004;20(5): 1341–1347. 24. Jankowsky JL, Melnikova T, Fadale DJ, et al. Environmental enrichment mitigates cognitive deficits in a mouse model of Alzheimer’s disease. J Neurosci 2005;25(21):5217–5224. 25. Barbelivien A, Herbeaux K, Oberling P, Kelche C, Galani R, Majchrzak M. Environmental enrichment increases responding to contextual cues but decreases overall conditioned fear in the rat. Behav Brain Res 2006;169(2):231–238. 26. Greenough WT. Experiential modification of the developing brain. Am Sci 1975;63(1):37–46. 27. Benefiel AC, Greenough W. Effects of experience and environment on the developing and mature brain: Implications for laboratory animal housing. ILAR J 1998;39(1):5–11. 28. Kempermann G, Kuhn HG, Gage FH. More hippocampal neurons in adult mice living in an enriched environment. Nature 1997;386(6624):493–495. 29. Young D, Lawlor PA, Leone P, Dragunow M, During MJ. Environmental enrichment inhibits spontaneous apoptosis, prevents seizures and is neuroprotective. Nat Med 1999;5(4):448–453. 30. Walsh RN. Effects of environmental complexity and deprivation on brain anatomy and histology: A review. Int J Neurosci 1981;12(1):33–51. 31. Rosenzweig MR, Bennett EL. Psychobiology of plasticity: Effects of training and experience on brain and behavior. Behav Brain Res 1996;78(1):57–65. 32. van Praag H, Kempermann G, Gage FH. Neural consequences of environmental enrichment. Nat Rev 2000;1(3):191–198. 33. Van de Weerd HA, Aarsen EL, Mulder A, Kruitwagen CL, Hendriksen CF, Baumans V. Effects of environmental enrichment for mice: Variation in experimental results. J Appl Anim Welf Sci 2002;5(2):87–109. 34. Rockman GE, Gibson JE, Benarroch A. Effects of environmental enrichment on voluntary ethanol intake in rats. Pharmacol Biochem Behav 1989;34(3):487–490. 35. Boinski S, Swing SP, Gross TS, Davis JK. Environmental enrichment of brown capuchins (Cebus apella): Behavioral and plasma and fecal cortisol measures of effectiveness. Am J Primatol 1999;48(1):49–68. 36. Moncek F, Duncko R, Johansson BB, Jezova D. Effect of environmental enrichment on stress related systems in rats. J Neuroendocrinol 2004;16(5):423–431. 37. Marashi V, Barnekow A, Ossendorf E, Sachser N. Effects of different forms of environmental enrichment on behavioral, endocrinological, and immunological parameters in male mice. Horm Behav 2003;43(2):281–292. 38. Klein SL, Lambert KG, Durr D, Schaefer T, Waring RE. Influence of environmental enrichment and sex on predator stress response in rats. Physiol Behav 1994;56(2):291–297. 39. Reed HJ, Wilkins LJ, Austin SD, Gregory NG. The effect of environmental enrichment during rearing on fear reactions and depopu-

40.

41.

42.

43.

44.

45. 46.

47.

48.

49. 50.

51.

52. 53.

54.

55. 56.

57. 58.

59.

60. 61.

62.

61

lation trauma in adult caged hens. Appl Anim Behav Sci 1993;36(1): 39–46. Overall KL, Dyer D. Enrichment strategies for laboratory animals from the viewpoint of clinical veterinary behavioral medicine: Emphasis on cats and dogs. ILAR J 2005;46(2):202–215. Benefiel AC, Dong WK, Greenough WT. Mandatory “enriched” housing of laboratory animals: The need for evidence-based evaluation. ILAR J 2005;46(2):95–105. Weed JL, Raber JM. Balancing animal research with animal wellbeing: Establishment of goals and harmonization of approaches. ILAR J 2005;46(2):118–128. Welberg L, Thrivikraman KV, Plotsky PM. Combined pre- and postnatal environmental enrichment programs the HPA axis differentially in male and female rats. Psychoneuroendocrinology 2006;31(5):553–564. Meaney MJ, Viau V, Bhatnagar S, et al. Cellular mechanisms underlying the development and expression of individual differences in the hypothalamic-pituitary-adrenal stress response. J Steroid Biochem Mol Biol 1991;39(2):265–274. De Kloet ER. Hormones and the stressed brain. Ann NY Acad Sci 2004;1018:1–15. Bartolomucci A, Palanza P, Sacerdote P, et al. Social factors and individual vulnerability to chronic stress exposure. Neurosci Biobehav Rev 2005;29(1):67–81. Bloomsmith MA, Brent LY, Schapiro SJ. Guidelines for developing and managing an environmental enrichment program for nonhuman primates. Lab Anim Sci 1991;41(4):372–377. Keeling ME, Alford PL, Bloomsmith MA. Decision analysis for developing programs of psychological well-being: A bias-for-action approach. In: Novak MA, Petto AJ, Eds. Through the Looking Glass. Washington, DC: American Psychological Association, 1991:57–65. Young RJ. Environmental Enrichment for Captive Animals. Oxford, UK: Blackwell Publishing, 2003. Newberry RC. Environmental enrichment: Increasing the biological relevance of captive environments. Appl Anim Behav Sci 1995; 44(2–4):229–243. Van de Weerd HA, Baumans V, Koolhaas JM, van Zutphen LF. Strain specific behavioural response to environmental enrichment in the mouse. J Exp Anim Sci 1994;36(4–5):117–127. Rawlins JM, Poerstel S, Coleman K. Utilization of toy devices by rhesus macaques. Contemp Top Lab Anim Sci 2003;42:123. Waitt C, Buchanan-Smith HM, Morris K. The effects of caretakerprimate relationships on primates in the laboratory. J Appl Anim Welf Sci 2002;5(4):309–319. Boice R. Burrows of wild and albino rats: Effects of domestication, outdoor raising, age, experience, and maternal state. J Comp Physiol Psychol 1977;91(3):649–661. Hutchinson E, Avery A, Vandewoude S. Environmental enrichment for laboratory rodents. ILAR J 2005;46(2):148–161. Cowan DP. Group living in the European rabbit oryctolaguscuniculus mutual benefit or resource localization. J Anim Ecol 1987;56(3):779–796. Balcombe JP. Laboratory environments and rodents’ behavioural needs: A review. Lab Anim 2006;40(3):217–235. Crawley JN, Belknap JK, Collins A, et al. Behavioral phenotypes of inbred mouse strains: Implications and recommendations for molecular studies. Psychopharmacology (Berl) 1997;132(2):107– 124. Van Loo PLP, Kruitwagen CLJJ, Koolhaas JM, Van de Weerd HA, van Zutphen LFM, Baumans V. Influence of cage enrichment on aggressive behaviour and physiological parameters in male mice. Appl Anim Behav Sci 2002;76(1):65–81. Joffe JM, Rawson RA, Mulick JA. Control of their environment reduces emotionality in rats. Science 1973;180(93):1383–1384. Van de Weerd HA, Van Loo PLP, Van Zutphen LFM, Koolhaas JM, Baumans V. Preferences for nest boxes as environmental enrichment for laboratory mice. Anim Welf 1998;7(1):11–25. Van de Weerd HA, Van Loo PLP, Van Zutphen LFM, Koolhaas JM, Baumans V. Strength of preference for nesting material as

62

63.

64. 65.

66.

67.

68.

69. 70.

71.

72.

73.

74. 75.

76. 77.

78.

79.

80.

81.

82. 83.

84. 85. 86.

SECTION II / GENERAL CONSIDERATIONS

environmental enrichment for laboratory mice. Appl Anim Behav Sci 1998;55(3–4):369–382. Shepherdson D. Stereotypic behavior: What is it and how can it be eliminated or prevented? J Assoc Br Wild Anim Keepers 1993;16:100–105. Wiepkema PR, Koolhaas JM. Stress and animal welfare. Anim Welf 1993;2:195–218. Renner MJ, Rosenzweig MR. Social interactions among rats housed in grouped and enriched conditions. Dev Psychobiol 1986;19(4): 303–313. Wemelsfelder F. Boredom and laboratory animal welfare. In: Rollin BE, Ed. The Experimental Animal in Biomedical Research, Vol. I, A Survey of Scientific and Ethical Issues for Investigators. Boca Raton, FL: CRC Press, Inc., 1990:243–272. Chu L-R, Garner JP, Mench JA. A behavioral comparison of New Zealand White rabbits (Oryctolagus cuniculus) housed individually or in pairs in conventional laboratory cages. Appl Anim Behav Sci 2004;85(1–2):121–139. Harris LD, Custer LB, Soranaka ET, Burge JR, Ruble GR. Evaluation of objects and food for environmental enrichment of NZW rabbits. Contemp Top Lab Anim Sci 2001;40(1):27–30. Carder B, Berkowitz K. Rats’ preference for earned in comparison with free food. Science 1970;167(922):1273–1274. Pham TM, Brene S, Baumans V. Behavioral assessment of intermittent wheel running and individual housing in mice in the laboratory. J Appl Anim Welf Sci 2005;8(3):157–173. Baumans V. Environmental enrichment for laboratory rodents and rabbits: Requirements of rodents, rabbits, and research. ILAR J 2005;46(2):162–170. Bazille PG, Walden SD, Koniar BL, Gunther R. Commercial cotton nesting material as a predisposing factor for conjunctivitis in athymic nude mice. Lab Animal 2001;30(5):40–42. Adams KM, Navarro AM, Hutchinson EK, Weed JL. A canine socialization and training program at the National Institutes of Health. Lab Anim 2004;33:32–36. Hubrecht RC. Enrichment in puppyhood and its effects on later behavior of dogs. Lab Anim Sci 1995;45(1):70–75. Odendaal JS, Meintjes RA. Neurophysiological correlates of affiliative behaviour between humans and dogs. Vet J 2003;165(3): 296–301. Fox MW, Stelzner D. Behavioural effects of differential early experience in the dog. Anim Behav 1966;14(2):273–281. Hubrecht RC, Serpell JA, Poole TB. Correlates of pen size and housing conditions on the behaviour of kennelled dogs. Appl Anim Behav Sci 1992;34(4):365–383. Wells DL, Hepper PG. A note on the influence of visual conspecific contact on the behaviour of sheltered dogs. Appl Anim Behav Sci 1998;60(1):83–88. Wells DL. A review of environmental enrichment for kennelled dogs, Canis familiaris. Appl Anim Behav Sci 2004;85(3–4):307– 317. Milgram NW, Head E, Zicker SC, et al. Learning ability in aged beagle dogs is preserved by behavioral enrichment and dietary fortification: A two-year longitudinal study. Neurobiol Aging 2005;26(1):77–90. Hubrecht RC. A comparison of social and environmental enrichment methods for laboratory housed dogs. Appl Anim Behav Sci 1993;37(4):345–361. Clark JD, Calpin JP, Armstrong RB. Influence of type of enclosure on exercise fitness of dogs. Am J Vet Res 1991;52(7):1024–1028. Campbell SA, Hughes HC, Griffin HE, Landi MS, Mallon FM. Some effects of limited exercise on purpose-bred beagles. Am J Vet Res 1988;49(8):1298–1301. Skinner BF. The Behavior of Organisms. New York: AppletonCentury-Crofts, 1938. Pryor K. Don’t Shoot the Dog: The New Art of Teaching and Training. New York: Simon & Schuster, 1999. Crockett CM. Psychological well-being of captive non-human primates: Lessons from the laboratory studies. In: Shepherdson D, Mellen J, Hutchins M, Eds. Second Nature: Environmental Enrich-

87. 88.

89.

90.

91.

92. 93. 94.

95.

96.

97.

98.

99.

100.

101.

102.

103.

104. 105.

106.

107.

108. 109.

ment for Captive Animals. Washington, DC: Smithsonian Institution Press, 1998:129–152. Reinhardt V. Pair-housing rather than single-housing for laboratory rhesus macaques. J Med Primatol 1994;23(8):426–431. Majolo B, Buchanan-Smith HM, Morris K. Factors affecting the successful pairing of unfamiliar common marmoset (Callithrix jacchus) females: Preliminary results. Anim Welf 2003;12(3):327– 337. Crockett CM, Bellanca RU, Bowers CL, Bowden DM. Groomingcontact bars provide social contact for individually caged laboratory macaques. Contemp Top Lab Anim Sci 1997;36(6):53–60. Schapiro SJ, Bloomsmith MA, Suarez SA, Porter LM. Effects of social and inanimate enrichment on the behavior of yearling rhesus monkeys. Am J Primatol 1996;40:247–260. Spring SE, Clifford JO, Tomko DL. Effect of environmental enrichment devices on behaviors of single- and group-housed squirrel monkeys (Saimiri sciureus). Contemp Top Lab Anim Sci 1997;36(3): 72–75. Reinhardt V. Pair-housing overcomes self-biting behavior in macaques. Lab Primate News 1999;38(1):4–5. Lutz CK, Novak MA. Environmental enrichment for nonhuman primates: Theory and application. ILAR J 2005;46(2):178–191. Novak MA. Self-injurious behavior in rhesus monkeys: New insights into its etiology, physiology, and treatment. Am J Primatol 2003;59(1):3–19. Gust DA, Gordon TP, Brodie AR, McClure HM. Effect of a preferred companion in modulating stress in adult female rhesus monkeys. Physiol Behav 1994;55(4):681–684. Gerber P, Schnell CR, Anzenberger G. Behavioral and cardiophysiological responses of common marmosets (Callithrix jacchus) to social and environmental changes. Primates 2002;43(3):201–216. Epley SW. Reduction of the behavioral effects of aversive stimulation by the presence of companions. Psychol Bull 1974;81(5): 271–283. Crockett CM, Bowers CL, Bowden DM, Sackett GP. Sex differences in compatibility of pair-housed adult longtailed macaques. Am J Primatol 1994;32(2):73–94. McMillan J, Maier A, Tully L, Coleman K. The effects of temperament on pairing success in female rhesus macaques. Am J Primatol 2003;60:95. Bernstein IS, Gordon TP, Rose RM. Aggression and social controls in rhesus monkey (Macaca mulatta) groups revealed in group formation studies. Folia Primatol (Basel) 1974;21(2):81–107. Schapiro SJ, Lee-Parritz DE, Taylor LL, Watson L, Bloomsmith MA, Petto A. Behavioral management of specific pathogen-free rhesus macaques: Group formation, reproduction, and parental competence. Lab Anim Sci 1994;44(3):229–234. Westergaard GC, Izard MK, Drake JH, Suomi SJ, Higley JD. Rhesus macaque (Macaca mulatta) group formation and housing: Wounding and reproduction in a specific pathogen free (SPF) colony. Am J Primatol 1999;49(4):339–347. Coe CL. Is social housing of primates always the optimal choice? In: Novak MA, Petto AJ, Eds. Through the Looking Glass. Washington, DC: American Psychological Association, 1991:78–90. Sapolsky RM. Stress in the wild. Sci Am 1990;262(1):116–123. Schapiro SJ, Bloomsmith MA. Behavioral effects of enrichment on singly-housed yearling rhesus monkeys: An analysis including three enrichment conditions and a control group. Am J Primatol 1995;35:89–101. Crockett CM, Bielitzki J, Carey A, Velez A. Kong toys as enrichment devices for singly-caged macaques. Lab Primate News 1989;28(2):21–22. Bloomsmith MA, Finlay TW, Merhalski JJ, Maple TL. Rigid plastic balls as enrichment devices for captive chimpanzees. Lab Anim Sci 1990;40(3):319–322. Wells DL. The influence of toys on the behaviour and welfare of kennelled dogs. Anim Welf 2004;13(3):367–373. Kessel-Davenport A. Cage toys reduce abnormal behavior in individually housed pigtail macaques. J Appl Anim Welf Sci 1998;1(3):227–234.

CHAPTER 8 / PSYCHOLOGICAL ENRICHMENT FOR ANIMALS IN CAPTIVITY

110. Lam K, Rupniak NM, Iversen SD. Use of a grooming and foraging substrate to reduce cage stereotypies in macaques. J Med Primatol 1991;20(3):104–109. 111. Line SW, Morgan KN, Markowitz H. Simple toys do not alter the behavior of aged rhesus monkeys. Zoo Biol 1991;10:473–484. 112. Novak MA, Kinsey JH, Jorgensen MJ, Hazen TJ. Effects of puzzle feeders on pathological behavior in individually housed rhesus monkeys. Am J Primatol 1998;46:213–227. 113. Bayne KAL, Dexter SL, Strange GM, Hill EE. Kong toys for laboratory primates: Are they really an enrichment of just fomites? Lab Anim Sci 1993;43(1):78–85. 114. Holmes SN, Riley JM, Juneau P, Pyne D, Hofing GL. Short-term evaluation of a foraging device for non-human primates. Lab Anim 1995;29(4):364–369. 115. Rawlins JM, Johnson JG, Coleman K. The effect of novelty on device use in female rhesus macaques. Contemp Top Lab Anim Sci 2004;43:96. 116. Brent L, Stone AM. Long-term use of televisions, balls, and mirrors as enrichment for paired and singly caged chimpanzees. Am J Primatol 1996;39:139–145. 117. Bloomsmith MA, Lambeth SP. Videotapes as enrichment for captive chimpanzees (Pan troglodytes). Zoo Biol 2000;19:541–551. 118. Harris LD, Briand EJ, Orth R, Galbicka G. Assessing the value of television as environmental enrichment for individually housed rhesus monkeys: A behavioral economic approach. Contemp Top Lab Anim Sci 1999;38(2):48–53. 119. Laule GE, Bloomsmith MA, Schapiro SJ. The use of positive reinforcement training techniques to enhance the care, management, and

120.

121.

122.

123.

124.

125.

126. 127.

63

welfare of primates in the laboratory. J Appl Anim Welf Sci 2003;6(3):163–173. Priest GM. Training a diabetic drill (Mandrillus leucophaeus) to accept insulin injections and venipuncture. Lab Prim News 1991;30:1–4. Schapiro SJ, Bloomsmith MA, Laule GE. Positive reinforcement training as a technique to alter nonhuman primate behavior: Quantitative assessments of effectiveness. J Appl Anim Welf Sci 2003;6(3):175–187. Klaiber-Schuh A, Welker C. Crab-eating monkeys can be trained to cooperate in non-invasive oral medication without stress. Primate Rep 1997;47:11–30. Laule GE, Thurston RH, Alford PL, Bloomsmith MA. Training to reliably obtain blood and urine samples from a young, diabetic chimpanzee (Pan troglodytes). Zoo Biol 1996;15:587–591. Bassett L, Buchanan-Smith HM, McKinley J, Smith TE. Effects of training on stress-related behavior of the common marmoset (Callithrix jacchus) in relation to coping with routine husbandry procedures. J Appl Anim Welf Sci 2003;6(3):221–233. Maier A, Mcmillan JL, Coleman K. Effects of positive reinforcement training on stereotypic behaviour in rhesus macaques. Folia Primatol 2004;75:392–393. Bloomsmith MA. Chimpanzee training and behavioral research: A symbiotic relationship. AAZPA/CAZPA 1992:403–410. McKinley J, Buchanan-Smith HM, Bassett L, Morris K. Training common marmosets (Callithrix jacchus) to cooperate during routine laboratory procedures: Ease of training and time investment. J Appl Anim Welf Sci 2003;6(3):209–220.

WELL-ESTABLISHED III MODELS Yeast, Worms, Flies, Sea Animals, and Birds

A

9

Integrated Network Modeling of Molecular and Genetic Interactions GREGORY W. CARTER, VESTEINN THORSSON,

ABSTRACT

AND

TIMOTHY GALITSKI

The primary objective of these methods is to identify functionally relevant genes and describe how they influence one another to produce cellular activity. In addition, this knowledge can be integrated with physical biochemistry to yield biomolecular models that carry out specific cellular functions. Such an integrated model facilitates validation of biological insights and identifies high-priority candidate genes for targeted therapeutic intervention. One strategy to achieve this integration is to first infer functional interactions between trait genes, and then use these relationships to guide the selection of biochemical paths that are putatively responsible for the observed phenotypes. This provides a systematic framework for integrating two complementary data types: genetic interactions and physical interactions. Genetic interactions are inferred by observing how genetic perturbations interact to affect phenotypes, and encode functional relationships such as activation, repression, and information flow.9 However, the biochemical interpretation of a genetic interaction is often ambiguous, frequently involving multiple molecular models and both direct and indirect mechanisms.10,11 At the same time, the biochemical interactions plentifully generated through highthroughput methods are often lacking in functional interpretation.12,13 Taking advantage of this complementarity by constraining the biochemical interpretation of genetic interactions by molecular wiring, we seek to construct biomolecular models that assign functional significance to physical interactions. This chapter outlines steps toward such an analysis. First, we outline a systematic analysis and classification of genetic interactions on a large scale. Second, we discuss methods to integrate genomic expression data and physical interaction data to generate molecular hypotheses of functional organization. Finally, we outline a systematic method to extend the two analyses in a predictive model for the control of a biological response in yeast.

In model organisms, systematic genetic perturbation and highthroughput data acquisition allow measurements of mutant phenotypes and detection of biomolecular interactions on a genome-wide scale. These data types are complementary in the sense that genetic interactions inferred from gene expression and other phenotype measurements can assign functional significance to the physical interactions between biomolecules. This chapter describes methods for the analysis of large-scale phenotype and expression data that employ genetic interactions to infer functional relationships between genes. Functional information is then used to direct the integration of physical interaction data to derive network models of cellular activity. These models, obtained from functional relationships but constrained by biochemistry, facilitate explicit and precise prediction of the effects of additional network perturbations. Key Words: Genetic interaction, Network, Genomics, Cell differentiation.

INTRODUCTION The identification of causal links between genetic variation and phenotype is central to the development of predictive, preventive, and personalized medicine. Many if not most phenotypes are determined by complex interactions among allelic variants and environmental factors. In biomedicine, these interacting elements are manifest as inherited and somatic genetic variants and polymorphisms, epigenetic effects on gene activity, environmental agents, and drug therapies including drug combinations. Development of new treatments for diseases with genetic susceptibilities will require not only the ability to genotype and classify patients on the basis of molecular fingerprints in tissues, but also an understanding of how genetic variants interact to affect clinical outcomes. The large-scale study of how genotype contributes to the control of phenotype has been greatly aided by advances in methods to both systematically perturb genes and collect phenotype data on a genomic scale.1–8 These high-throughput methods are designed to produce large quantitative data sets, requiring parallel development of computational and numerical modeling methods to interpret the output in terms of biological function.

GENETIC INTERACTION NETWORKS A genetic interaction is the phenotypic manifestation of how two alleles combine to jointly affect cellular activity. This classical method of analysis is based on comparing the phenotypes of strains with specific allelic variations, and thus is most readily performed using targeted perturbations in inbred strains of model organisms. Primary examples are the use of the deletion mutant library for Saccharomyces cerevisiae,1 RNAi technology in Candida elegans14 and Drosophila melanogaster,15 as well as

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

67

68

SECTION III / WELL-ESTABLISHED MODELS

curated data repositories such as FlyBase.16 The properties and uses of genetic interactions learned with model organisms are of interest in the broader context of outbred populations that carry multiple alleles. The genetic basis of human disease susceptibility, in which polygenic interaction effects are often preeminent, is naturally the most urgent application.17 Genetic interactions are defined for a chosen phenotype in specific environmental conditions, so that the lone variables are the specific genetic perturbations (i.e., allele forms) carried by the strains being compared. The logic is as follows. Consider two genes, A and B, mutations of which yield observable phenotypes. Taking cell growth as an example phenotype, imagine a null mutation of A inhibits growth while a null mutation of B enhances growth. The growth rate of a double mutant, with null mutations on both A and B, is also observed to be inhibited. In this case the A mutant is said to be epistatic to the B mutant, as the A mutant phenotype does not change in the B-null background.* Such an interaction is often interpreted as evidence that A acts downstream of B, in that its knockout masks any modifications that occur further upstream of the phenotypic output.9 So while this observation cannot, when taken alone, offer any biochemical explanation, it establishes a clear functional relationship between the two genes. Thus the genetic interaction can be considered as providing a comprehensive view of their coordinated activity, encompassing every step in cellular processing between genotype and phenotype. Classical epistasis is one of many possible modes of genetic interaction. For instance, high-throughput screens in yeast have been developed to detect synthetic interactions,2 which are manifest when a pair of genes that does not show phenotypic effects when mutated individually exhibits an altered phenotype when the mutations are combined in a single strain. With the proper experimental design, the modes of genetic interaction can be systematically analyzed and formally classified.8 To this end, consider a genotype X, and its associated phenotype observation PX. The phenotype could be a numerical measurement or any other observation that can be clearly compared across mutant genotypes (e.g., slow vs. standard vs. fast growth). If genotypes are labeled by mutant alleles, a set of four phenotype observations can be assembled that defines a genetic interaction: PA and PB for the A and B mutant alleles, PAB for the AB double mutant, and PWT for the wild type. The relationship between these four measurements defines a genetic interaction; for example, PAB = PA < PWT < PB described an epistatic interaction while PAB < PWT = PA = PB represents synthesis. It is immediately clear that some of these interactions are symmetric under an exchange of genes A and B (e.g., synthetic) while others are not (e.g., epistatic). There are a total of 75 distinct inequalities that can be constructed from four phenotypes.8 These inequalities were recently cataloged and classified as one of nine interaction modes, including epistasis and synthesis.8 To test the applicability of this classification method over 1800 strains of systematic single and double gene perturbations were phenotyped for invasive growth, a cell differentiation response that occurs under specific environmental conditions. A software

*The term epistasis is sometimes used as a generic term meaning genetic interaction. Here we use it in the narrow sense of one mutant completely masking the effect of another.

FLO8(gf) TEC1(gf) MID2(lf) STE20(gf)

STE12(gf) BEM1(gf)

GLN3(gf)

RGS2(lf)

CDC42(gf) EGT2(lf)

PBS2(lf)

YJL142C(lf)

HSL1(lf) HOG1(lf)

YAP1(lf)

ISW1(lf)

FKH2(lf) SFL1(lf)

Figure 9–1. Networks of mutual information in patterns of genetic interaction show cliques. Nodes represent perturbed genes, gf indicates a gain-of-function allele, and lf indicates a loss-of-function allele. Edges connect gene pairs with significant mutual information in their patterns of genetic interaction. (Reprinted from Drees et al.8)

application called PhenotypeGenetics, created as a plugin for the Cytoscape software platform,18,19 was used to systematically construct a genetic interaction network from the high-throughput, quantitative phenotype data. All nine interaction modes were found in significant numbers, illustrating that a spectrum of genetic interaction effects is present in complex biological systems. Specific alleles were found to interact in a particular mode with neighbor genes of coherent biological function, leading to hypotheses on regulatory and pathway organization. Largescale patterns of mutual information were also extracted from the data set, and groups of genes with significant mutual information between them formed network cliques corresponding to physical pathways (Figure 9–1). The genetic interaction patterns represent the biological system as a map of information flow from specific genetic perturbations to quantifiable phenotype effects.

DIRECTED DATA INTEGRATION The invasive-growth network described in the previous section encodes functional information inferred from genetic interactions. We next consider a method to integrate these functional relationships with physical interactions in order to identify candidate molecular mechanisms that control cellular activity. In this section, we outline a strategy of data integration in which the analysis of genomic expression data directs the construction of regulatory networks. By analyzing the effects of specific genetic perturbations on genomic expression, it is possible to identify specific genes that coordinate control of phenotypes at the transcriptional level. Moreover, gene expression can itself be viewed as a quantitative phenotype that is a suitable platform for studying complex phenomena such as genetic interactions.20 Genomic expression measurements have many distinct advantages as a basis for quantitative

CHAPTER 9 / INTEGRATED NETWORK MODELING OF MOLECULAR AND GENETIC INTERACTIONS

analysis. DNA microarray technology provides a well-established platform for systematic data collection and many statistical methods now exist that identify groups of coexpressed genes. Sets of genes obtained can be systematically queried for common function in increasingly comprehensive annotation databases such as Gene Ontology.21 Furthermore, the perturbations and measurements of genes introduce a concrete, molecular basis for further analysis, and large-scale interaction databases now make it possible to place these biomolecules in a network context. We took advantage of these resources in a study of the RascAMP signaling pathway in yeast.22 This pathway has been implicated in filamentous growth,23 cell proliferation, and glycolysis.24–26 In yeast, the pathway centers on the activation of adenylate cyclase (Cyr1) by GTP-bound Ras2 and Ras1 proteins, which in turn facilitates the synthesis of cAMP. We perturbed key pathway elements and collected genomic expression data, with the intent of tracing the observed expression responses to the perturbation(s) that caused them. Specifically, we created strains with exogenously controlled levels of cAMP, dominant-active and dominantnegative alleles of Ras2, and genetic perturbations of the GTPase activating proteins Ira1 and Ira2. The intent was to infer a detailed map of the activity of these pathway elements, ranging from shared effects resulting from multiple perturbations to highly specific effects resulting from one or two perturbations. We wanted to identify and classify signals regardless of global magnitude, detecting not only quantitatively large signals but also fainter (yet coherent) expression effects. These faint effects may be of particular biological interest, possibly identifying functional differences between multiple perturbations of a single gene or two near-homologous genes. Expression patterns specifically related to phenotypes can also be quantitatively minor.27,28 To disentangle signals in the data, we performed singular value decomposition (SVD) on the global data set to identify overlap-

IRA1

IRA2

RIM11 SRB4

NAP1 YAP6

CIN5

GTS1

SWI4 TYE7 GLN3

9+ Figure 9–2. Regulatory network derived from perturbations of IRA1 and IRA2 genes, mapping putative biomolecular pathways for information flow to a set of genes sharing an expression component (box). The gene set, which is statistically enriched in Ty element transposons, is labeled “9+” to denote that it is the ninth SVD mode and the genes express the expression pattern with positive coefficients. Circular nodes represent individual proteins; black, nondirectional edges represent protein–protein interactions; gray, directional edges represent protein–DNA interactions. (Adapted from Carter et al.22)

69

ping sets of coexpressed genes, each weighted by global significance.29,30 To test the presumption that these genes were cofunctional and coregulated, we queried gene sets for statistical enrichment of annotated functions and enrichment in targets of known transcription factors. Both were found for the majority of gene sets. With SVD we were also able to identify the group of experimental perturbations (i.e., microarray conditions) that specifically caused the expression pattern shared by each set of genes. This provided both molecular origins (the perturbations) and targets (the enriched transcription factors and hence the coexpressed genes) of regulatory signals. By querying physical interaction databases, we linked the former to the latter via molecular-interaction paths. The resulting series of linked subnetworks maps multiple pathways of information flow through a dense signaling network to targets throughout the genome (Figure 9–2). They are explicit hypotheses of biomolecular mechanisms for signal transduction, obtained through data integration directed by functional relationships.

SYSTEM GENETICS The functional information derived from genetic interaction analysis of phenotype is a powerful means of mapping relationships between genes in a pathway. However, many phenotypes are difficult to quantify and cannot yet be assayed with high throughput. This limits efforts to systematically exploit genetic interactions with quantitative methods. At the same time, we have demonstrated how analysis of microarray data for genetic perturbations can facilitate data integration and molecular network modeling. Thus we sought to combine genetic interactions analysis, numerical data decomposition, and functionally directed data integration to model the network control of gene expression and associated phenotypes. We used a data-driven, linear decomposition of our expression data matrix to determine genetic influences from seed genes to genome-wide transcripts as well as cross-influences of the seed genes on one another’s activity. Matrix decomposition methods such as singular value decomposition (SVD)22,30 and generalized Network Component analysis (gNCA)31 have proved successful in disentangling multiple overlapping quantitative signals in microarray data. The mathematical modeling was combined with physical interaction databases to infer (1) the degree to which certain transcription factors influence the expression level of every transcript in the genome, (2) the degree to which these transcription factors influence the regulatory activity of one another and hence cause genetic interactions, (3) the biochemical mechanisms that transmit these influences, and (4) how these biochemical mechanisms are organized into the biomolecular network that specifically controls the phenotype. Our decomposition can be illustrated with the simplified case of two genes A and B, called seed genes since they are the starting point of the analysis, that influence the expression of two genes X and Y. For a strain background labeled with superscript S, consider a linear pair of equations for gene expression: X S = x0 + xA gAS + xB gBS Y S = y0 + yA gAS + yB gBS

(9–1)

The parameters xA, xB, and x0 represent contributions to the expression of X from the gene A, gene B, and the remainder of

70

SECTION III / WELL-ESTABLISHED MODELS

the genetic background (similarly for gene Y). These are independent of the genotype. The coefficients gAS and gBS are the inferred activity levels of the seed genes A and B in the strain background S, and are independent of the transcript measurements. Influences between seed genes (observed as genetic interactions) can be systematically inferred from changes in activity levels of one in a strain in which the other has been deleted, e.g., in a deletion strain of gene B, gAB < gWT A would evince a positive influence from gene B on the activity of gene A. Rather than substituting transcript level data for these activities (as in regression methods) these model-derived parameters conceptually include all levels of gene control from initiation of transcription to protein localization, modification, and degradation. The system of equations can be expanded to model an arbitrary number of expression values and seed genes by systematically adding parameters. They can be cast in matrix form and the solution can be represented as a network of influences, as illustrated in Table 9–1. These matrices can be expanded to model numerous observables (expression of genes X, Y, Z, . . .) under single and

pairwise perturbations of multiple genes (A, B, C, . . .), simply by adding appropriate matrix rows and columns. For example, three seed genes would be written as X X ⎛X ⎜ Y WT Y A Y B ⎜ WT ZA ZB ⎜Z ⎜⎝


⎛ x0 x A x B ⎜ y0 y A yB =⎜ ⎜ z0 z A z B ⎜⎝


WT

A

B

X C X AB X AC X BC ⎞ Y C Y AB Y AC Y BC ⎟ ⎟ Z C Z AB Z AC Z BC ⎟ ⎟



⎠ xC ⎞ ⎛ 1 1 1 1 yC ⎟ ⎜ gWT 0 g AB gCA A ⎟ • ⎜ WT zC ⎟ ⎜ gB gBA 0 gBC ⎟⎠ ⎜⎝ WT gC gCA gCB 0


1 0 0 gCAB

1 ⎞ g ABC ⎟ ⎟ 0 ⎟ ⎟ 0 ⎠

1 0 gBAC 0

(9–2) This procedure results in the decomposition of an expression data matrix D into two matrices: (1) an influence matrix, X, of coefficients for the influence of the seed genes on target genes and (2) a genotype matrix, G, of inferred activity levels for the

Table 9–1 Outline of mathematical modelinga 1. Model all possible influences (xA, xB, yA, yB) from genes A and B on the expression of genes X and Y

XWT = x0 + xA + xB Y WT = y0 + yA + yB

xA

2. Add coefficients (gA, gB) representing possible effects of A on activity of B, and vice versa

X = x0 + xA g Y WT = y0 + yA g

WT A WT A

WT

xB

+ xB g + yB g

3. Model effects of a deletion of gene A (similar for deletion of B); gA becomes 0; gBA represents the activity of gene B in the A deletion strain; faded lines represent possible influences lost

4. Model effects of double perturbation (deletion of genes A and B); faded lines represent possible influences lost

X = x0 + xB g YA = y0 + yB g

A B A B

xB

A

B

xB

yB

yA

X

Y

A

B

AB

xB

X Expression data

a

yB

yA

Y

xA

5. Rewrite equations as a matrix decomposition of the expression data

Y B

X xA

X = x0 YAB = y0

yB

yA

X A

WT B WT B

xA

A

B

A

wt

⎛X ⎝⎜ Y wt

XA YA

XB YB

Influence matrix

X AB ⎞ ⎛ x0 ⎟ =⎜ Y AB ⎠ ⎝ y0

xA yA

yA

yB

Y

Genotype 1 ⎛ 1 xB ⎞ WT 0 ⎟⎠ • ⎜ g A yB ⎜ WT ⎝ gB gBA

matrix 1 1⎞ g AB 0⎟ ⎟ 0 0⎠

Modeling strategy illustrated for the simplified case of two seed genes (A and B) influencing the expression of two genes (X and Y).

CHAPTER 9 / INTEGRATED NETWORK MODELING OF MOLECULAR AND GENETIC INTERACTIONS

seed genes in each genetic background. This is concisely written: D = X·G

(9–3)

Thus, the more direct influences from the seed genes to target genes are separated quantitatively from the indirect effects that involve a second seed gene via a genetic interaction. The structure of the genotype matrix [Eq. (9–2)] is fixed by the genetic composition of the laboratory strains. Activity levels of null alleles are fixed at zero and wild-type activity is set equal to one in all cases (gene-by-gene variation is encoded in the first column of the influence matrix). Other genotype matrix elements are unknown a priori. To set a scale for the inferred regulatory activities, we define the wild-type activities to be equal to one (gA = gB = . . . = 1) and calculate the remaining activities as changes relative to wild type under perturbations of other seed genes (gAB, gBA, gCAB, etc.). The form of matrix G guarantees the existence of a unique best-fit solution due to the strict arrangement of ones and zeros required by the genotypes (i.e., the rows of matrix G are linearly independent and cannot be transformed to yield a similar format). In the decomposition of microarray data, the number of data points in Eq. (9–3) far exceeds the number of model parameters and a least-squares best-fit solution can be found. From the genotype matrix G in Eq. (9–3), we can infer quantitative cross-influences between the seed genes that generate genetic interactions. These correspond to influences on inferred regulatory activity, rather than the gene expression influences encoded in the matrix X. They will allow us to make predictions for perturbations of path genes, with the modified influences experimentally manifest as altered genetic interactions. This is a further dimensional reduction of the genotype matrix G. Starting again with the case of two seed genes, A and B, we can write for their activity levels: A = A0 + mAB B B = B0 + mBA A

(9–4)

The variables A and B define generalized activity levels of the seed genes, and the parameters A0 and B0 represent basal input not directly due to genes A or B. The mij account for influences between A and B. Self-influences, such as mAA, are not included since they cannot be numerically distinguished from the basal input. The model can be readily generalized to the case of N perturbed genes. For a vector of gene activities, we replace {A, B} with g = {g1, g2, . . . , gN} and write gi = g0i + mij gj

(9–5)

where g0 is a vector of base activity and the mij form is an N × N matrix encoding the influence of the ith gene on the jth gene. Equation (9–5) can be solved for the activities and we find the vector solution gWT = (1 − m)−1 · g0

(9–6)

where 1 is the N × N identity matrix. The vector {1, gWT} is the first column of the genotype matrix G. In this formulation, the deletion of a seed gene requires setting both its base activity and its influences on other seed genes to zero. This corresponds to replacing the appropriate entry in g0 with zero and the appropriate column in m with zeros. This can be achieved by rewriting Eq. (9–6) in terms of a diagonal base

71

activity matrix, G0, formed by placing the elements of the vector g0 along the diagonal, and a scaled influence matrix with elements Mij = mij/(g0)i. Defining the vector 1 = {1, 1, . . . , 1, 1} of length N, we then have gWT = [(G0)−1 − M]−1 · 1

(9–7)

In this form, a deletion of gene A is modeled by taking the limit as (G0)AA → 0, which means setting the basal activity of that gene to zero. The resulting gA (with a 1 prepended) corresponds to the second column of the matrix G. Multiple deletions are modeled by taking multiple zero limits for entries of the diagonal matrix G0. This effectively removes all traces of the deleted genes from the system. Note that by after fixing gWT = {1, 1, 1, . . . ,1} we can find a solution for the matrix elements of M and the base activities G0 using the matrix elements of the solution for G as described above. Since this is a further dimensional reduction, reducing N 2(N − 1)/2 parameters to N 2, this generally requires a best fit solution. To summarize, the linear influences decomposition assigns quantitative values to the influence of each seed gene on (1) the expression of every gene (encoded in X) and (2) the activity of the other seed genes (encoded in M). These influences amount to a global map of functional information flows of specified magnitude and sign (positive or negative). A key use of the influence map is to serve as a template for integrating physical interaction data, in order to generate specific biomolecular hypotheses for regulatory activity. One strategy for this is to begin by selecting significant influences (i.e., the strongest ones). An example selection criterion is to use crossvalidation to determine all influences that are highly unlikely to be nonzero.28 Then, for each of these, a candidate molecular pathway is constructed that transmits the influence, using the integration of physical interaction data as previously described. In this way, networks can be constructed from physical interactions that were specifically selected based on the presence of numerical influences derived from the data. They propose specific biomolecular hypotheses of information flow in the network. Furthermore, perturbation of network elements predicts quantifiable changes in gene expression, since the corresponding numerical influence would be modified. As a test of this modeling approach, we recently applied it to the control of filamentous growth in yeast. Certain strains of S. cerevisiae grow either as a round single-cell yeast form, or as a pathogenic, adhesive, invasive, filamentous form (also referred to as pseudohyphal development).23 Many fungal pathogens of humans and plants exhibit similar dimorphism.32 In response to environmental cues, the pathogenic chains of elongated cells adhere to host tissues, evade immune responses, and penetrate barriers. The invasive-growth phenotype previously discussed can be viewed as one part of this response. We chose five filamentation-related transcription factors as our seed genes and collected gene expression data for the 16 strains necessary to infer all genetic interactions between them: the wild type, 5 single knockouts, and 10 double knockouts. We then combined limited phenotype data with genomic expression data (thousands of measurements per strain) to infer a network that controls filamentous growth. We identified a large set of genes with differential expression and performed the linear influences decomposition on the expression data (a 1863 × 16 data matrix). We then determined which

72

SECTION III / WELL-ESTABLISHED MODELS

(A)

(B)

TEC1

CUP9 YAP6

MSN2 PHD1

GAC1 SOK2 KSS1 SWE1 CIN5 PCL2

(C)

MGA1 MOT3

ROX1

MGA1 MOT3 SKN7

TEC1

CUP9

SKN7

TEC1

CUP9 YAP6

HSF1 CRZ1

MSN2 PHD1

SKN7

KSS1

YKL171W FES1

STE12

DDR48

STE12

DDR48

DDR48

Figure 9–3. Data integration example: modeling how TEC1, CUP9, and SKN7 control the expression of gene DDR48. (A) Network of physical interactions connecting the four genes from high-throughput data sets. This network is too dense and disorganized to identify functional pathways. Edges represent interactions as protein–protein (black, nondirectional), protein phosphorylation (black, directional),

and protein–DNA (gray, directional). (B) Network of inferred influences using genetic influences decomposition. Bold edges indicate the direction of positive and negative influences (as labeled) and intensity indicates magnitude. (C) Integrated network constructed from the subset of physical pathways in (A) that are specific candidates for transmission of influences in (B). (Adapted from Carter et al.28)

influences were significant and constructed putative biochemical networks controlling the expression of each gene. The strategy is illustrated in Figure 9–3 with a network for the transcriptional regulation of the gene DDR48, which encodes an ATPase involved in stress response, cell wall organization, and flocculation.33 We used the network model to predict genomic expression effects of additional genetic perturbations and tested these predictions by constructing additional combinatorial knockout strains and collecting microarray data. This served as a direct test of our genetic interaction modeling and data integration methods. We made predictions by removing (set to zero) numerical influences whenever the paths from influencer to influenced were broken under the downstream gene deletion. As a control, we made parallel predictions using a simpler linear model without genetic interactions. Our model provided a general improvement in fit across all genes. When assessed over thousands of expression measurements the improvement was highly significant, providing empirical evidence for both the importance of genetic interactions and the applicability of our modeling technique.28 In addition to microarray data, we also phenotyped each strain for filamentous growth. We then performed SVD on the genomic expression data30 and identified a set of genes with an expression component correlated to the phenotype. The genes that strongly exhibit this expression component were a quantitative proxy for the phenotype, even though this component was quantitatively not the most dominant pattern in the data. These genes are statistically enriched in targets of eight transcription factors, of which six were known to have filamentous-growth-related phenotypes. Subsequently, we found that deletions of the other two have filamentation phenotypes. We were able to determine which of our seed genes had strong expression influences on the phenotype-proxy gene set. By following the methods previously described to integrate physical interaction data, we connected these influencers to genes in the set in a biomolecular network. This constituted a candidate network for the regulation of filamentation, involving both known and novel regulators. Furthermore, from the topology of the network we were able to make systematic and precise predictions for the phenotypes of additional combinatorial perturbations.

These predictions proved to be very accurate,28 compared with predictions based a large training set of interactions.8 We correctly predicted the phenotypes of all 13 of the novel double-knockout strains we constructed, compared to the training set expectation of seven correct predictions (significance of p = 0.0002). Furthermore, we were able to correctly predict the precise phenotype inequalities of six of the 13 strains, compared to an expectation of only two in the training set (p = 0.009).

DISCUSSION The construction of predictive bionetworks has proved to be a substantial challenge due in part to the difficulty of extracting functionally relevant interactions from diverse high-throughput data types. With the increasing abundance of molecular interaction data, there are often numerous possible pathways of information flow between two genes.34 Many recent works have sought to distill functionally important information by detecting systematic congruence in multiple large data sets.10,35–39 These approaches have demonstrated substantial success in classifying genes according to functionality and identifying probable candidates for genetic interaction. In contrast, our goal was to infer functional relationships to drive network modeling. By using genetic influences decomposition of microarray data to guide data integration, our procedure assigned functional significance to specific physical interactions. This allowed us to make precise, testable predictions for novel combinatorial perturbations of specific genes.28 These models implicated new regulators and regulatory relationships. The network we obtained for the filamentous growth phenotype in yeast contained many genes known to be involved in filamentation, and the network also implicated new regulators of filamentation that were verified experimentally. In addition to implicating genes, our approach was often able to correctly infer relationships between the genes. The accuracy of the interaction network was evident in our broad success in predicting expression profiles and phenotypes for novel combinatorial perturbations. A central concept in our approach was the assumption of linearity in quantitative relationships between genes. This allowed us to efficiently identify the effects of genetic interactions in the data and enabled systematic mapping of how the expression of

CHAPTER 9 / INTEGRATED NETWORK MODELING OF MOLECULAR AND GENETIC INTERACTIONS

each gene is influenced by a genetic perturbation. Although the linear approach was sufficient to predict the effects of novel pairwise gene deletions, we note that it has the potential to oversimplify complicated regulatory mechanisms. In particular, the nonlinearity inherent in many posttranscriptional and feedback mechanisms often leads to behavior too complex to be adequately modeled with static linear relationships. In such cases, the networks obtained from our approach might best be used as a starting point for dynamic modeling. With relatively few network elements connected by quantitative relationships, these networks are ideally suited for ordinary differential equation (ODE) and stochastic modeling.40

ACKNOWLEDGMENTS We thank our collaborators for their contributions to this work: I. Avila-Campillo, B.L. Drees, G.R. Fink, B. Marzolf, C. Neou, M.Z. Raymond, A.W. Rives, S. Rupp, S. Prinz, P. Shannon, and J.P. Shelby. We also thank I. Shmulevich, R.J. Taylor, and D. Galas for discussions and P. Troisch for contributions. This work was supported in part by NIH Grant P50 GM076547. G.W.C. was supported in part by NSF Grant FIBR-0527023. T.G. is a recipient of a Burroughs Wellcome Fund Career Award in the Biomedical Sciences.

REFERENCES 1. Tong AH, Evangelista M, Parsons AB, Xu H, Bader GD, Page N, Robinson M, Raghibizadeh S, Hogue CW, Bussey H, et al. Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 2001;294(5550):2364–2368. 2. Tong AH, Lesage G, Bader GD, Ding H, Xu H, Xin X, Young J, Berriz GF, Brost RL, Chang M, et al. Global mapping of the yeast genetic interaction network. Science 2004;303(5659):808– 813. 3. Fraser AG, Kamath RS, Zipperlen P, Martinez-Campos M, Sohrmann M, Ahringer J. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 2000;408(6810): 325–330. 4. Tewari M, Hu PJ, Ahn JS, Ayivi-Guedehoussou N, Vidalain PO, Li S, Milstein S, Armstrong CM, Boxem M, Butler MD, et al. Systematic interactome mapping and genetic perturbation analysis of a C. elegans TGF-beta signaling network. Mol Cell 2004;13(4):469– 482. 5. Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B, Bangham R, Benito R, Boeke JD, Bussey H, et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 1999;285(5429):901–906. 6. Strausberg RL, Schreiber SL. From knowing to controlling: A path from genomics to drugs using small molecule probes. Science 2003;300(5617):294–295. 7. Swedlow JR, Goldberg I, Brauner E, Sorger PK. Informatics and quantitative analysis in biological imaging. Science 2003;300(5616): 100–102. 8. Drees BL, Thorsson V, Carter GW, Rives AW, Raymond MZ, AvilaCampillo I, Shannon P, Galitski T. Phenotype and the interaction of genetic perturbations. Genome Biol 2004;6(4):R38. 9. Avery L, Wasserman S. Ordering gene function: The interpretation of epistasis in regulatory hierarchies. Trends Genet 1992;8(9): 312–316. 10. Zhang LV, King OD, Wong SL, Goldberg DS, Tong AH, Lesage G, Andrews B, Bussey H, Boone C, Roth FP. Motifs, themes and thematic maps of an integrated Saccharomyces cerevisiae interaction network. J Biol 2005;4(2):6. 11. Kelley R, Ideker T. Systematic interpretation of genetic interactions using protein networks. Nat Biotechnol 2005;23(5):561–566. 12. Galitski T. Molecular networks in model systems. Annu Rev Genomics Hum Genet 2004;5:177–187.

73

13. Carter GW. Inferring network interactions within a cell. Brief Bioinform 2005;6(4):380–389. 14. Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, Kanapin A, Le Bot N, Moreno S, Sohrmann M, Welchman DP, Zipperlen P, Ahringer J. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 2003;421: 231–237. 15. Boutros M, Kiger AA, Armknecht S, Kerr K, Hild M, Koch B, Haas SA, Consortium HF, Paro R, Perrimon N. Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 2004;303: 832–835. 16. Drysdale R. Phenotypic data in FlyBase. Brief Bioinform 2001;2:68–80. 17. Hartman JL IV, Garvik B, Hartwell L. Principles for the buffering of genetic variation. Science 2001;291:1001–1004. 18. Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T. Cytoscape: A software environment for integrated models of biomolecular interaction networks. Genome Res 2003;13:2498–2504. 19. Online at http://galitski.systemsbiology.net. 20. Van Driessche N, Demsar J, Booth EO, Hill P, Juvan P, Zupan B, Kuspa A, Shaulsky G. Epistasis analysis with global transcriptional phenotypes. Nat Genet 2005;37(5):471–477. 21. Online at http://www.geneontology.org. 22. Carter GW, Rupp S, Fink GR, Galitski T. Disentangling information flow in the Ras-cAMP signaling network. Genome Res 2006;16(4):520–526. 23. Gimeno CJ, Fink GR. The logic of cell division in the life cycle of yeast. Science 1992;257:626. 24. Thevelein JM. The RAS-adenylate cyclase pathway and cell cycle control in Saccharomyces cerevisiae. Antonie Van Leeuwenhoek 1992;62:109–130. 25. D’Souza CA, Heitman J. Conserved cAMP signaling cascades regulate fungal development and virulence. FEMS Microbiol Rev 2001;25:349–364. 26. Jones DL, Petty J, Hoyle DC, Hayes A, Ragni E, Popolo L, Oliver SG, Stateva LI. Transcriptome profiling of a Saccharomyces cerevisiae mutant with a constitutively activated Ras/cAMP pathway. Physiol Genomics 2003;16:107–118. 27. Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, Puigserver P, Carlsson E, Ridderstrale M, Laurila E, et al. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 2003;34(3):267–273. 28. Carter GW, Prinz S, Neou C, Shelby JP, Marzolf B, Thorsson V, Galitski T. Prediction of phenotype and genomic expression for combinations of mutations. Mol Syst Biol 2007;3:96. 29. Weaver DC, Workman CT, Stormo GD. Modeling regulatory networks with weight matrices. Pac Symp Biocomput 1999;4(1): 112–123. 30. Alter O, Brown PO, Botstein D. Singular value decomposition for genome-wide expression data processing and modeling. Proc Natl Acad Sci USA 2000;97:10101–10106. 31. Yang YL, Suen J, Brynildsen MP, Galbraith SJ, Liao JC. Inferring yeast cell cycle regulators and interactions using transcription factor activities. BMC Genomics 2005;6(1):90. 32. Lengeler KB, Davidson RC, D’Souza C, Harashima T, Shen WC, Wang P, Pan X, Waugh M, Heitman J. Signal transduction cascades regulating fungal development and virulence. Microbiol Mol Biol Rev 2000;64(4):746–785. 33. Tonouchi A, Fujita A, Kuhara S. Molecular cloning of the gene encoding a highly expressed protein in SFL1 gene-disrupted flocculating yeast. J Biochem (Tokyo) 1994;115(4):683–688. 34. Vidal M. Interactome modeling. FEBS Lett 2005;579(8):1834– 1838. 35. Bader GD, Heilbut A, Andrews B, Tyers M, Hughes T, Boone C. Functional genoimcs and proteomics: Charting a multidimensional map of the yeast cell. Trends Cell Biol 2003;13:344–356. 36. Gunsalus KC, Ge H, Schetter AJ, Goldberg DS, Han JD, Hao T, Berriz GF, Bertin N, Huang J, Chuang LS, et al. Predictive models

74

SECTION III / WELL-ESTABLISHED MODELS

of molecular machines involved in Caenorhabditis elegans early embryogenesis. Nature 2005;436(7052):861–865. 37. Sachs K, Perez O, Pe’er D, Lauffenburger DA, Nolan GP. Causal protein-signaling networks derived from multiparameter single-cell data. Science 2005;308(5721):523–529. 38. Zhong W, Sternberg PW. Genome-wide prediction of C. elegans genetic interactions. Science 2006;311(5766):1481–1484.

39. Workman CT, Mak HC, McCuine S, Tagne JB, Agarwal M, Ozier O, Begley TJ, Samson LD, Ideker T. A systems approach to mapping DNA damage response pathways. Science 2006;312(5776): 1054–1059. 40. Kaern M, Elston TC, Blake WJ, Collins JJ. Stochasticity in gene expression: From theories to phenotypes. Nat Rev Genet 2005;6:451–464.

10

The Sponge as a Model of Cellular Recognition XAVIER FERNÀNDEZ-BUSQUETS

ABSTRACT

AGGREGATION ASSAYS WITH CELLS AND SYNTHETIC BEADS Sponges are the most primitive present-day multicellular animals. They lack organs, but their bodies consist of a few classes of cells that are responsible for specialized functions such as food intake, epithelium formation, and self–nonself recognition, among others. In 1907, Wilson2 pioneered the use of sponges as model animals for the study of cell adhesion, describing the existence of species-specific reaggregation of marine sponge cells that had previously been mechanically dissociated by passing them through a fine cloth. After disaggregation the cells slowly settled down and started moving; upon contact they generally adhered and larger aggregates were formed as new encounters continued to occur, eventually reorganizing functional miniature sponges.3 When dissociated cells of differently colored sponge species were mixed and allowed to aggregate, the cells of one species combined with each other, but not with those from a different species. This phenomenon is analogous to the organ-specific cell adhesion events that represent the basis of clinically important processes such as cancer metastasis and graft rejection. Sponge cells associate species specifically through multivalent interactions of carbohydrate structures on a type of extracellular proteoglycan termed aggregation factor (AF),4–6 the slime Aristotle was referring to. Based on their molecular structure, AFs have been related to hyalectans5: large, extracellular aggregating modular proteoglycans. But unlike hyalectans, aggregation factors do not possess any of the main glycosaminoglycan (GAG) types described in higher animals; instead, they have complex and repetitive acidic carbohydrate motifs different from those found in classical proteoglycans and mucins,7 which include novel acidresistant and acid-labile carbohydrate domains, large and branched pyruvylated oligosaccharides,8 and other previously unknown structures.6,9–11 In the marine sponge Microciona prolifera the proteoglycan molecule, Microciona AF (MAF), binds cell membrane receptors via Ca2+-independent interactions of small 6-kDa glycans (g6). Larger 200-kDa glycans (g-200) self-interact through calcium-dependent associations (Figure 10–1C and D). This bifunctional structure is common to all sponge AFs studied so far and represents one of the first specific cell adhesion systems that were necessary for the evolution of Metazoans. In aggregation assays performed with synthetic beads coupled to AFs purified from different sponge species,12,13 the beads sorted out and associated only with those carrying AFs from the same species, providing conclusive evidence that AFs are the molecular entity responsible for species-specific cell aggregation.

Sponges, the simplest extant Metazoans, have been traditionally used as models to study cell adhesion, since their abundant extracellular matrix allows a mild cell dissociation and the recovery of functionally active macromolecular structures. Dissociated sponge cells quickly reaggregate in a species-specific manner, differentiate, and reconstruct tissue, providing a simple model to investigate the molecular basis of animal intercellular recognition systems. Here, we review the application of sponges as biotools for the study of three cell recognition processes that are relevant for biomedical research. First, the species-specific association of sponge cells is discussed in the context of the role ascribed to carbohydrates in adhesion-related phenomena implicated, among other processes, in tumor metastasis. Second, we will summarize the current knowledge about a sponge proteoglycan that has been proposed as a model for the investigation of the mechanical properties of extracellular matrix proteoglycans in calcium-dependent cell adhesion events, whose impairment can lead to severe disorders such as Alzheimer’s disease and Marfan syndrome. Finally, the self–nonself recognition reactions in sponges are presented as a window to the early period in the evolution of histocompatibility systems, and the corresponding molecular and cellular events will be compared with the processes and cells known to be involved in innate immunity and in placental implantation. Potential important applications of sponges in the search for new pharmaceuticals and in key biomedical areas such as stem cell research are just around the corner awaiting the development of sponge cell cultures. Key Words: Cell adhesion, Extracellular matrix, Glycosaminoglycans, Invertebrate histocompatibility, Porifera, Proteoglycans, Self–nonself recognition, Single-molecule force spectroscopy.

SPECIES-SPECIFIC CELL ADHESION IN SPONGES AS A MODEL OF ORGAN-SPECIFIC CELL ADHESION “Sponges grow spontaneously either attached to a rock or on sea-beaches, and they get their nutriment in slime: a proof of this statement is the fact that when they are first secured they are found to be full of slime.” From Aristotle’s History of Animals, 350 BC.1

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

75

SECTION III / WELL-ESTABLISHED MODELS

▲

76

Figure 10–1. The aggregation factor of the marine sponge M. prolifera as a model for the study of proteoglycan structure–function relationships. (A) In its native form, MAF has the structure of a sunburst where the ring is formed by ∼20 units of the MAFp3 protein (open circles), each noncovalently linked to a unit of the MAFp4 protein (an MAF “arm”). The solid circles forming MAFp4 represent repeated domains within the protein. (B) If the ring of MAF were open, the resulting structure is analogous to a classical proteoglycan, with MAFp3 and MAFp4 in place of link protein and proteoglycan monomer, respectively. (C) Model of the MAF interactions responsible for species specificity of cell adhesion: carbohydrates on MAFp4 bind receptors on the cell membrane, whereas the g-200 glycan on MAFp3 self-interacts in a calcium-dependent manner. For clarity, both MAF molecules are represented as linearized. (D) Detail of the interaction between the two subunits marked by asterisks (* and **) in (C). (E) Scheme of an SMFS experiment to study the adhesive interactions between two MAF molecules. For clarity, both molecules are represented as linearized. (F) Typical SMFS approach-retract curve obtained in experiments performed with native MAF, consistent with the representation from (E). The numbers from 1 to 13 correspond to the breaking of the individual g-200-g-200 interactions represented in (E) as the AFM tip is retracting from the surface. The 100-nm scale bar refers to parts (E) and (F) only. (Adapted from Garcia-Manyes et al.18)

Glycoprotein subunits of MAF obtained through dissociative denaturing procedures bind Microciona cells, but with decreasing affinity correlating linearly with the decreasing size of the subunits,14 suggesting polyvalency in the cell binding site. A small glycopeptide of 10 kDa obtained after trypsin digestion of MAF showed very little affinity for homotypic cells in its monomeric form, but reconstitution of binding affinity in the same order of magnitude as the native molecule could be obtained by polymerizing the glycopeptide fragment.15 Eventually, both MAF–cell and MAF–MAF binding affinities were recovered upon chemical crosslinking of protein-free glycans isolated from the AF complex into large multivalent structures.6,9,16 This indicated an active role for the carbohydrate moiety of MAF in the aggregation of sponge cells. In a final series of experiments it was shown that glycan-coated beads aggregated according to their species of origin, i.e., the same way as live sponge cells did.17 Live cells also demonstrated species-selective binding to glycan-coated surfaces. These findings confirmed for the first time the existence of highly specific recognition between surface glycans, a process that may have significant implications in cellular interactions. ROLE OF CARBOHYDRATES IN CELL ADHESION g200 is not a common GAG, as suggested by its elevated fucose content and its resistance to usual GAG-digesting enzymes.6 Preliminary nuclear magnetic resonance analyses performed with intact g-200 chains indicated such a structural diversity that the possible existence of a backbone containing a basic repetitive oligosaccharide unit could not be determined.11 This complex structure of g-200, in contrast to the much simpler linear repetitive chains of chondroitin sulfate, heparan sulfate, or hyaluronan, can provide the basis for its highly selective adhesive properties in 10 mM Ca2+, as illustrated by a strong intraspecies-specific binding,18 weaker interspecies-specific interactions,17 and very small adhesion forces with other unrelated GAGs.18 Both the strength and the specificity of calcium-dependent carbohydrate– carbohydrate interactions might be guaranteed by polyvalency, by

CHAPTER 10 / THE SPONGE AS A MODEL OF CELLULAR RECOGNITION

compositional and architectural differences between carbohydrates, and by the arrangement of the carbohydrate chain in a three-dimensional context, where calcium ions provide not only electrostatic forces but can contribute via coordinative forces to glycan superstructures.19 Specific carbohydrate–carbohydrate interactions are rarely reported in biologically relevant situations such as cell recognition. However, carbohydrate structures have immense structural diversity, a ubiquitous distribution in vertebrate and invertebrate tissues, and are associated with the cell surface, as required of cell recognition molecules.20 Carbohydrate–carbohydrate interactions are characterized by relatively weak forces that, when multimerized, can be easily potentiated by orders of magnitude, representing a highly versatile form of cell adhesion given the extraordinary plasticity of their structures.21 Among the few known examples of carbohydrate self-recognition proposed to be specific in biological processes are the multivalent binding of Lewisx epitopes involved in the first steps of embryogenesis22 and in cancer metastasis,23,24 glycolipid–glycolipid interactions controlling cell adhesion, spreading, and motility,25,26 and self-interactions of the glycan portion of sponge proteoglycan carbohydrates leading to speciesspecific cell adhesion.17,19 The species specificity of cell recognition mediated by sponge proteoglycans can be a useful model to study other adhesion processes at structural levels above individual cells. Sponges do not have organs, and therefore the molecular machinery regulating the recognition between cells from different sponge species might be based on the same principles that rule the interactions between cells from different organs in higher animals. Adhesion molecules are essential to motility by interacting with bound and soluble substrates within the extracellular matrix (ECM). Proteoglycans bound to the plasma membrane mediate the polyvalent interaction of the cell with ECM constituents and with molecules from neighboring cell surfaces. Given their direct involvement in cell–cell and cell–ECM interactions, proteoglycans are likely to be implicated in the regulation of cell movement, but to what degree the carbohydrate moiety affects this process is only partially understood. Cadherins are a family of Ca2+-dependent adhesion molecules that bind cells via homophilic interactions, and cells expressing distinct cadherins aggregate separately when mixed in culture.27 Cadherins are responsible for the cell sorting that is necessary to distribute different cell types to their proper positions during development. During embryogenesis, the expression of different cadherins is spatiotemporally regulated, and correlates with events that involve cell aggregation or disaggregation. Alterations in cadherin expression or function occur frequently during carcinogenesis and tumoral angiogenesis.28,29 Another group of cell adhesion molecules includes those related to the immunoglobulin (Ig) superfamily, which generally have Ca2+-independent heterophilic binding.30 AF-promoted cell adhesion in sponges is based on carbohydrate structures with both Ca2+-dependent homophilic binding and Ca2+-independent heterophilic interactions with cell surface receptors, suggesting that proteoglycan-linked glycans can have important functions in cell adhesion and recognition, comparables to those assigned to cadherins and to molecules of the Ig superfamily. Metastasis, the process by which a primary malignancy establishes distant and discontiguous disease, clearly involves carbohydrate-mediated cell adhesion.31 Not all tumors are capable of

77

metastasis, and those that succeed in it rely on a series of steps that involve cell dissociation, motility, and subsequent readhesion.32 CD44 is a widely distributed cell surface glycoprotein that binds to ECM components containing hyaluronic acid (HA).33 This permits tumor attachment and migration toward degraded areas rich in HA. CD44 expression is associated with aggressive melanomas and it is an indicator of poor prognosis.34 As we will see below, antibodies raised against CD44 specifically target a sponge cell type of elevated motility.35 Study of the essential role that carbohydrates play in sponge cell recognition, motility, and specific adhesion might contribute to understand the participation of carbohydrate structures in the complex processes leading to cancer spreading.

SPONGE AGGREGATION FACTORS AS A MODEL FOR THE STUDY OF PROTEOGLYCAN STRUCTURE–FUNCTION RELATIONSHIPS The biological roles assigned to proteoglycans are highly diversified, ranging from relatively straightforward mechanical functions to effects on more dynamic processes such as cell adhesion and motility, to complex and still poorly understood tasks in cell differentiation and development.36–39 Angiogenesis, axon guidance and synapse development, metastasis, and patterning events are just some examples of processes that require finely tuned interactions between cells or between cells and the ECM where proteoglycans are involved.40–44 Efforts aimed at a better understanding of proteoglycan structure and function are often hampered by the difficulties in their isolation from vertebrate tissues. The purification of proteoglycans is often complicated by (1) limited source quantities, (2) the necessity of chaotropic solvents, proteases, and high temperatures for efficient extraction, (3) their large molecular size, and (4) a lack of defined functions to enable purity to be assessed.45 Marine sponge proteoglycans are relatively small and can be extracted in considerable amounts while preserving their biological activity by following a mild protocol that essentially consists of dissociating the sponge tissue in calcium-free sea water and spinning down the cells and other debris. The resulting supernatant contains aggregation factor as the main component in such quantities and purity that it can be directly used for cell aggregation assays and other functional experiments. For structural determinations, this preparation has to be centrifuged at high speed to pellet the AF that is finally purified in a cesium chloride density gradient. Concentrated AF solutions (≥1 mg/ml) are highly resistant to proteolysis and can be stored at 4°C for months without signifi cant loss in their specific cell aggregation capacity. STRUCTURAL ANALYSES AND SINGLE MOLECULE FORCE SPECTROSCOPY STUDIES The supramolecular structure of MAF was elucidated by using immunochemical and electrophoretic procedures, combined with atomic force microscopy (AFM) imaging.46 Twenty units from each of two N-glycosylated proteins, MAFp3 and MAFp4, form the central ring and radiating arms of MAF, respectively, stabilized by a hyaluronidasesensitive component.46 Each of the 20 arms is attached to one of the 20 globular structures in the ring in a 1:1 stoichiometry (Figure 10–1A), with an estimated molecular mass for the whole molecule of 2 × 107 Da. Each MAFp3 ring unit carries on average one copy of the g200 glycan involved in homologous self-interactions between

78

SECTION III / WELL-ESTABLISHED MODELS

aggregation factor molecules (Figure 10–1C and D), whereas each MAFp4 unit carries about 50 copies of the g-6 glycan that binds cell surface receptors.46 MAFp3 is a highly polymorphic 35-kDa protein, as deduced from direct cDNA sequencing of several different allelic forms and from restriction fragment length polymorphism analysis of genomic DNA isolated from different individual sponges.47,48 MAFp4 has an estimated molecular mass of ∼400 kDa,48 is also polymorphic, contains putative transmembrane regions, and has a stretch sharing 30% homology with the cytoplasmic domain of the Na+–Ca2+ exchanger,49 a membrane protein responsible for the maintenance of low intracellular Ca2+ levels. Both MAFp3 and MAFp4 contain abundant Ca2+-binding sites and glycosylation motifs. If the MAF circular core were open, the resulting linear structure would be remarkably similar to hyalectans50 (Figure 10–1A and B), although the building blocks of both molecules lack sequence homologies. MAFp4 is analogous to the glycosylated core protein of the proteoglycan monomer, and carries the N-linked g-6 glycan instead of O-linked GAGs. MAFp3 is the sponge counterpart of the link protein, both molecules having very similar sizes. Like link protein, MAFp3 is Nglycosylated and contains eight cysteines involved in disulfide bonds. Unlike link protein, though, MAFp3 does not contain an Ig-like domain. During the past decade, single molecule force spectroscopy (SMFS) has developed into a highly sensitive tool for the investigation of single biomolecule interactions.51 Most SMFS experiments use either optical tweezers or AFM to measure dissociation forces of single ligand–receptor complexes in the piconewton range. AFM-based SMFS has the capacity to detect interactions as weak as individual hydrogen bonds. The molecular binding partners are attached to the nanoscale force sensor and to a sample holder by covalent chemistry. A critical step in the design of an SMFS experiment is the crosslinking of biomolecules to surfaces, which can introduce undesired reactive groups or steric constraints resulting in the alteration of the biological interaction under study. In the particular case of AFs, their abundant sulfate groups provide a very strong quasicovalent binding to gold surfaces, thus allowing immobilization in the absence of chemical modification, and without affecting the binding sites.18 When the AFM sensor and holder are brought into close contact, a specific bond between the individual molecules can form. By increasing the distance between the two surfaces again, the molecular bond is loaded under an external force until it finally breaks, yielding the molecular dissociation force. Upon systematical variation of the externally applied load and monitoring the mechanistic elasticity of the complex, information about the kinetic reaction rates, the mean lifetime, the equilibrium rate of dissociation, dissociation length, and the energy landscape of the interaction can be derived. SMFS studies performed with spongederived proteoglycans of their calcium-dependent interactions18 have revealed functional intermolecular domains (Figure 10–1E) that can contribute to many adhesive and elastic ECM interactions. The multiplicity of individual binding sites with a mean force of ∼250 pN for each adhesion event (Figure 10–1E and F) confers a high degree of modulability as required in most biological interactions, in contrast to the higher stability of a single, strong bond. This modular elongation mechanism, be it intramolecular or intermolecular, has been proposed to be a general strat-

egy for conveying toughness to natural fibers and adhesives.52 Sponge proteoglycan extension curves, then, derive from the existence of intermolecular adhesion domains that through the summation of multiple single binding sites provide strong adhesion forces for the resulting polymer.18 The cooperativity of abundant, relatively weak intermolecular carbohydrate adhesion domains provides a molecular basis for some of the functions of modular proteoglycans. IMPLICATION OF PROTEOGLYCANS IN EXTRACELLULAR MATRIX-RELATED PATHOLOGIES Proteoglycans are among the first ECM constituents to be produced during embryonic development and are often aberrantly expressed in a variety of inherited and acquired disorders. One of the major pathological features of Alzheimer’s disease is the presence of plaques composed of β-structured fibrils made up of amyloid-β peptide (Aβ).53 Nucleation of Aβ can occur by self-assembly or by heterogeneous nucleation resulting from seeding Aβ onto non-Aβ elements present in the ECM, such as proteoglycans.54 One of the most common nonenzymatic posttranslational protein modifications is the reaction of reducing sugars (or of other carbohydrates resulting from the fragmentation of diverse polysaccharides) with nucleophilic amino acid side chains such as those found in arginine and lysine. Subsequent restructuring, oxidations, and dehydrations generate a group of heterogeneous compounds termed advanced glycation endproducts (AGEs).55 AGEs have been found in pathological protein deposits such as the senile plaques characteristic of Alzheimer’s disease or the β2-microglobulin deposits in hemodialysis patients.56 Given their participation in the regulation of diverse growth factors and cytokines through receptor-mediated mechanisms, it has been proposed that AGE-modified proteins are involved in the pathology of several age-related diseases.57 In vitro, AGE formation accelerates the growth of β-amyloid aggregates through covalent crosslinking of Aβ monomers.58 The extracellular accumulation of AGEs can be caused by an accelerated oxidation of glycosylated proteins and proteoglycans.59 Glycosylated proteins and, especially, proteoglycans (whose carbohydrate content can be higher than 50%) produce an amount of radicals almost 50 times higher than that generated by nonglycosylated proteins. Proteoglycans are associated with and are likely play a fundamental role in the deposition of amyloid fibrils in all amyloid diseases.60 Fibril formation and the lateral aggregation of Aβ in vitro are increased by GAGs,61 in a pH-dependent effect.62 Therefore the interaction of GAGs with amyloid fibrils might be based on electrostatic interactions similar to those involved in the calcium-mediated aggregation of AFs. MAF has been shown to induce a structural transition in Aβ from random coil toward highly stable fibrillar β-sheet structures as detected by circular dichroism spectroscopy,63 and to increase Aβ-induced toxicity of nerve growth factor-differentiated PC-12 cells in the absence of Ca2+. The addition of Ca2+ to MAF-Aβ aggregates resulted in a moderate attenuation of toxicity possibly due to a reduction in Aβ–cell interactions caused by extensive lateral aggregation of the MAF–Aβ complexes. Such aggregation might be seeded by the self-association of MAF triggered by calcium. These results indicate that Aβ is generally susceptible to proteoglycan-mediated aggregation and fibril formation. Sponge proteoglycans, thus, represent a simple model system that can be used to examine potential drugs for the treatment of amyloid-related disorders.

CHAPTER 10 / THE SPONGE AS A MODEL OF CELLULAR RECOGNITION

Microfibrils are evolutionarily ancient macromolecular assemblies of the ECM formed by the protein fibrillin. They have unique extensible properties that endow vascular and other tissues with long-range elasticity. In humans, mutations in the principal structural component of microfibrils, fibrillin-1, are linked to the inheritable disease Marfan syndrome, which is characterized by severe cardiovascular, skeletal, and ocular defects.64 Fibrillin-1 is a calcium-binding protein and it has been found that variations in its calcium-binding properties may be important for microfibril assembly and for the biomechanical properties of microfibrils within tissues.65 The fibrillin-1 domain with the highest recorded affinity for calcium is located in the region of the protein where mutations (including those that specifically disrupt calcium binding) cause the most severe form of disease, neonatal Marfan syndrome. Proteoglycans have important, but still poorly understood structural interactions with microfibrils, and they may contribute to their integration into the ECM.66

ALLOGENEIC RECOGNITION IN SPONGES AS MODEL OF HISTOINCOMPATIBILITY REACTIONS GRAFTING EXPERIMENTS, IMMUNOCYTOCHEMICAL ANALYSES, AND GENETIC STUDIES When tissues from different individuals of a given sponge species are brought into contact, they either fuse or reject through cellular events similar to those observed in vertebrate grafts.67 There is, though, a peculiarity of the sponge self–nonself recognition system that so far has not been described in other phyla, which is the possible involvement of proteoglycans in histoincompatibility reactions. In M. prolifera, sponge fragments can be easily grafted, thus allowing the design of tissue histocompatibility experiments that yield important information about the cell types involved in the process.35 In a typical grafting experiment, 2-cm-long sponge papillae are pushed together on a 0.5-mm-thick stainless steel insect pin, keeping the tissues under seawater at all times. The pins are finally stuck into the underside of a Styrofoam rack and left to float on a tank with running seawater at 20°C. As soon as 2 h after the graft set-up, massive cell migration toward the zone of contact is evident. The study of the sponge cell types involved in this phenomenon has shed new light on our understanding of the evolution of early histocompatibility systems. Polyclonal antibodies raised against MAFp3 are specific markers for archeocytes, the sponge stem cells.35 Archeocytes are mobilized upon allogeneic contact (between tissues from two individuals of the same species), and they accumulate in the contact zone. A second type of cell that also migrates profusely to allograft interfaces, the gray cells, is specifically recognized by monoclonal antibodies raised against the HA receptor CD44.35 In allograft contact areas an observed decrease in the intensity of archeocyte staining is accompanied by an increase in the number of gray cells, which are the most adhesive cell type in M. prolifera.68 A higher adhesion would slow down cell movement and aggregation, contributing to the nonfusion characteristic of allografts. Besides this cell adhesion-related role of gray cells in allograft interfaces, other evidence suggests a more direct immunological function for this cell type, which has been proposed to be the sponge immunocyte.69,70 In contrast, the contact zone of

79

sponge isografts (grafts made between genetically identical tissue fragments) does not accumulate cells. Here, the absence of a massive gray cell migration would permit the cell movements and aggregation necessary for the final outcome of isografts, which invariably end up in the fusion of the interacting tissues. This differential behavior implies the existence of a genetically based system of individual-specific cell signaling molecules. Although the precise nature of these self–nonself recognition markers has not been established yet, genetic studies point at MAF or MAFrelated entities as one of the elements of the sponge histocompatibility system.47,48 Due to the presence in sponge tissue of a potent nuclease activity, a special protocol had to be devised for the purification of high-quality sponge genomic DNA49: 0.1 ml from a pellet of freshly dissociated sponge cells is added to a 2-ml tube to 0.9 ml of a buffer containing 4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7, 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol, and gently mixed by immersion until the viscous solution becomes homogeneous. The proteins are then extracted with a 1:1 mixture of phenol/chloroform equilibrated with TE buffer (10 mM Tris–HCl, pH 8, 1 mM EDTA). After a second extraction with chloroform/isoamyl alcohol 24:1, the DNA contained in the water phase is ethanol precipitated and stored at 4°C in TE, pH 7.5. Southern blot analysis of a significant number of sponges that were subjected to grafting experiments revealed that each genetically distinct sponge individual has a different set of genomic DNA coding sequences for MAFp3 and MAFp4.47,48 Such interindividual variability, also observed in the carbohydrate moiety of MAF,18,47 matches the allelic complexity of the vertebrate major histocompatibility complex (MHC) and represents an early form of self-recognition. An evolutionary connection between cell adhesion and histocompatibility systems, however, has yet to be demonstrated. A prominent domain implicated in cell adhesion, the Ig domain, exists in several copies in the large basement membrane proteoglycan.71 Similar domains are functionally important structures in most Ca2+-independent adhesion molecules such as the neural cell adhesion molecule (NCAM),72 the intercellular adhesion molecules (ICAMs),73,74 and other neural or immune system-associated adhesion molecules. No obvious Ig-like domains have been identified in the cDNA-deduced MAF proteins, although the repeats found in MAFp4 exhibit interesting resemblances.48 SELF–NONSELF RECOGNITION IN SPONGES AS AN ANCESTOR OF IMMUNE SYSTEMS Natural killer (NK) cells are a subset of granular lymphocytes that express high levels of CD44 upon activation and mediate efficient MHC-unrestricted T cell receptor-independent lysis following binding of CD44.33 The cross-reactivity of sponge gray cells with anti-CD44 antibodies, together with other morphological and functional similarities, suggests that deeper links between NK cells and sponge allogeneic reactions are possible. First, carbohydrates are crucial to the functions of NK cells75: NK cells can be identified and subdivided into functionally distinct subsets on the basis of the expression of lectin-like receptors.76 Similarly, species-specific adhesion of sponge cells has a strict dependence on finely tuned carbohydrate interactions.67 Second, NK cells are thought to be the remnants of a primeval immune system and are generally considered to be the evolutionary precursors of T cells.77 In the human fetus, NK cells are the first lymphocyte lineage to appear,78 followed by γδT cells

80

SECTION III / WELL-ESTABLISHED MODELS

and αβT cells, in accordance with the controversial concept that ontogeny recapitulates phylogeny. NK-like cells have been suggested to exist in invertebrates like the snail,79 a tunicate,80 an earthworm,81 and the leech.82 Finally, in the allorecognition system of invertebrates no prior sensitization is necessary. The presence of self MHC class I molecules protects target cells from lysis mediated by NK cells,75 verifying the “missing self” hypothesis that states that NK cell targets become susceptible to NK cell killing if they lack sufficient expression of self MHC molecules. Thus, unlike T cells, which recognize the presence of non-self, NK cells recognize the absence of self.83 This observation prompted Burnet84 to conclude that the primary mechanism of invertebrate allorecognition is based on self-recognition and any cell that lacks self antigens will be immediately recognized as foreign, a view that was later supported by investigations on the allogeneic responses of the colonial tunicate Botryllus schlosseri.85 Some aspects of this invertebrate system appear to be retained in higher vertebrates. There are many features of the immunology of allogeneic reactions in sponges that more closely resemble the immunological relationship between the animal fetus and its mother than between vertebrate allograft and host.86 While contact with allogeneic cells in clinical transplantation is an artificial situation, in the crowded marine environment direct cellular contact among unrelated members of sessile invertebrate species occurs as naturally as the intermingling of allogeneic cells in placental implantation. In the latter case there is a well-defined boundary dividing invasive trophoblast and maternal decidua that mimics the collagen boundary separating two unrelated sponges, a situation very different from the extensive cell killing typical of vertebrate allograft rejection. In implantation, NK cells are the predominant cell type in deciduas and T cells are sparse; similarly, invertebrate allorecognition is mediated by NK-like cells, whereas vertebrate allorecognition is mediated by T and B cells. Finally, NK cells kill target cells deficient in MHC class I antigens, unlike vertebrate graft rejection where nonself antigens are detected; as mentioned above, current evidence suggests that sponge nonself recognition detects the absence of self antigens.35 The development in early animals of the ability to discriminate self from nonself ensured the maintaining of individuality. It is possible that such a primitive form of allorecognition has been conserved to play a role in mammalian reproduction, which is the only natural situation in which allogeneic cells come into contact in vertebrates.

FUTURE PERSPECTIVES: DEVELOPMENT OF STABLE SPONGE CELL CULTURES, STEM CELL RESEARCH, AND BIODISCOVERY OF NEW PHARMACEUTICALS Sponges possess totipotent cells, the archeocytes, that can differentiate into the rest of the cell types. Most important, other sponge cell types do spontaneously dedifferentiate into archeocytes, a phenomenon observed in naturally occurring processes such as tissue regression and remodeling, gemmule formation, and trauma induced by tissue removal.87 Understanding the mechanism by which specialized sponge cells can return to an undifferentiated state will contribute to the quest for a reliable and ethically acceptable source of unlimited amounts of stem cells for therapeutic applications.

The main obstacle for the firm establishment of sponges as a generally accepted model laboratory animal is the current lack of stable sponge cell cultures. Interest in the development of sponge cell lines grew in parallel with the realization that sponges are an exceptional source of metabolites with potential pharmacological activities. Because of their longer evolutionary history, marine organisms have a greater molecular diversity than do their terrestrial counterparts. Among the numerous groups of marine invertebrates sponges rank first as the leading source of natural products in terms of the biogenetic diversity of secondary constituents and the sheer number of compounds isolated.88 Furthermore, sponges have a high strike rate, especially for cytotoxic compounds: of the investigated marine sponge species, >10% has exhibited cytotoxic activity, suggesting production of potential medicinals. This percentage is considerably higher than that found for other marine animals (2%), terrestrial plants (97% depletion of the DA neurons in the SNc and up to 80% in the ventral tegmental area (VTA). A more restricted, progressive, partial lesion of the nigrostriatal pathway can also be achieved by injecting the 6-OHDA into the striatum.4 DAergic terminals take up the toxin and retrogradely transport it to the cell bodies in the SNc. The extent of the partial lesion can range from a 30% to 90% loss of DA neurons of the SNc, dependent upon the number of striatal injection sites and the quantity of 6-OHDA administered.6 A four-site striatal lesion gives rise to about an 80–90% loss of the DA neurons in the SNc, but only 15% of VTA neurons are affected.4 Partial lesions can also be achieved by targeting the MFB with lower concentrations of the toxin,5 although this strategy is typically more variable. In addition, 6-OHDA has also been directly injected in the SNc, resulting in substantial degeneration of the nigrostriatal pathway, but this lesion method is less commonly used. BEHAVIORAL TESTS FOR UNILATERAL 6-HYDROXYDOPAMINE LESIONS The unilateral denervation produces a number of deficits on the side contralateral to the lesion, displayed

as spontaneous circling toward the (ipsilateral) lesioned side, reduced use of the contralateral forelimb, and a neglect of the contralateral side. A very wide range of behavioral tests has been described in the nearly four decades since 6-OHDA came into widespread use. We describe below a selected range of behavioral tests in widespread use, which have been found by ourselves and others to be sensitive, objective, and easily quantified. The list is of course not exhaustive, and other tests can be found elsewhere.1,6–8 Rotational Behavior Circling behavior is the most frequently used behavioral test, largely due to its simplicity.9 It is affected differentially by the administration of dopaminomimetics, the direction and nature of the behavior being related to their respective modes of action. The number of rotations can be counted manually, although commonly this is done with the use of automated rotometers. The output is the total number of rotations in either direction over a predetermined period of time, which can also be expressed as net rotations. Some days after receiving a unilateral lesion, rats exhibit a tendency to turn during spontaneous locomotion to the (ipsilateral) side of the lesion. This ipsilateral bias is markedly enhanced by activating the animal, for example, by exposure to mildly stressful stimuli or injection with a stimulant drug. Thus, amphetamine will increase DA release on the intact side to a greater extent than the DA-depleted lesioned side, and will enhance marked turning toward the side ipsilateral to the lesion. Significant asymmetry can be induced by amphetamine with more than a 30% loss of the DA neurons in the SN or a 40–50% depletion of the DA content in the striatum. In contrast, direct acting dopamine receptor agonists will induce a contralateral circling behavior by acting predominantly on the DA receptors on the postsynaptic striatal neurons, made supersensitive by the deafferenting lesion. More extensive lesions are required to induce high levels of rotation with apomorphine. Many studies have reported a close correlation between the rates of amphetamine and apomorphine-induced rotation and the extent of denervation, such that rotation scores can be used to determine the extent of lesions on an animal-by-animal basis, allowing exclusion from further study of animals with inadequate lesions and allocation of the remainder to matched groups prior to subsequent investigations. Generally, 14 days postlesion, in excess of five ipsilateral rotations per minute induced by d-amphetamine sulfate (2–5 mg/kg ip or sc in saline) measured over 90 min is used to indicate >90% denervation. Similarly, over 90% dopamine depletion is assumed from >5 contralateral rotations per minute following the administration of apomorphine (0.5 mg/kg sc in 0.02% ascorbic acid) over 40–60 min. It should be noted that apomorphine can sensitize the circling response and should be avoided if the animals are intended for use in studies of striatal sensitivity; in addition, compared to amphetamine, apomorphine is less responsive to dopamine replacement measures. Compounds inducing contralateral rotations, such as direct acting DA agonists and l-DOPA, are predicted to have antiparkinsonian effects, while a reduction in amphetamine-induced ipsilateral rotations is frequently used to demonstrate improved DAergic function on the lesioned side following cell transplantation or treatment with viral vectors.10,11 Spontaneous Forelimb Test (Cylinder Test) The cylinder test evaluates forelimb akinesia by determining the animals’ ability to support their body weight against the wall of a glass cylinder during explorative behavior. The cylinder test was first

CHAPTER 34 / ANIMAL MODELS OF PARKINSON’S DISEASE

described by Schallert and Lindner12 and further modified by Schallert and Tillerson.7 The animals are placed in a transparent glass cylinder (approximately 20 cm in diameter for rats and 10 cm for mice) and videotaped; two mirrors are placed at a 90° angle behind the cylinder to allow visualization of the whole surface. An observer blinded to the treatment of the animals counts the number of independent weight-bearing touches made with each forelimb, usually expressed as percent touches with the lesioned paw of the total performed. A normal animal will, on average, achieve a score of 50% (Figure 34–2A, “normal”), while animals with DA depletion of the nigrostriatal fibers will exhibit a marked reduction in the use of the contralateral paw (Figure

Figure 34–2. Photographic demonstration of (A) the cylinder test of forepaw asymmetry. A “normal” rat will use both forepaws to explore the cylinder, but a 6-OHDA “lesion” will cause a predominant use of the paw on the ipsilateral side to the lesion. (B) The stepping test, in which normal rats (A, A′, A″) will make adjusting steps with the weight-bearing forepaw as it is moved across a surface, but a lesioned animal (B, B′, B″) will not. (C) A rat in the pawreaching “staircase” apparatus.

315

34–2A, “lesion”). To improve the sensitivity of this test Schallert and Tillerson7 describe a more detailed analysis where the first touches during rearing and landing and differential counting of simultaneous versus lateralized paw use are separately scored. There are several advantages of the cylinder test, in that no drug administration is required, food restriction is not necessary, and there is no or limited handling by the experimenter, reducing the influence of external parameters. However, especially where repeat testing is necessary, the animals can become habituated and more reticent to explore the cylinder. To encourage a good performance, the test should be carried out in the latter part of the light cycle, and the following can also be applied: (1) turning the light off and/or switching the light on and off two or three times (requires a video camera with infrared recording capability), (2) mildly shaking the cylinder for a few seconds, (3) removing the animals from the cylinder for 10–30 sec and returning them, and (4) exposing the animals to female/male odors or mild stressors. Stepping Test The stepping test forces the use of individual forelimbs and determines the rats’ ability to perform adjusting steps when moved sideways. The design was first described by Schallert et al.13 and further defined by Olsson et al.14 The experimenter supports the animal lightly round the waist, restraining the hind limbs and the forelimb that is not being tested. The animal is lowered to the table surface and the free forepaw is allowed to provide placing support. The animal is then moved sideways over a 90 cm surface, from one edge to the other, over about 5 sec, during which time the tested paw is touching the surface (Figure 34–2B). The number of steps, while the animal is moved over the surface, is counted. The test is repeated twice per day for three consecutive days for each forepaw in both a forehand and backhand direction. Prior to testing, a habituation trial period of 3–7 days is required to ensure that the animals perform the test properly and to give consistent results. A normal rat will perform about 9–13 adjusting steps in the forehand direction and a few more, 12–16, in the backhand direction. Rats with extensive 6-OHDA lesions of the nigrostriatal pathway (>80%) show a marked reduction in adjusting steps for the contralateral forepaw to only 0– 24,14,15 (Figure 34–2B, B′). Stepping in the backhand direction tends to be much less affected than stepping in the forehand direction, and may be only marginally impaired in rats with less completed intrastriatal 6-OHDA lesions. Intermediate 6-OHDA lesions, 50–80%, will make intermittent forelimb steps, while a lesion of less than 40% will not significantly alter forelimb stepping.4,15 In conducting this test it should be noted that a subjective element is introduced when there is direct interaction between experimenter and animal. This makes comparing the results between different experimenters and studies difficult and the same experimenter should conduct all the testing within a study. Other factors such as strain used, testing environment, and stress can also influence the rats’ performance. Skilled Forelimb Use in the Staircase Test This test, also called the paw reaching test, was introduced by Montoya et al.16 to independently evaluate individual forepaws in a skilled test and is based on the animals’ ability to successfully retrieve and eat sugar pellets with different levels of difficulty. The apparatus is generally a transparent Plexiglas box with a central platform separating two staircases such that the animal can reach down into the staircase on each side only with the forepaw on the same side (Figure 34–2C). This configuration allows the animal’s performance to be assessed separately for the two forelimbs without

316

SECTION IV / MODELS FOR SPECIFIC PURPOSES

having to restrain the animal. Each staircase has a well on each step in which a predefined number of sugar pellets is placed (between 2 and 10 are used in different laboratories). The animals are placed in the box and allowed to collect and eat the pellets for 10– 15 min.4,16 The number of pellets taken from each well and the number of displaced (“missed”) pellets are counted, giving the total number of successfully eaten pellets for each forepaw. For the animals to perform well in this task they should be food deprived prior to starting the test (1–3 days) and be maintained at around 90% of their body weight during the testing period; sugar pellets can be introduced as food during this time. On the first day of testing, the animals generally explore the test box and begin to associate the task with the sugar pellets, and with repeated sessions on subsequent days begin to learn the task. The time required for training varies between groups of animals and can be up to 2 weeks in older rats before a stable performance is reached. When more than 50% of DA is lost in the SNc and greater than 80% in the striatum4,17,18 the animals start to show significant deficits. Complete unilateral 6-OHDA lesions reduce performance in this test by about 50–70% taken pellets and 30–50% eaten pellets compared to intact rats.14,16,17,19,20 It should be noted that lesioned animal usually improve on their intact paw and retrieve up to 10% more pellets than intact control animals. The test can also be used as a forced paw choice test, where sugar pellets are placed only on one side of the staircase, forcing the use of the paw on that side.21 Other less widely used but validated tests include the rotarod and forelimb-placing test. The rotarod is a rotating cylinder on which the rats (or mice22) are trained to walk. There are two main approaches: the first uses a fixed-speed protocol in which rats perform on set speeds (increments between 4 and 40 rpm) and their time to stay on the drum at different speeds is measured. In the second approach accelerating paradigms have the rod increasing in speed (4–44 rpm) at a fixed rate (increasing over 90– 300 sec)23–25; the time at which the rat falls off the drum is recorded and can be directly related to a speed. A comparative study between the two protocols found that the fixed-speed assay was more predictive of therapeutic efficacy, but accelerating protocols could be used more efficiently and easily to test lesion extent.23 Again, animals do require some training on this apparatus to reach a stable performance in the task before any testing can be carried out, but this can be as little as one or two training sessions.25 These tests were primarily developed in rats, but have been adapted for other species, most notably mice and primates. Thus, unilateral nigrostriatal lesions induce similar postural and rotation asymmetries in mice, although their size typically makes it easier to monitor rotation by direct observation or using automated video tracking devices than mechanical rotometers.22 Similarly, mice show performances akin to rats in staircase and other paw-reaching tasks with the apparatus scaled down to be suitable for the smaller species.26 Whereas the effects of DAergic lesions in large primates have been more extensively explored using peripheral administration of MPTP (see below), the brains of small New World marmoset monkeys are sufficiently consistent to allow stereotaxic positioning using external reference landmarks to produce reliable nigrostriatal lesions with 6-OHDA, which, as in rats, produce a range of lateralized motor and sensorimotor impairments. Rotation is less consistent because these monkeys typically move in a three-dimensional space, but contralateral postural and head-turning biases are prominent and easy to measure by video recording.27 Moreover, Marshall, Ridley, and

Kendall have developed primate analogues of the staircase test in which the monkeys reach through slots behind transparent vertical baffles that constrain them to use just one arm in reaching to retrieve palatable foods from staircase wells behind the barrier.28,29 In each of these tasks, marmosets exhibit lateralized changes in response to unilateral nigrostriatal lesions and to pharmacological and cellular therapeutic interventions directly comparable to rats.30,31

1-METHYL-4-PHENYL-1,2,3,6-TETRAHYDROPYRIDINE A byproduct of the illicit manufacture of a pethidine analogue, MPTP was identified as the cause of rapidly developing parkinsonism in a group of drug addicts in the United States in the early 1980s.32 Postmortem analysis revealed that MPTP had induced a self-perpetuating cell death of DAergic neurons in the SNc, with few other anatomical changes. It was found to have similar effects in nonhuman primates;33–35 this has developed into what is currently considered the best available model for PD, providing both a similar behavioral profile and neuropathology. MPTP has also been used in a wide range of other species from the mouse to the worm and zebra fish. In a series of enzyme steps, non-DAergic cells such as glia and serotonergic neurons convert MPTP into the active metabolite MPP+. It is then released into the synaptic space and subsequently taken up by DAergic neurons through the DA transporter. Although the mechanism through which it induces selective DAergic cell death has not been fully elucidated, it is known to act as an inhibitor of complex I of the mitochondrial respiratory chain, producing a decrease in tissue ATP content and an increase in oxidative stress. 1-METHYL-4-PHENYL-1,2,3,6-TETRAHYDROPYRIDINETREATED PRIMATE The behavioral effects of MPTP have been well characterized in various nonhuman primates including the squirrel, rhesus, and cynomolgus monkey, and the common marmoset.33–35 The progression of the degeneration can be controlled by the paradigm of administration. Most protocols use a relatively acute regime of administration for convenience, but a more prolonged administration of regular small doses of weeks or months is required to reproduce the slow progressive deterioration and the absence of spontaneous recovery seen in patients. The mode of administration to give a bilateral lesion can be subcutaneous, intraperitoneal, intravenous, or intramuscular. For example, a common acute bilateral paradigm in marmosets is the subcutaneous administration of MPTP at 2 mg/kg over 5 days.34 With this regime the nigrostriatal DAergic cell loss is upward of 90%, representing late stage PD. After a few days of treatment the primates show akinesia, limb rigidity, reduced vocalization in the marmosets, and reduced grooming. Although resting tremor is not common, it can occasionally be seen as a whole body tremor. A regime of 1 mg/kg for 3 days has also been described recently in the marmoset, in which a subthreshold DA loss produces more limited behavioral deficits that more closely represent presymptomatic to early stage disease states.36 The more chronic treatment protocols that have been described use between 0.2 and 4.5 mg/kg MPTP repeatedly over weeks or months.37–39 However, there appears to be an optimum treatment regime to create a stable cell death, as recovery of the DAergic losses has been described with protocols of 29 days,37 while with 15 days a reportedly nonrecoverable DAergic deficiency is induced.38 MPTP is highly toxic, and the literature reports a number of cases of permanent neurological impairment in laboratory person-

CHAPTER 34 / ANIMAL MODELS OF PARKINSON’S DISEASE

nel working with it.40 Supreme care should therefore be taken when working with MPTP, typically to category III standard, to avoid self-administration or any contact with animal products during the administration period and for a washout period of a few days afterward. The animals require careful observation over several weeks following administration and hand feeding is advisable until the animals can eat unaided and sustain their body weight. Occasionally antiparkinsonian medication such as lDOPA is given to increase their ability for self-care. The bilateral MPTP-treated primate model is commonly used to screen pharmaceuticals for their antiparkinsonian potential. Following treatment, animals are observed either in their home cages or in specialized locomotor cages to independently determine their activity levels, while subjective rating scales for activity and disability are also used. Several different scales are used by different research groups, each with its own criteria and grading system, often based on clinical rating scales such as the UPDRS (United Parkinson’s Disease Rating Scale). Many of these scales include criteria such as bradykinesia, freezing, rigidity, tremor, alertness, reaction to external stimuli/interaction, posture, balance/ coordination, and vocalization (for comparisons of different scales see Imbert et al.41). In Old World primates such as the macaque and rhesus monkeys, the toxin may be administered though the intracarotid artery, giving a predominantly unilateral syndrome. This surgery is more invasive but, as with bilateral versus unilateral lesion with 6-OHDA, the unilateral lesion results in improved survival and requires less posttreatment care. The behaviors are evaluated using rating scales similar to the bilateral model but there are individual scores for the gross motor skills for each limb. The fine motor skills of each forelimb can also be evaluated in food retrieval tasks.42,43 1-METHYL-4-PHENYL-1,2,3,6-TETRAHYDROPYRIDINETREATED MOUSE Rats have a relative insensitivity to MPTP that may be the result of a species-specific metabolism or sequestration of MPP+.44 Very high doses are required to induce DAergic cell death compared to mice and the administration of guanethidine is required to prevent peripheral catecholamine release and mortality.45 Mice, however, are susceptible to the effect of MPTP and have provided a useful small animal model of MPTP-induced parkinsonism. There are caveats to this model, as mice are not as sensitive as primates to the toxin and susceptibility is also hugely variable between mouse strains. Furthermore, with some treatment regimes, cell death is not complete and the mice can show a spontaneous functional recovery. This is more often seen with acute paradigms, while longer-term treatment may provide a more stable DAergic loss. The toxin is usually injected subcutaneously (although it can be given via other routes) typically in the range of 2–45 mg/kg/day for up to 10 days (for a full summary see Seledis et al.46). As in the primate, this produces a bilateral lesion and therefore the behavioral tests that are applied are distinct from those used for the unilateral 6-OHDA lesioned rodents.

BEHAVIOR TESTS WITH THE 1-METHYL-4PHENYL-1,2,3,6-TETRAHYDROPYRIDINETREATED MOUSE ACTIVITY Depending on the paradigm of administration, MPTP-treated mice may develop a hypokinesia that can be assessed using simple tests of locomotor activity and catalepsy.

317

The simplest test of akinesia involves placing the animal of a flat surface and timing how long it takes to move all four limbs (akinesia), while for catalepsy the animals are placed with their fore or hind limbs supporting them on an elevated block or a raised bar, and the latency to restore normal posture is recorded.1 More generally, activity can be monitored in a large square open field (typically 100 cm × 100 cm). In the manual version of the task, the field is divided into squares by lines on the floor and the number of squares entered in a fixed time interval is recorded by direct observation. Frequently, the total counts are subdivided into central squares and squares adjacent to the wall of the open field, as an index of exploratory activity (more anxious animals will spend more time at the edge). The open field test can be readily automated by mounting an array of infrared beams to traverse the open field or a standard cage. The number of beam breaks is used to calculate the time spent locomoting and the total distance traveled. In addition, the location and pattern of beam breaks can also be interpreted, i.e., whether the central beams are broken or just in the peripheral areas of the cage, or if single beams are repeatedly broken, indicating stereotypic behaviors, and with a decline of counts over time taken as an index of “habituation.” Higher beams can also be used to measure vertical rearing activity, as a second measure of exploration. As mice are nocturnal, better results are achieved if they are tested during the dark cycle, as their spontaneous activity is higher. Placing a novel object in a center square of the open field and recording the rise in the time spent and number of entries in the squares immediately adjacent to the object can further assay exploration more directly. POLE TEST The pole test47 is very sensitive to changes in nigrostriatal function as it requires maneuvering and grip function. The mouse is placed head up near the top of a vertical pole (1 cm in diameter, approximately 50 cm in height) and the time to rotate 180° and time to descend are used as measures of motor function. Similarly, tests of grip strength are also sensitive to changes in nigrostriatal DA. The rotarod, as described above, in the 6-OHDA rat model has been very widely used for assessing motor abnormalities in mutant mice.1,48 However, MPTP has been reported to have variable effects on the rotarod,46 and the use of different protocols in the administration of the toxin as well as in the ways the rotarod test is conducted makes comparisons difficult. Other tests used in the evaluation of motor function in the MPTP-treated mouse include swim tests (set in a narrow tank of water at 22–25°C for 30 min) assessing active swimming versus passive floating over a period of time both in the presence and absence of an escape platform, grip strength, and apomorphine-induced climbing.

DYSKINESIA IN ANIMAL MODELS OF PARKINSON’S DISEASE The gold standard for pharmacological dopamine replacement in PD is l-DOPA, the metabolic precursor to DA. This is a highly effective treatment, but within 10 years of initiating l-DOPA therapy abnormal involuntary movements (AIMs), known as dyskinesia, develop in most patients limiting its use.49 Thus, a major area of research using animal models of PD has involved identifying the cause of dyskinesia and evaluating the potential of new compounds to elicit, exacerbate, or reduce this side effect. DYSKINESIA TESTING IN THE 6-HYDROXYDOPAMINELESIONED RAT The ability of drugs to induce contralateral rotations in the 6-OHDA-lesioned rat is taken as an indicator of

318

SECTION IV / MODELS FOR SPECIFIC PURPOSES

Table 34–1 The amplitude ratings for the abnormal involuntary movement scale in rodents given in addition to scores of durationa Score

Axial

Limb

Orolingual

1

Persistent deviation of the head to a 30˚ angle

2

Lateral deviation of the head and neck at an angle between 30˚ and 60˚ Lateral deviation of greater than 60˚ without loss of balance Twisting of the head and neck such that balance is lost

Small movements of the paw and distal forelimb around a fixed position Low-amplitude movements of the distal and proximal forelimb Translocation of the whole limb with the shoulder muscle Vigorous limb and shoulder movements to their maximum extent

Chewing and repeated jaw movements With tongue protrusion

3 4

N/A N/A

a

Axial and limb movements are scored 1–4 and orolingual 1–2.

their antiparkinsonian potential, but with repeated administration the circling in response to l-DOPA changes in profile.50 This sensitivity of the locomotor response was considered an indicator of dyskinesiogenic potential. However, this model has been further developed by the extensive characterization of the simultaneous development of abnormal involuntary movement of the torso, fore and hind limbs, and orolingual area; this is considered to be rodent correlates of the l-DOPA-induced dyskinesia seen in patients.51,52 Rating scales have been devised for these behaviors based on their duration and the magnitude to which they are expressed. To evaluate these behaviors, rats are individually observed in sawdust-free, transparent cages without food or water. Each animal is injected with the compound of interest at 1 min intervals and then observed at regular intervals (10–30 min) for 1–2 min each and rated. Some researchers prefer to take fewer time points and video the behaviors, permitting scores of additional parameters such as head nodding, and also to distinguish between choreic and dystonic movements of the forelimb. However, care should be taken to ensure that enough time points are evaluated as differences between single time points could be attributed to shifts in the time course, rather than a change in the severity of the response. There is a three-tier 1–4 point rating scale based on the duration, class, and amplitude of dyskinetic behaviors, as follows for the duration of behaviors of each class: 1. The behavior is present for less than half the observation time. 2. The behavior is present for more than half but not all the time. 3. The behavior is continuously present but interruptible by external stimuli, e.g., a sharp rap on the outside of the cage. 4. The behavior is uninterruptible by external stimuli. Scores for amplitude in each category are listed in Table 34– 1.20 Duration and amplitude ratings are combined to give an overall score of severity for each category at each time point, which were accumulated to give a cumulative severity score. The same methodology and rating scales can also be applied to 6-OHDA-lesioned mice.53 DYSKINESIA TESTING THE 1-METHYL-4-PHENYL1, 2, 3, 6-TETRAHYDROPYRIDINE-TREATED MARMOSET The MPTP-treated primate has been extensively used to determine the neural mechanisms underlying the development of lDOPA-induced dyskinesia and to assess the dyskinesiogenic potential of new compounds in addition to the antiparkinsonian

activity. The dyskinesia takes the form of choreic movements of the fore and hind limbs, athetosis, and dystonic posturing and a separate dyskinesia rating scale based on the clinical UPDRS is used. The scoring occurs at repeated intervals (generally every 30 min), often for the duration of drug action, on several days during the treatment regime. A rater blind to the treatment received by each animal, as these are subjective scales, should carry these out.

CHOOSING THE RIGHT MODEL UNILATERAL VERSUS BILATERAL LESIONS 6-OHDA and to a certain extent MPTP can produce either unilateral or bilateral lesions. PD generally affects patients on both sides, although the symptoms are generally worse on one side. In the early stages of disease, PD is often observed to be asymmetric with greater symptoms on one side, but the degree of lateralization observed in extensive unilateral lesions in animals is seldom seen, with both sides affected significantly in advanced disease. Therefore the bilateral models can be seen as more closely representative of the human disease. However, generally the bilateral lesions are more problematic in terms of the health of the animal. Extensive bilateral lesions in rats, whether induced by intraventricular or intracerebral injections of 6-OHDA, typically result in profound inanition, with the animals failing to eat, drink, or groom spontaneously, requiring prolonged and intensive care to maintain basic health status.54 By contrast, both primates and rodents with unilateral lesions can feed and drink adequately to sustain their own body weight and maintain basic health, although MPTP-treated primates with severe bilateral lesions will often require feeding by hand for the first few weeks following treatment and may require antiparkinsonian medication. Partial, bilateral, striatal, and nigral 6-OHDA lesions have been used successfully and the rats are then able to sustain themselves,55,56 but because the deficit is then bilateral, a different combination of behavior tests is then used including open field and operant tests such as the reaction time task (this is described in more detail elsewhere).8,56 In terms of the behavioral tasks that can be used to assess motor function and recovery, in the bilateral primate models especially, qualitative observational parameters are predominantly used to evaluate changes in motor function. In contrast, the tests that utilize the asymmetry of the unilateral models generally provide a more quantitative assessment. A further advantage to the unilateral model is that it enables comparisons of the physiological events in damaged and intact hemispheres, allowing functional deficits to be related to physiological changes. The intact

CHAPTER 34 / ANIMAL MODELS OF PARKINSON’S DISEASE

side may therefore be used as an internal control, but caution must be exercised, as there are some changes to the intact side as a result of the lesion. 6-HYDROXYDOPAMINE VERSUS 1-METHYL-4-PHENYL1,2,3,6-TETRAHYDROPYRIDINE MPTP is a highly dangerous, lipophilic toxin and special care has to be taken in all aspects of handling and injecting it and when cleaning out the cages during and shortly after the toxin-administration period. 6-OHDA should also be handled with care, but the main concern, as detailed above, is in keeping the toxin fresh. MPTP (in the bilateral model) can be given as a simple systemic injection and in the unilateral model by peripheral intracarotid injection, but 6-OHDA must be centrally injected by stereotactic surgery, which requires a greater level of skill and equipment, is more time consuming, and because of the accuracy of stereotactic placement adds an additional degree of variability. Ultimately, however, central injection of either toxin is required to achieve extensive unilateral lesion in the absence of any depletion on the contralateral side. Using the modes of administration described above, both 6OHDA and MPTP are relatively selective for the DAergic nigrostriatal pathway. Indeed, although 6-OHDA can also be taken up by and can kill noradrenergic neurons, there is only sparse noradrenergic innervation of the striatum such that these lesions should have little impact. If this is a concern, desipramine can be administered 30 min prior to surgery, which will block the noradrenaline transporter, thereby increasing the selectivity for DAergic neurons. The actions of MPTP and 6-OHDA on the serotonin system have been debated, and both toxins have been claimed to leave the serotonin system unaffected, induce serotonergic sprouting, or decrease levels of serotonin in the striatum following DAergic denervation.57–60 This may depend on the mode of administration in the case of 6-OHDA (striatal vs. mfb) or the time course of treatment for MPTP.37,61,62 One of the pathological hallmarks of PD is the presence of intracellular inclusions termed Lewy bodies, now known to be attributable to the abnormal aggregation of the α-synuclein protein.63 The administration of 6-OHDA does not result in the development of inclusions. However, MPTP has been reported to induce protein accumulation in the SN of aged primates that resembles some features of Lewy bodies.64–66 RODENT VERSUS PRIMATE The main considerations in deciding between rodent and primate models are both ethical and practical. Both legislative and welfare considerations require use of the least advanced species to address the specific experimental hypotheses. Moreover, primates are more expensive and have limited availability, and the level of care is higher than for rodents as they require larger and more specialized housing facilities.67 In addition, larger equipment for recording activity is also required, while rodents are easier to handle, house, and care for. Nevertheless, some experimental questions can be addressed only in the larger primate brain. A major advantage of the use of nonhuman primates, especially in the testing of pharmaceuticals, is their phylogenetic similarity to humans and the expression of PD pathology. Rodents do not have a distinguishable caudate and putamen, which are instead combined in a singular structure, the neostriatum, and thus in this regard nonhuman primates have a closer structural arrangement to humans. Aside from the pathology, the expression of parkinsonian symptoms in primates is more similar to the movement disorder in humans, and so has greater face validity in preclinical studies. Indeed many of the movement

319

disorder rating scales used in primates are adapted from those used clinically to assess patients. By contrast, while many of the behaviors observed in rodents are less clearly recognizable as “parkinsonism,” there are multiple behavioral tasks that can be applied in order to probe different components of the underlying motor deficits such as neglect versus motor function. When comparing rodent species, rats are larger, more docile, and more amenable to both surgical manipulation and behavioral analysis than are mice. Nevertheless, there has been greater interest in recent years to developing mouse models also, because of the greater numbers of mutant strains and the opportunities provided by new transgenic and knockout technologies to probe specific genetic and molecular influences on degeneration and behavior. OTHER TOXIN MODELS One of the earliest toxins to be used in PD research is reserpine, a toxin that induces the release of catecholamines from their stores in the nerve terminals. This induces a hypokinetic state in most rodents and drugs can then be administered systemically or into selected brain regions through implanted cannulas. Reduced akinesia and increased activity and grooming can be observed when drugs are administered systemically, or the induction of a locomotor side bias when drugs are injected unilaterally. However, unlike the disease state, the depletion of the catecholamine stores and thus the resulting behavioral deficits are only temporary, and the stores are replenished and locomotor activity restored over a few days. It is therefore difficult to investigate experimental deficits such as dyskinesia that accumulate over time, or to evaluate chronic therapeutic treatments against a stable baseline. Furthermore, there are no morphological effects on the DAergic neurons, no permanent cell death or damage is observed, and no inclusions are documented. There is no selectivity for nigrostriatal as opposed to mesolimbic DAergic neurons, and other monoamine neurotransmitters are also severely depleted. Despite these failings, this model has been successfully used to evaluate pharmacotherapeutics and was fundamental in demonstrating the efficacy of l-DOPA in restoring motor function following DA depletion.68 In the past few years, models have been proposed based on rotenone and paraquat, two agricultural chemicals that have both been associated with an increased incidence of PD. However, these models produce a high mortality and decidedly variable results, and the induced degeneration is not specific to the SNc.69–72 More recently, abnormalities of protein processing by the proteosome have been identified as a possible mechanism for inducing PD and the accumulation of proteins such as α-synuclein. McNaught et al.73 reported that chronic administration of PSI, a synthetic peptide aldehyde that inhibits proteasomal function, reproduced both the motor and pathological consequences of PD in rats. They describe specific degeneration of dopaminergic neurons, proteinous deposits akin to Lewy bodies, in addition to bradykinesia, tremor, and rigidity. However, although similar results have been reported by other researchers,74 equally many have not been able to reproduce these findings.75,76 Each of these models still has to be fully and reliably characterized before being used to evaluate therapeutic or neuroprotective approaches to the treatment of PD.

CONCLUSIONS 6-OHDA and MPTP are the tools most frequently used to induce selective and effective degeneration of the DAergic nigrostriatal pathway in experimental animals, and thereby model the

320

SECTION IV / MODELS FOR SPECIFIC PURPOSES

primary pathology and symptoms of PD in the laboratory. In deciding which model to use, the focus should be on first selecting the most appropriate model and, more importantly, the behavioral tests most appropriate for that model for the specific experimental hypotheses or preclinical strategies to be evaluated. The alternatives will be weighed according to a range of criteria involving dimensions of practicality, efficiency, validity, and animal welfare. Generally, several tests are used to determine the generality of results and the extent and limitations of their application. Treatments may not necessarily improve function in all of them. For example, transplants of DAergic tissue produce improved forepaw asymmetry in the cylinder test but not in the more complex pawreaching task. However, while the 6-OHDA-lesioned rat and MPTP-treated primate are well-established models of PD that have provided considerable insight into the disease, it should be remembered that both still have significant limitations and the search continues for a model that can not just reproduce the neuropathology and symptoms of the disease, but also recreate more faithfully the progressive pathogenic aspects of the disease.

ACKNOWLEDGMENT The authors would like to thank Bengt Mattson for providing the figures.

REFERENCES 1. Meredith GE, Kang UJ. Behavioral models of Parkinson’s disease in rodents: A new look at an old problem. Mov Disord 2006;21(10): 1595–1606. 2. Ungerstedt U. 6-Hydroxy-dopamine induced degeneration of central monoamine neurons. Eur J Pharmacol 1968;5(1):107–110. 3. Glinka YY, Youdim MB. Inhibition of mitochondrial complexes I and IV by 6-hydroxydopamine. Eur J Pharmacol 1995;292(3–4): 329–332. 4. Kirik D, Rosenblad C, Bjorklund A. Characterization of behavioral and neurodegenerative changes following partial lesions of the nigrostriatal dopamine system induced by intrastriatal 6-hydroxydopamine in the rat. Exp Neurol 1998;152(2):259–277. 5. Visanji NP, O’Neill MJ, Duty S. Nicotine, but neither alpha4beta2 nor alpha7 nAChR subtype selective agonists, protects against a partial 6-hydroxydopamine lesion of the rat nigrostriatal tract. Neuropharmacology 2006;51(3):506–516. 6. Schwarting RK, Huston JP. The unilateral 6-hydroxydopamine lesion model in behavioral brain research. Analysis of functional deficits, recovery and treatments. Prog Neurobiol 1996;50(2–3):275–331. 7. Schallert T, Tillerson JL. Intervention strategies for degeneration of dopamine neurons in parkinsonism: Optimising behavioural assessment of outcome. In: Innovative Models of CNS Disease: From Molecule to Therapy. Clifton, NJ: Humana Press, 1999:131–151. 8. Deumens R, Blokland A, Prickaerts J. Modeling Parkinson’s disease in rats: An evaluation of 6-OHDA lesions of the nigrostriatal pathway. Exp Neurol 2002;175(2):303–317. 9. Ungerstedt U, Arbuthnott GW. Quantitative recording of rotational behaviour in rats after 6-hydroxy-dopamine lesions of the nigrostriatal dopamine system. Brain Res 1970;24(3):485–493. 10. Kirik D, Georgievska B, Burger C, Winkler C, Muzyczka N, Mandel RJ, Bjorklund A. Reversal of motor impairments in parkinsonian rats by continuous intrastriatal delivery of L-dopa using rAAV-mediated gene transfer. Proc Natl Acad Sci USA 2002;99(7):4708–4713. 11. Dunnett SB, Bjorklund A, Stenevi U, Iversen SD. Behavioural recovery following transplantation of substantia nigra in rats subjected to 6-OHDA lesions of the nigrostriatal pathway. II. Bilateral lesions. Brain Res 1981;229(2):457–470. 12. Schallert T, Lindner MD. Rescuing neurons from trans-synaptic degeneration after brain damage: Helpful, harmful, or neutral in recovery of function? Can J Psychol 1990;44(2):276–292.

13. Schallert T, De Ryck M, Whishaw IQ, Ramirez VD, Teitelbaum P. Excessive bracing reactions and their control by atropine and LDOPA in an animal analog of parkinsonism. Exp Neurol 1979;64(1): 33–43. 14. Olsson M, Nikkhah G, Bentlage C, Bjorklund A. Forelimb akinesia in the rat Parkinson model: Differential effects of dopamine agonists and nigral transplants as assessed by a new stepping test. J Neurosci 1995;15(5Pt. 2):3863–3875. 15. Chang JW, Wachtel SR, Young D, Kang UJ. Biochemical and anatomical characterization of forepaw adjusting steps in rat models of Parkinson’s disease: Studies on medial forebrain bundle and striatal lesions. Neuroscience 1999;88(2):617–628. 16. Montoya CP, Campbell-Hope LJ, Pemberton KD, Dunnet SB. The “staircase test”: A measure of independent forelimb reaching and grasping abilities in rats. J Neurosci Methods 1991;36(2–3):219– 228. 17. Barneoud P, Parmentier S, Mazadier M, Miquet JM, Boireau A, Dubedat P, Blanchard JC. Effects of complete and partial lesions of the dopaminergic mesotelencephalic system on skilled forelimb use in the rat. Neuroscience 1995;67(4):837–848. 18. Lee CS, Sauer H, Bjorklund A. Dopaminergic neuronal degeneration and motor impairments following axon terminal lesion by instrastriatal 6-hydroxydopamine in the rat. Neuroscience 1996;72(3): 641–653. 19. Nikkhah G, Duan WM, Knappe U, Jodicke A, Bjorklund A. Restoration of complex sensorimotor behavior and skilled forelimb use by a modified nigral cell suspension transplantation approach in the rat Parkinson model. Neuroscience 1993;56(1):33–43. 20. Winkler C, Bentlage C, Nikkhah G, Samii M, Bjorklund A. Intranigral transplants of GABA-rich striatal tissue induce behavioral recovery in the rat Parkinson model and promote the effects obtained by intrastriatal dopaminergic transplants. Exp Neurol 1999;155(2): 165–186. 21. Dobrossy MD, Dunnett SB. Training specificity, graft development and graft-mediated functional recovery in a rodent model of Huntington’s disease. Neuroscience 2005;132(3):543–552. 22. Iancu R, Mohapel P, Brundin P, Paul G. Behavioral characterization of a unilateral 6-OHDA-lesion model of Parkinson’s disease in mice. Behav Brain Res 2005;162(1):1–10. 23. Monville C, Torres EM, Dunnett SB. Comparison of incremental and accelerating protocols of the rotarod test for the assessment of motor deficits in the 6-OHDA model. J Neurosci Methods 2006;158(2): 219–223. 24. Lundblad M, Vaudano E, Cenci MA. Cellular and behavioural effects of the adenosine A2a receptor antagonist KW-6002 in a rat model of l-DOPA-induced dyskinesia. J Neurochem 2003;84(6):1398–1410. 25. Rozas G, Guerra MJ, Labandeira-Garcia JL. An automated rotarod method for quantitative drug-free evaluation of overall motor deficits in rat models of parkinsonism. Brain Res Brain Res Protoc 1997;2(1):75–84. 26. Baird AL, Meldrum A, Dunnett SB. The staircase test of skilled reaching in mice. Brain Res Bull 2001;54(2):243–250. 27. Annett LE, Rogers DC, Hernandez TD, Dunnett SB. Behavioural analysis of unilateral monoamine depletion in the marmoset. Brain 1992;115(Pt. 3):825–856. 28. Kendall AL, Rayment FD, Torres EM, Baker HF, Ridley RM, Dunnett SB. Functional integration of striatal allografts in a primate model of Huntington’s disease. Nat Med 1998;4(6):727–729. 29. Marshall JW, Ridley RM. Assessment of functional impairment following permanent middle cerebral artery occlusion in a non-human primate species. Neurodegeneration 1996;5(3):275–286. 30. Annett LE, Martel FL, Rogers DC, Ridley RM, Baker HF, Dunnett SB. Behavioral assessment of the effects of embryonic nigral grafts in marmosets with unilateral 6-OHDA lesions of the nigrostriatal pathway. Exp Neurol 1994;125(2):228–246. 31. Eslamboli A, Cummings RM, Ridley RM, Baker HF, Muzyczka N, Burger C, Mandel RJ, Kirik D, Annett LE. Recombinant adenoassociated viral vector (rAAV) delivery of GDNF provides protection against 6-OHDA lesion in the common marmoset monkey (Callithrix jacchus). Exp Neurol 2003;184(1):536–548.

CHAPTER 34 / ANIMAL MODELS OF PARKINSON’S DISEASE

32. Langston JW, Ballard P, Tetrud JW, Irwin I. Chronic parkinsonism in humans due to a product of meperidine-analog synthesis. Science 1983;219(4587):979–980. 33. Burns RS, Chiueh CC, Markey SP, Ebert MH, Jacobowitz DM, Kopin IJ. A primate model of parkinsonism: Selective destruction of dopaminergic neurons in the pars compacta of the substantia nigra by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Proc Natl Acad Sci USA 1983;80(14):4546–4550. 34. Jenner P, Rupniak NM, Rose S, Kelly E, Kilpatrick G, Lees A, Marsden CD. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism in the common marmoset. Neurosci Lett 1984;50(1–3): 85–90. 35. Langston JW, Forno LS, Rebert CS, Irwin I. Selective nigral toxicity after systemic administration of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) in the squirrel monkey. Brain Res 1984;292(2): 390–394. 36. Iravani MM, Syed E, Jackson MJ, Johnston LC, Smith LA, Jenner P. A modified MPTP treatment regime produces reproducible partial nigrostriatal lesions in common marmosets. Eur J Neurosci 2005; 21(4):841–854. 37. Russ H, Mihatsch W, Gerlach M, Riederer P, Przuntek H. Neurochemical and behavioural features induced by chronic low dose treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the common marmoset: Implications for Parkinson’s disease? Neurosci Lett 1991;123(1):115–118. 38. Bezard E, Dovero S, Prunier C, Ravenscroft P, Chalon S, Guilloteau D, Crossman AR, Bioulac B, Brotchie JM, Gross CE. Relationship between the appearance of symptoms and the level of nigrostriatal degeneration in a progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaque model of Parkinson’s disease. J Neurosci 2001;21(17):6853–6861. 39. Colosimo C, Granata R, Del Zompo M, Piccardi MP, Perretta G, Albanese A. Chronic administration of MPTP to monkeys: Behavioural morphological and biochemical correlates. Neurochem Int 1992;20(Suppl.):279S–285S. 40. Langston JW, Ballard PA Jr. Parkinson’s disease in a chemist working with 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine. N Engl J Med 1983;309(5):310. 41. Imbert C, Bezard E, Guitraud S, Boraud T, Gross CE. Comparison of eight clinical rating scales used for the assessment of MPTPinduced parkinsonism in the Macaque monkey. J Neurosci Methods 2000;96(1):71–76. 42. Emborg ME, Ma SY, Mufson EJ, Levey AI, Taylor MD, Brown WD, Holden JE, Kordower JH. Age-related declines in nigral neuronal function correlate with motor impairments in rhesus monkeys. J Comp Neurol 1998;401(2):253–265. 43. Collier TJ, Steece-Collier K, Kordower JH. Primate models of Parkinson’s disease. Exp Neurol 2003;183(2):258–262. 44. Sundstrom E, Samuelsson EB. Comparison of key steps in 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in rodents. Pharmacol Toxicol 1997;81(5):226–231. 45. Giovanni A, Sonsalla PK, Heikkila RE. Studies on species sensitivity to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Part 2: Central administration of 1-methyl-4-phenylpyridinium. J Pharmacol Exp Ther 1994;270(3):1008–1014. 46. Sedelis M, Schwarting RK, Huston JP. Behavioral phenotyping of the MPTP mouse model of Parkinson’s disease. Behav Brain Res 2001;125(1–2):109–125. 47. Ogawa N, Hirose Y, Ohara S, Ono T, Watanabe Y. A simple quantitative bradykinesia test in MPTP-treated mice. Res Commun Chem Pathol Pharmacol 1985;50(3):435–441. 48. Crawley JN. Behavioral phenotyping of transgenic and knockout mice: Experimental design and evaluation of general health, sensory functions, motor abilities, and specific behavioral tests. Brain Res 1999;835(1):18–26. 49. Ahlskog JE, Muenter MD. Frequency of levodopa-related dyskinesias and motor fluctuations as estimated from the cumulative literature. Mov Disord 2001;16(3):448–458. 50. Papa SM, Engber TM, Kask AM, Chase TN. Motor fluctuations in levodopa treated parkinsonian rats: Relation to lesion extent and treatment duration. Brain Res 1994;662(1–2):69–74.

321

51. Lundblad M, Andersson M, Winkler C, Kirik D, Wierup N, Cenci MA. Pharmacological validation of behavioural measures of akinesia and dyskinesia in a rat model of Parkinson’s disease. Eur J Neurosci 2002;15(1):120–132. 52. Cenci MA, Tranberg A, Andersson M, Hilbertson A. Changes in the regional and compartmental distribution of FosB- and JunB-like immunoreactivity induced in the dopamine-denervated rat striatum by acute or chronic L-dopa treatment. Neuroscience 1999;94(2): 515–527. 53. Lundblad M, Usiello A, Carta M, Hakansson K, Fisone G, Cenci MA. Pharmacological validation of a mouse model of l-DOPAinduced dyskinesia. Exp Neurol 2005;194(1):66–75. 54. Zigmond MJ, Stricker EM. Proceedings: The effects on ingestive behaviors of damage to central dopamine-containing neurons. Psychopharmacol Bull 1974;10(3):40–41. 55. Sakai K, Gash DM. Effect of bilateral 6-OHDA lesions of the substantia nigra on locomotor activity in the rat. Brain Res 1994;633(1–2):144–150. 56. Amalric M, Moukhles H, Nieoullon A, Daszuta A. Complex deficits on reaction time performance following bilateral intrastriatal 6OHDA infusion in the rat. Eur J Neurosci 1995;7(5):972–980. 57. Karstaedt PJ, Kerasidis H, Pincus JH, Meloni R, Graham J, Gale K. Unilateral destruction of dopamine pathways increases ipsilateral striatal serotonin turnover in rats. Exp Neurol 1994;126(1):25–30. 58. Balcioglu A, Zhang K, Tarazi FI. Dopamine depletion abolishes apomorphine- and amphetamine-induced increases in extracellular serotonin levels in the striatum of conscious rats: A microdialysis study. Neuroscience 2003;119(4):1045–1053. 59. Takeuchi Y, Sawada T, Blunt S, Jenner P, Marsden CD. Effects of 6-hydroxydopamine lesions of the nigrostriatal pathway on striatal serotonin innervation in adult rats. Brain Res 1991;562(2):301–305. 60. Guerra MJ, Liste I, Labandeira-Garcia JL. Effects of lesions of the nigrostriatal pathway and of nigral grafts on striatal serotonergic innervation in adult rats. Neuroreport 1997;8(16):3485–3488. 61. Perez-Otano I, Herrero MT, Oset C, De Ceballos ML, Luquin MR, Obeso JA, Del Rio J. Extensive loss of brain dopamine and serotonin induced by chronic administration of MPTP in the marmoset. Brain Res 1991;567(1):127–132. 62. Pifl C, Schingnitz G, Hornykiewicz O. Effect of 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine on the regional distribution of brain monoamines in the rhesus monkey. Neuroscience 1991; 44(3):591–605. 63. Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert M. Alpha-synuclein in Lewy bodies. Nature 1997;388(6645): 839–840. 64. Kowall NW, Hantraye P, Brouillet E, Beal MF, McKee AC, Ferrante RJ. MPTP induces alpha-synuclein aggregation in the substantia nigra of baboons. Neuroreport 2000;11(1):211–213. 65. Gibb WR, Terruli M, Lees AJ, Jenner P, Marsden CD. The evolution and distribution of morphological changes in the nervous system of the common marmoset following the acute administration of 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Mov Disord 1989;4(1): 53–74. 66. Forno LS, DeLanney LE, Irwin I, Langston JW. Similarities and differences between MPTP-induced parkinsonism and Parkinson’s disease. Neuropathologic considerations. Adv Neurol 1993;60:600– 608. 67. Eslamboli A. Marmoset monkey models of Parkinson’s disease: Which model, when and why? Brain Res Bull 2005;68(3):140–149. 68. Utley JD, Carlsson A. Relative effects of L-DOPA and its methyl ester given orally or intraperitoneally to reserpine-treated mice. Acta Pharmacol Toxicol (Copenh) 1965;23(2):189–193. 69. Brooks AI, Chadwick CA, Gelbard HA, Cory-Slechta DA, Federoff HJ. Paraquat elicited neurobehavioral syndrome caused by dopaminergic neuron loss. Brain Res 1999;823(1–2):1–10. 70. Corasaniti MT, Strongoli MC, Rotiroti D, Bagetta G, Nistico G. Paraquat: A useful tool for the in vivo study of mechanisms of neuronal cell death. Pharmacol Toxicol 1998;83(1):1–7. 71. Höglinger GU, Oertel WH, Hirsch EC. The rotenone model of parkinsonism—the five years inspection. J Neural Transm 2006; (Suppl.)70:269–272.

322

SECTION IV / MODELS FOR SPECIFIC PURPOSES

72. Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT. Chronic systemic pesticide exposure reproduces features of Parkinson’s disease. Nat Neurosci 2000;3(12):1301–1306. 73. McNaught KS, Perl DP, Brownell AL, Olanow CW. Systemic exposure to proteasome inhibitors causes a progressive model of Parkinson’s disease. Ann Neurol 2004;56(1):149–162. 74. Schapira AH, Cleeter MW, Muddle JR, Workman JM, Cooper JM, King RH. Proteasomal inhibition causes loss of nigral tyrosine hydroxylase neurons. Ann Neurol 2006;60(2):253–255.

75. Manning-Bog AB, Reaney SH, Chou VP, Johnston LC, McCormack AL, Johnston J, Langston JW, Di Monte DA. Lack of nigrostriatal pathology in a rat model of proteasome inhibition. Ann Neurol 2006;60(2):256–260. 76. Kordower JH, Kanaan NM, Chu Y, Suresh Babu R, Stansell J 3rd, Terpstra BT, Sortwell CE, Steece-Collier K, Collier TJ. Failure of proteasome inhibitor administration to provide a model of Parkinson’s disease in rats and monkeys. Ann Neurol 2006;60(2): 264–268.

35

Transgenic Animal Models of Neurodegenerative Diseases ANTHONY W.S. CHAN AND YUKSEL AGCA

ABSTRACT

gene transfer technology. Parkinson’s disease (PD), Alzheimer’s disease (AD), and Huntington’s disease (HD) are the most common NDs and affect more than six million people in the United States. Compared to HD, which is caused by the extension of the polyglutamine [poly(Q)] tract at the 5′ region of the huntingtin (htt) gene, PD and AD are more complex.1–6 Extensive effort has been made to determine the genetic and environmental components that lead to complex genetic disorders such as PD and AD.7,8 Candidate genes are often identified in a small number of families that are scattered around the globe.3,7,9 Although candidate genes have been identified, no single gene is unanimously linked to PD and AD. The latest discovery of the leucine-rich repeat kinase 2 (LRRK2) gene has the greatest link to familial parkinsonism (FP), which contributes to only 2–10% of these cases.1,2,9,10 Several mutations have also been identified in the Alzheimer precursor protein (APP) gene that are linked to the development of AD.3,11–13 However, no single mutation leads to neuropathology and cognitive behavioral declines in animal models equivalent to that seen in human patients.3,10 Transgenic animal technology has revolutionized animal modeling, which becomes a very useful tool for elucidating the link between genetics and human diseases.3,10,14–18 The development of a new animal model is not only important for a better understanding of human diseases, but also important for the development of diagnostic tools and treatments.18,19 Unfortunately, animals that carry genetic defects similar to those found in patients do not develop identical patterns.20–21 This suggests fundamental differences between human and other mammalian species such as rodents and nonhuman primates (NHPs).19 Physiological differences include life span, brain complexity, cellular metabolism, and endocrine and reproductive function, which could be the result of genetic redundancy or variations in biochemical pathways.22–24 Hence, a good animal model is not only dependent on the genetic constitution of the transgene but also the genetic and physiological background of the animal species.25 It is particularly important for NDs because of the complexity in genetic composition, the unknown neuropathological mechanisms, and the gradual decline in cognitive and behavioral function. For example, most of the transgenic HD mice do not develop identical neuropathology. Although brain size was reduced by 20%, specific neuronal cell loss was not found in the striatal region or cortex.26 A similar phenomenon was also observed in AD and PD mice. Due to physiological differences between species, the rat has certain advantages over the mouse because of brain

Neurodegenerative diseases are complex disorders involving neuropathological and psychiatric alterations. Transgenic mice expressing mutant human Alzheimer precursor protein (βAPP), α-synuclein, and mutant huntingtin genes have been developed for Alzheimer’s, Parkinson’s, and Huntington’s disease (AD, PD, and HD), respectively. However, none of the existing mouse models completely represents the pathology of these diseases, including neuronal loss, cerebral atrophy, widespread neurofibrillary tangles, and neuropil threads in AD, formation of Lewy bodies and the extensive loss of dopaminergic neurons in PD, and neuronal loss and aggregate formation in HD. Although rat models have been shown to be superior to mouse models because of the brain size and the availability of more sophisticated behavioral testing, a common disadvantage in rodent models is the lack of neurodegeneration. Furthermore, rodents have relatively smaller brains and thus provide a limited behavioral repertoire for assessing transgene-associated structural and cognitive deficits. Nonhuman primates are ideally suited for modeling the neuropathology of neurodegenerative diseases because of their biological proximity to humans, size, and age-related development of lesions. Thus the development of a transgenic nonhuman primate model of neurodegenerative diseases holds greater promise for success in the discovery of diagnoses, treatments, and cures than approaches using other animal species. Nevertheless, it is undeniable that rodent models contribute significantly to the understanding of neurodegenerative diseases and the development of cures. A transgenic nonhuman primate model will be able to mimic human conditions not only physiologically but also genetically. However, the development of transgenic rodent and nonhuman primate models of neurodegenerative diseases is equally important for unraveling the mystery of neurodegeneration and the development of early diagnosis and cures. Key Words: Neurodegenerative disease, Neurodegeneration, Animal model, Transgenic rodent, Transgenic nonhuman primates.

INTRODUCTION Animal models of neurodegenerative diseases (NDs) have been developed quickly in the past decades because of the advancement in molecular genetics and the latest development of From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

323

324

SECTION IV / MODELS FOR SPECIFIC PURPOSES

size, neurocircuitry, and availability of cognitive–behavioral testing.12,13,27–31 Additionally, NHPs are considered the best animal models, especially for NDs, because of the complex correlation between aging and progressive neurodegeneration, which leads to deleterious alterations in behavior and psychiatric status associated with diseases such as AD, PD, and HD in humans.19,32,33 NHPs are the only animal model providing accessibility to a wide range of cognitive–behavioral testing including the possibility for high-resolution brain imaging such as functional magnetic resonance imaging (fMRI) and positron emission tomography (PET).19,32 Thus, a transgenic animal with a genetic alteration that leads to human inherited NDs might best mimic patient conditions; this could increase our understanding of disease development, thereby paving the way for the development of cures.32 Besides the selection of target genes and animal species, promoter is also an important factor for the development of a transgenic model. Promoter regulates the expression of disease-related genes, which affects the etiological pattern of the animals.3,10,16 The R6/2 mouse model of HD has good representation in neuropathology and motor deficits; however, mutant htt expressed under the control of the human htt promoter results in an excessive decline in health and sudden death with no known cause.34–36 On the other hand, transgenic mice that carry mutant htt with 82Q under the control of the prion protein promoter have neuronspecific pathology with distinctive stages of disease development, which mimics the course of HD progression.37 These studies suggest that promoter is important in determining disease development, neuropathology, and cognitive–behavioral patterns in transgenic animal models. Here we will focus on animal model development of AD, PD, and HD as a representative of NDs. Our goal is to provide a brief overview of currently available animal models and new methodology for creating transgenic rat and NHP models.

METHODS FOR CREATING TRANSGENIC ANIMALS The method for creating transgenic animals has a significant impact on the application of the disease model.14,18 In general, transgenic animals can be categorized by the expression pattern, which includes (1) overexpression, (2) gene targeting: knockin, knockout, and knockdown, (3) conditional expression, and (4) inducible expression. Although the expression pattern could be manipulated by strategies such as the tetracycline-inducible system, the Cre-lox conditional expression system, and artificial chromosome (AC), an exogenous gene construct has to be inserted into the target cell genome in order to achieve long-term expression. Additionally, there are three fundamental criteria determining the success of a transgenic animal: (1) germline transmission, (2) stable expression, and (3) all body cells must carry the transgene.38 Two exceptions are chimeric mice generated by blastocyst injection of genetically modified embryonic stem cells and local gene transfer such as stereotaxic viral injection in a selected brain region.33,39–41 Several methods have been used to generate transgenic animals. Among these methods, pronuclear microinjection (PI) is the most commonly used method in creating transgenic mice and rats. A similar method has also been used to generate transgenic livestock; however, somatic cell nuclear transplantation (SCNT; cloning), a recent development, is slowly replacing PI in livestock because more versatile genetic manipulation could be achieved.

Table 35–1 Focal transgenesis versus germline transgenesis Focal transgenesis

Germline transgenesis

Number of transgenic cells Expression pattern

Regional and limited Regional

Inheritance Timeline

No 1–6 months

All body and germ cells Whole body (dependent on promoter) Yes 6–24 months (dependent on species)

SCNT will not be discussed in detail because it is not commonly used in ND models. Compared to PI, viral vectors such as retrovirus and lentivirus have been developed with high efficiency in generating transgenic animals.14,42,43 Retroviral gene transfer was first described by Jaenisch and colleagues in 1976. However, retrovirus was not widely used for the production of transgenic animals because of biosafety concerns. Not until the mid-1990s was a replication-defective pseudotyped retroviral vector developed.44,45 Pseudotyped retroviral vectors have the advantage of achieving a high titer and broad host cell range, which are the two major limitations of conventional retroviral vector systems. Chan and colleagues have successfully generated transgenic cattle and the first transgenic monkey using pseudotyped retrovirus at high efficiency.14,42 However, due to the life cycle of retroviruses, efficient gene transfer could be achieved only in actively dividing cells.14,42 The recent development of the lentiviral vector has led to a new era of in vivo genetic manipulation.46,47 Nondividing cells could be transfected efficiently using pseudotyped lentivirus.46,48 Replication-defective lentivirus is derived from HIV-1 with most of the viral structural genes removed. Vesicular stomatitis virus envelope glycoprotein protein G (VSVG) pseudotyped lentivirus is widely used in neurological research because gene transfer in neuronal cell types that are not actively dividing could be achieved at high efficiency.49 Stereotaxic injection of lentivirus at selected brain regions provides an efficient46,48 and effective method for studying gene function in a relatively short time compared to germline transgenesis43,47,50 (Table 35–1). There are significant differences between focal and germline transgenesis. Focal transgenesis is limited to a small region in the brain and is able to transfect only a limited number of neuronal cell types in an individual. Therefore, each model has to be generated independently and variation between animals is expected. This technique offers the opportunity for an in vivo gene function study in species that are highly relevant to humans but for which efficient gene transfer techniques are not available for the creation of transgenic models such as rat and NHPs.14,19 The development of transgenic rat and NHP models of NDs will also be discussed.

TRANSGENIC MOUSE MODEL OF NEURODEGENERATIVE DISEASES The transgenic mouse is the most commonly used and diversified animal model in NDs.3,4,10,15,16,36,37 Extensive information on the mouse genetic background is important when developing an

CHAPTER 35 / TRANSGENIC ANIMAL MODELS OF NEURODEGENERATIVE DISEASES

ND model. Mice with different genetic backgrounds have been shown to be a crucial factor for the future development of a cognitive–behavioral pattern in a transgenic mouse model.25 Additionally, well-established techniques in handling gametes, embryos, and surrogates, inexpensive and relatively limitless supplies, and short generation times allow a more versatile application of the transgenic mouse model than models of other species.19 Therefore, transgenic rodents, developed with genetic defects similar to those seen in patients, are widely used in biomedical research. Various genetic manipulation strategies are possible only in mice but not in species such as rats or NHPs.19 Besides the overexpression of a transgene, downregulation by RNAi or microRNA, or conditional expression of a target gene, disease-related genes could be altered in embryonic stem cells by recombination techniques followed by blactocyst injection. However, the resulting chimeric mice require an additional breeding procedure in order to establish a genetically defined transgenic line. Although mouse cloning technology has not been widely used in ND, the prospect of an identical mouse model is expected to have significant impact. Transgenic mouse models of AD, PD, and HD have also been developed with various promoters, which include tissue- and cell type-specific promoters such as the prion protein (PrP) and neuron-specific enolase (NSE) promoter, the thymus-1 minigene (thy-1), a viral and housekeeping gene promoter such as cytomegalovirus (CMV), ubiquitin, platelet-derived growth factor (PDGF) promoters, and endogenous promoters such as the htt promoter. AD mouse models are generated primarily by the overexpression of wild-type APP or APP with mutations at positions K670N and M671L (Swedish), E639Q (Dutch), D694N (Iowa), and V717F (London) that favors Aβ processing.3,10,51 These mutants are important factors for achieving Aβ levels sufficient to lead to amyloid deposition.3 Additionally, presenillin 1 (PS1) and 2 (PS2) affect the activity of -secretase, which results in the alteration of the Aβ42 level.10,13 Several transgenic mice with PS1 mutations have no significant effect on Aβ deposition. However, the coexpression of APP Swedish or London mutations and the PS1 mutant, a significant increase of Aβ42, and amyloidosis at an early age were observed compared to mice with APP mutations alone.3 A double mutant mouse (PS1 M146L/APP K670N/ M671L) has plaque formation by 3 months of age compared to 9–12 months in an APP K670N/M671L mouse.3 Besides APP and PS1, overexpression of wild-type or mutant tau protein in mice has led to various degrees of neurofibrillary pathology, which is highly related to the number of tau repeats.3,10 A triple transgenic mouse model with the combination of mutant APP, PS1, and tau has resulted in an early onset at 3 months of age of plaque formation, positive for phosphorylated tau and neurofibrillary tangles.52,53 A transgenic mouse with triple APP mutations (Swe/Dut/Iowa) has also been generated with early-onset and robust amyloid accumulation.51 Animal model targeting modifiers of amyloid pathology have also been developed. These include overexpression and knockout models of apolipoprotein E (ApoE) and BACE1.54–57 An increase in the level of ApoE results in enhancement of amyloid deposition,55 whereas knockout of ApoE results in a decrease of deposition in transgenic mice.54 A similar result was observed in BACE1. Consistently, a mouse deficient in low-density lipoprotein receptor-related protein (LRP), an ApoE receptor, has an increase in

325

amyloid deposition.10 However, transgenic mice overexpressing TGF-β1 have an increase in vascular deposition and a decrease in plaque deposition. These animal models suggest how complicated the neuropathology in AD is and these modifiers play an influential role in amyloid pathology in APP mice. In fact, ApoE is considered one of the risk factors for AD. Several candidate genes including PARK 1 (α-synuclein), 2 (Parkin), 5 (UCH-L1), 6 (PINK1), 7 (DJ1), and 8 (LRRK2) have been identified and are related to PD.1,7,9 Among these genes, α-synuclein is the first linked to PD. Several mutations of αsynuclein have been identified and are sporadically distributed at low percentages in familial PD cases. Overexpression of both wild-type and mutant α-synuclein has resulted in a decrease of dopamine and dopamine transporter with behavioral impairment.4,27 However, no significant difference was found when different promoters were used, which includes tyrosine hydroxylase (TH), PDGF, and the Thy-1 promoter.4 On the other hand, the parkin gene is related to familial autosomal recessive juvenile PD.1,7,9,27 Loss of parkin function results in an abnormal accumulation of its substrate such as glycosylated α-synuclein.15 A variation in neuropathology was also found in the deletion of exons 2, 3, and 7 of the parkin gene. Some common features include no loss of nigrastriatal DA neurons and behavioral impairments that were observed in exon 3 and 7 deletions but were undetectable in exon 2-deleted mice.15 Mouse models with DJ-1 knockout, reduced Nurr1, and the loss of Pitx3-aphakia have also been generated recently. Variations in neuropathology and behavioral impairments suggest that Nurr1 and Pitx3-aphakia mice are well suited for studying later stages of AD because of the loss of nigrastriatal dopaminergic neurons, whereas DJ-1 mice may be best for the study of early-onset parkinsonism. Genetically HD is less complex than AD and PD.2,7,30 The extended glutamine tract at the 5′ region of exon 1 of htt has been identified and linked to HD.58–60 Variations in neuropathology and behavioral decline are highly correlated with the length of the poly(Q) in patients as well as in HD mice.58,60–62 In addition to the poly(Q) tract, promoter, which regulates the expression of mutant htt, is also important in the development of animal models. Although mice are not the perfect model for humans, HD mice present features that are highly relevant to HD and are great tools in understanding the etiology and the development of cures.19,32 Although the function of htt has not been clearly determined, htt knockout is embryonic lethal and knockin mice do not have neuropathology identical to that seen in HD patients. Systemic overexpression of mutant htt is lethal with a board spectrum of HD phenotypes including motor impairment and neuropathology. However, the cause of death in R6/2 mice, one of the most popular HD mouse models,61 is not directly linked to the deteriorating effect of HD but to complications that lead to physiological dysfunction. Although most of the PD, AD, and HD mice present certain features of cognitive–behavioral decline and motor deficit, which lead to further insight on the molecular basis of neurodegenerative diseases, highly sensitive testing for in-depth analysis in mice is very limited. Therefore, the need for an animal model that allows longitudinal studies such as noninvasive imaging analysis as well as access to more accurate testing methodology for evaluating cognitive–behavioral and motor impairment are important in unraveling the etiology of the disease and developing diagnostic tools and cures.

326

SECTION IV / MODELS FOR SPECIFIC PURPOSES

TRANSGENIC RAT MODEL OF NEURODEGENERATIVE DISEASES

PRONUCLEAR MICROINJECTION METHOD FOR THE GENERATION OF TRANSGENIC RATS

For over a century the rat has been the model of choice for studies of many human diseases. Several hundred rats lines including inbred strains, congenics, and mutant rats exist. Unfortunately, germline transgenic rats are less available to biomedical researchers. In the context of neurological science, the rats have served as important animals for the study of neural development and disease, neurodegeneration, addiction, and behavior. There are several reasons for the wide use of rats in biomedicine: (1) the availability of extensive baseline data that make the interpretation of the experimental results more meaningful, (2) their larger size makes many live operations feasible, and (3) certain physiological similarities of the rat to humans. Although transgenic mice have proven to be important models for human diseases, there are many cases in which rats are superior models due to metabolic similarities and larger body size including larger brain for the development of neurosurgical interventions. In some instances, transgenic rats provided a more appropriate phenotype when compared to mice bearing the same transgene.63 For example, rats are a superior animal model for studying autoimmune diseases such as rheumatoid arthritis.64 Transgenic rats have also been very useful in transplantation research, in which microsurgery is an essential procedure. However, there are currently only a handful of transgenic rat lines compared to transgenic mouse lines. This is mainly due to inefficient gene transfer methods and limited understanding of gamete and embryo manipulation skills in the rat.65 In recent years, little advancement has been made in rat germline modification techniques; further improvement in gene transfer technology will ensure the wider application of transgenic rats as human disease models. Fortunately, genomic resources for the better utilization of rat models of human disease are growing rapidly.66 With the completion of the Human Genome Project, there is an unprecedented need for animal models to determine gene functions and to test new therapeutic strategies aimed at curing human disease. There is no doubt that the recent completion of the rat genome sequence further improves the utility of the rat as a model organism. Therefore, genetically modified rats will play a pivotal role in those advances since they offer many unique advantages as models of human disease. Despite the availability of remarkable new genomic tools, the development of genetically modified rats is still in its early stages and complex genetic manipulation such as gene targeting is not currently available.

The generation of transgenic rats by PI often requires a large number of embryo donor and recipient animals. The procedure is labor intensive, and requires significant technical skill. Moreover, the production of transgenic rats by PI is inefficient due, in part, to difficulties associated with observing the pronuclei and a high postinjection lysis rate (30–35%). Rat zygotes are often not synchronized as they are in mice and thus finding the appropriate timing for visualization and injection of the pronucleus is timely and labor intensive. To generate five or six transgenic founder animals, about 800–1000 embryos have to be injected if outbred strains such as Sprague–Dawley and Wistar (see Table 35–2) are used. These shortcomings are magnified when creating transgenic rats in an inbred background such as Fischer 344 and Lewis, primarily due to their suboptimal response to superovulation, higher sensitivity to microinjection, and lower gene transfer efficiency. Due to low efficiencies many investigators initially create transgenic rats using outbred stocks, i.e., Sprague–Dawley, and subsequently backcross them to the desired inbred genetic background using a congenic approach. This approach is not only expensive due to animal housing, but also time consuming, requiring at least 10 generations of backcrossing.67,68 Since the significance of genetic background in animal disease models cannot be discounted, the availability of a simple and reliable methodology to create transgenic rats with desired genetic background is urgently needed.

GENERATION OF TRANSGENIC ANIMALS USING VIRAL VECTORS Since the production of transgenic rats by PI is inefficient, the development of alternative gene delivery method(s) is critical for the advancement of transgenic rat models of NDs. An alternative approach to generating germline transgenic animals is the microinjection of viral vectors into the perivitelline space (PVS) of zygotes or MII oocytes. This method appears to yield a much higher transgenic rate (Table 35–2) compared to the PI method and overcomes many of the problems associated with the highly inefficient PI method in the rat. In addition, lentivirus-mediated delivery of small interfering RNA (siRNA) into cycling, noncycling mammalian cells, embryonic stem cells, zygotes, and their derivatives has been successfully demonstrated.69,70 The combination of lentiviral gene transfer and siRNA technology has further accelerated the need for rat disease models that require the suppression or the loss of gene function.

Table 35–2 Transgenic efficiency using pronuclear microinjection of plasmid DNA or perivitelline space microinjection of lentiviral vector in the rat Method of injection Pronuclear microinjectiona Perivitelline space microinjectionb a b

Based on Charreau et al.63 Unpublished data.

Number of zygotes injected

Postinjection survival (%)

Number of embryo transfers

Number of transgenic founder

Overall (number injected/ number transgenic (%))

800–1000 30–40

65–70 90

25–27 2–3

5–6 8–10

0.6 25–30

CHAPTER 35 / TRANSGENIC ANIMAL MODELS OF NEURODEGENERATIVE DISEASES

An alternative to the transgenic rat model, lentiviral gene transfer, has led to the possibility of focal transgenesis. Lentivirus is injected into a selected region of the brain and gene function could then be studied. Focal transgenesis has been widely used in ND research because it is fast and effective in determining gene function. Functional studies of α-synuclein and parkin in PD, APP, and tau in AD rat models have been reported, which accelerate the understanding of gene function and the role of genes in disease development.11,27,33,41,71

TRANSGENIC RAT MODEL OF NEURODEGENERATIVE DISEASES Only limited numbers of transgenic rat models of NDs were developed in recent years because of the low efficiency. Most of these transgenic rats were generated by the traditional PI method. A transgenic rat model of NDs was first reported in 1998.13 The PS1 gene was introduced into rats and a significant increase of apoptosis in primary cortical neurons was observed.13 Transgenic rat models that carry various combinations of APP and PS1 mutations have also been developed. However, no senile plaque of aggregated Aβ was observed in these rats.29 In addition to an AD model, a truncated rat htt cDNA with 52 CAG repeats under the control of the rat htt promoter was microinjected into the pronucleus of rat zygote.31 These rats exhibit adult-onset HD with typical histopathological alterations.31 In vivo imaging including MRI and PET was also performed in these rats and longitudinal studies could be accomplished. Although several rat models of HD were developed afterward, the development of ND rat model remains slow.30,71 So far, no transgenic rat model of PD has yet been reported and the search for candidate genes remains controversial. Only recently has lentivirus been shown to be highly efficient in generating transgenic animals.43,47,50 Compared to PI, lentiviral gene transfer results in a much higher transgenic rate (Table 35– 2). Although transgenic rat lines remain primarily generated by PI, the versatile application of lentivirus has thrived in recent years not only in focal gene transfer but also in the creation of a transgenic animal model.33,41,47,49,50,72,73 We have generated several transgenic rat lines at a high rate as summarized in Table 35–2. A brief but precise protocol will be discussed in the following section.

GENERATION OF TRANSGENIC RAT USING LENTIVIRUS RAT SUPEROVULATION Female rats (28–30 days old) are superovulated using SC implantation of 8 units of follicle-stimulating hormone (FSH), introduced via Alzet miniosmotic pumps, and followed by an intraperitoneal (ip) injection of 15 units of luteinizing hormone (LH) approximately 50–52 h after FSH implantation.74 To obtain zygotes, donors will be mated with male rats right after LH injection. RAT EMBRYO COLLECTION Superovulated female rats are euthanized by CO2 inhalation 20–24 h post-LH injection. The oviducts are excised and placed into a culture dish containing TL-HEPES supplemented with 1 mg/ml hyaluronidase (Sigma Chemical Co., St. Louis, MO) and 4 mg/ml bovine serum albumin (BSA) (Sigma Chemical Co., St. Louis, MO). The extended, translucent oviductal ampulla is dissected to release the clutch of zygotes into the solution in a culture dish. Zygotes are then incubated with hyaluronidase for about 5 min to enable dissociation

327

and removal of the cumulus cells. An inverted microscope is used to evaluate zygotes for the presence of polar bodies, pronuclei, and sperm tail to verify fertilization. The cumulus-free zygotes are washed by transferring them through three drops of hyaluronidase-free TL-HEPES before being placed into 40 µl culture drops of KSOM under oil in culture dishes, and placed in an incubator (humidified 5% CO2/95% N2) at 37°C. The zygotes are subjected to lentiviral injection via PVS 1–2 h after collection. SURGICAL EMBRYO TRANSFER To obtain pseudopregnant embryo recipient rats, 8- to 10-week-old Sprague–Dawley female rats (HSD: Sprague Dawley® SD®) are synchronized by ip injection of 40 µg gonadotropin-releasing hormone (GnRH) analog des-Gly10 [d-Ala6] ethylamide (Sigma L4513) and with mature, vasectomized Sprague–Dawley males 4 days after hormone injection. For verification of mating and hence pseudopregnancy, the female rats are removed the following morning and checked for the presence of a vaginal plug. For embryo transfer (ET), the recipient rats are anesthetized by injecting the ketamine:xylazine mixture intramuscularly (im) into the biceps femoris muscle. Approximately 5 min after the injection, the dorsal and lateral skin is clipped and the surgery site prepared with three alternate scrubs of betadine and alcohol. The skin of the dorsum is sagittally incised (10–15 mm in length) on the midline at the level of the paralumbar fossa. The skin incision is then rolled laterally to superimpose the paralumbar fossa. The muscle layers and the peritoneal wall are opened via incision and spread to create a window about 8–12 mm in length that permits retraction of the ovarian fat pad. The ovary is gently grasped with a tissue forceps and retracted/extracted until the ovary, the ovarian bursa, and the oviduct are exposed and visualized using a stereomicroscope at 10–15×. A few drops (∼25 µl) of epinephrine (VEDCO, St. Joeseph, MO; 1 mg/ml) are sprinkled over the bursa to reduce bleeding. The bursa is gently dissected using watchmaker forceps to gain access to the infundibulum. PVS-injected embryos are loaded into a small column consisting of a few microliters of phosphate-buffered saline (PBS) within a 150-µmdiameter transfer pipette. The transfer pipette is inserted into the infundibulum, 8–10 injected zygotes are discharged into the oviduct, and then the ovary is carefully replaced into the peritoneal cavity. After repeating the procedure on the contralateral side, the skin is closed using wound clips (Fisher Scientific, Pittsburgh, PA). LENTIVIRAL VECTOR CONSTRUCTION The gene of interest is inserted into a lentiviral vector backbone such as “pFUW”43 [F, flap; U, ubiquitin promoter; G, green fluorescent protein; W, woodchuck hepatitis virus posttranscriptional regulatory element (WRE)]. In brief, pFUW is a self-inactivating vector that is composed of the WRE in order to increase the transcription level and minimize the position effect. Additionally, an HIV flap element was inserted between the 5′ LTR and the internal promoter to increase the titer of the virus. COLLECTION OF AND CONCENTRATION OF VECTOR 293FT cells are plated into a 10-cm tissue culture plate the day before the cotransfection with p∆8.9 and pVSV-G into a 293FT packaging cell (Invitrogen, Inc.). Culture medium is collected at 48 h posttransfection for 3 consecutive days at 24-h intervals. The supernatant is centrifuged at 25,000 × g for 90 min. The vector pellet is resuspended and aliquoted in 50 µl of 0.1× Hanks’ balanced salt solution and kept frozen at −80°C. All procedures were performed inside a Biosafety level II cabinet. All of

328

SECTION IV / MODELS FOR SPECIFIC PURPOSES

the plasticware and instruments that have contacted the vector are decontaminated with 10% bleach or autoclaved before disposal.38 LENTIVIRAL INJECTION The procedure for lentiviral injection in the PVS is performed as described for NHP oocytes in the next section. GENOTYPING Two weeks after delivery, tailsnips are taken from all potential founder rat pups, DNA extracted, and genotyped using an appropriate primer set by polymerase chain reaction (PCR). Furthermore, Southern blot analysis is performed to determine copy numbers of the transgene of interest.

CURRENT STATUS OF THE TRANSGENIC NONHUMAN PRIMATE MODEL OF NEURODEGENERATIVE DISEASES NDs are complex disorders in which declines in neuropathology and psychiatric alterations are highly related to aging. Although transgenic mouse and rat models of neurodegenerative diseases such as AD, PD, and HD with certain clinical features have been created, a limited neuropathological pattern such as neurodegeneration is the major drawback of these animals. Chemical induction of HD and PD in NHPs has also been reported with similar neuropathological damage and psychiatric alteration; however, the role of genetic defects cannot be identified in the course of disease development. Due to the close relationship between human and NHPs, the development of transgenic NHP models will be able to mimic human conditions not only physiologically but also in terms of the genetic defects that lead to AD, PD, or HD. Therefore, disease onset and development could then be elucidated with the aid of a transgenic NHP model. Additionally, high-resolution brain imaging technology such as MRI and PET and cognitive–behavioral testing can be carried out in the NHP simultaneously, which is not possible in other animal models. A high degree of similarity in motor repertoire between NHPs and humans allows direct comparison of the neurological phenotype between the NHP model and patients. Because of this, a transgenic NHP is expected to have a significant impact on ND research. However, due to the difficulty in creating a transgenic NHP, an alternative strategy has been developed to bypass the creation of a germline transgenic monkey and to study gene function at a selected brain region. Focal transgenesis (stereotaxic delivery of a viral vector) is one of the most popular techniques in neuroscience because of the latest development of a lentiviral vector that allows efficient gene transfer in inactive cells such as neurons.33,41 Although focal transgenesis is fast and efficient, the role of the gene defect will be restricted to a selected brain region and a selected group of neurons, and its role beyond neurological functions may be easily overlooked. Therefore, focal transgenesis is not the solution for a germline transgenic animal that carries the genetic defect in all body cells, which best mimics patient conditions. Although both strategies are unique and are very useful in ND research, careful interpretation of results should be taken when using these animal models. Focal transgenesis by the injection of lentivirus has been widely used to study disease-related gene function in rodents and NHPs.33,41 However, there is only one report of the creation of a transgenic NHP.14 Although a transgenic NHP is not commonly used in the ND model, the potential impact of the transgenic NHP model in ND research could be considerable because of the great

similarity to humans including motor repertoire, brain structure and function, and genome constitution. Here we have a brief protocol for the generation of a transgenic NHP model of NDs. Preparation of a lentiviral vector was previously described when the transgenic rat model was discussed.

GENERATION OF TRANSGENIC NONHUMAN PRIMATES FOLLICLE STIMULATION Hyperstimulation of female rhesus monkeys exhibiting regular menstrual cycles is induced with exogenous gonadotropins. In brief, subcutaneous injection of a GnRH antagonist (Antide; Serono Inc.) and twice daily injection of r-hFSH (Serono Inc.) for 6 days is followed by twice daily injections of r-hFSH and r-hLH (Serono Inc.). On day 7 of the stimulation, ultrasonography is performed and r-hCG is administered for induction of ovulation when follicles of 3–4 mm in diameter are observed. FOLLICULAR ASPIRATION BY LAPAROSCOPY Follicular aspiration is performed at 27 h post-hCG. Oocytes are aspirated from follicles using a needle suction device lined with Teflon tubing. Multiple individual follicles are aspirated via continuous vacuum, and collection tubes are immediately transported to a dedicated primate oocyte/zygote laboratory for oocyte recovery and evaluation of the maturation stage. COLLECTION AND EVALUATION OF RHESUS OOCYTES The contents of each collection tube are diluted in TALP-HEPES supplemented with 2 mg/ml hyaluronidase. Oocytes are rinsed and then transferred to preequilibrated CMRL medium prior to the evaluation of maturation. MII-arrested oocytes, exhibiting expanded cumulus cells, a distinct PVS, and a first polar body, are selected for PVS injection and subsequent fertilization. IN VITRO MATURATION Oocytes are matured in modified CMRL-1066 containing 20% heat-inactivated fetal bovine serum (FBS; Hyclone Laboratories Inc., Logan, UT) supplemented with 5 µg/ml ovine FSH and 10 ng/ml ovine LH for up to 36 h in 50-µl drops of medium under saline-equilibrated silicone oil at 37°C in a humidified atmosphere of 5% CO2 in air. INFECTION OF MATURE RHESUS OOCYTES/ZYGOTES WITH THE VSVG PSEUDOTYPE MII-arrested oocytes and fertilized oocytes are selected for PVS injection followed by in vitro fertilization in oocytes. VSVG pseudotype solution is loaded into the injection needle by micropipette, mounted onto a micromanipulator, and connected to a microinjector. After microinjection, the oocytes are returned to the maturation drop and fertilized by intracytoplasmic sperm injection (ICSI), whereas the zygote is returned to the culture medium. COLLECTION, PREPARATION, AND HANDLING OF RHESUS SPERM Rhesus males of proven fertility are trained to routinely produce semen samples by penile electroejaculation. After serial washes, the sperm samples are counted and diluted to a concentration of 20 × 106 sperm/ml. Sperm suspensions are incubated at 37°C under 5% CO2 in air for 6 h, at which point 1 mM caffeine and 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) is added for the final hour to stimulate hyperactivation. FERTILIZATION BY INTRACYTOPLASMIC SPERM INJECTION A single sperm is aspirated tail-first into an injection needle from the sperm-polyvinylpyrrolidone (PVP) to the oocytecontaining drop. Oocytes are immobilized with the polar body at

CHAPTER 35 / TRANSGENIC ANIMAL MODELS OF NEURODEGENERATIVE DISEASES

the 12 o’clock position and the injection needle is inserted through the zona into the cytoplasm at the 3 o’clock position. The oolemma is breached by gentle cytoplasmic aspiration and the sperm will then be released into the oocyte. ASSESSMENT OF SUCCESSFUL FERTILIZATION AND IN VITRO EMBRYO CULTURE Fertilization is assessed within 3–6 h by detection of the second polar body using RC optics. Pronuclei are assessed between 12 and 16 h post-ICSI. After completion of the first cleavage division (24–28 h post-ICSI), two-cell embryos are cocultured in CMRL + 10% FBS (Hyclone Laboratories, Inc., Logan, UT) on a Buffalo rat liver cell (BRL 1442; ATCC, Rockville, MD) in 100 µl drops overlaid with oil. Embryos are selected at the 4- to 16-cell stage for transfer into selected surrogates. SELECTION AND PREPARATION OF SURROGATE FEMALES Rhesus females with normal menstrual cycles and natural cycles similar to those of the egg donor are screened as potential surrogate mothers. Blood samples, beginning on day 8 of the menstrual cycle (day 1 is the first day of menses), are collected and analyzed daily for serum progesterone and estrogen. Surgical ET is performed on day 2 or 3 following ovulation by transferring two four- to eight-cell embryos into the oviduct of the recipient. RHESUS EMBRYO TRANSFER BY LAPAROTOMY Surgical ET is performed by mid-ventral laparotomy. The oviduct is cannulated using a Tomcat catheter containing two four- to eightcell stage embryos in HEPES-buffered TALP, containing 3 mg/ml BSA. Embryos are expelled from the catheter in about 0.05 ml of medium while the catheter is withdrawn. The catheter is flushed with medium after removal from the female to ensure that the embryos are successfully transferred. CONFIRMATION OF PREGNANCY To confirm embryonic implantation, blood samples are collected daily and analyzed for serum estrogen and progesterone concentrations. If hormone levels indicate a possible pregnancy, the pregnancy is confirmed by a transabdominal ultrasound on day 35 posttransfer. INFANT CARE The pregnant animal will be monitored daily for the 150 days of pregnancy because rhesus monkeys occasionally experience preeclampsia, and newborns can be lost due to complications at birth. Therefore, cesarean sections may be performed. Babies are weighed and measured, and their head circumference recorded; they are kept in an incubator and raised in the nursery facility if needed.

REFERENCES 1. Morris HR. Trends in molecular medicine: Genetics in Parkinson’s disease. Ann Med 2005;37:86–89. 2. Jain S, Wood NW, Healy DG. Molecular genetic pathways in Parkinson’s disease: A review. Clin Sci 2005;109:355–364. 3. Higgins GA, Jacobsen H. Transgenic mouse models of Alzheimer’s disease: Phenotype and application. Behav Pharmacol 2003;14: 419–438. 4. Fernagut PO, Chesselet MF. Alpha-synuclein and transgenic mouse models. Neurobiol Dis 2004;17:123–130. 5. Huntington’s Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. Cell 1993;72:971–983. 6. Yu ZX, Li SH, Evans J, Pillarisetti A, Li H, Li XJ. Mutant huntingtin causes context-dependent neurodegeneration in mice with Huntington’s disease. J Neurosci 2003;23(6):2193–2202. 7. Abeliovich A, Beal MF. Parkinsonism genes: Culprits and clues. J Neurochem 2006;99(4):1062–1072.

329

8. Pardo LM, van Duijn CM. In search of genes involved in neurodegenerative disorders. Mutat Res 2005;592:89–101. 9. Gasser T. Genetics of Parkinson’s disease. Curr Opin Neurol 2005;18:363–369. 10. Spires TL, Hyman BT. Transgenic models of Alzheimer’s disease: Learning from animals. NeuroRx 2005;2:423–437. 11. Bass B, Olanow CW, Kordower JH. Gene transfer of trophic factors and stem cell grafting as treatments for Parkinson’s disease. Neurology 2006;66:S89–S103. 12. Vercauteren FGG, Clerens S, Roy L, Hamel N, Arckens L, Vandesande F, Alhonen L, Janne J, Szyf M, Cuello AC. Early disregulation of hippocampal proteins in transgenic rats with Alzheimer’s diseaselike mutations in amyloid precursor protein and presenilin 1. Mol Brain Res 2004;132:241–259. 13. Czech C, Lesort M, Tremp G, Terro F, Blanchard V, Schombert B, Carpentier N, Dreisler S, Bonici B, Takashima A, Moussaoui S, Hugon J, Pradier L. Characterization of human presenilin 1 transgenic rats: Increased sensitivity to apoptosis in primary neuronal cultures. Neuroscience 1998;87(2):325–336. 14. Chan AW, Chong KY, Martinovich C, Simerly C, Schatten G. Transgenic monkeys produced by retroviral gene transfer into mature oocytes. Science 2001;291(5502):309–312. 15. Fleming SM, Fernagut PO, Chesselet MF. Genetic mouse models of Parkinsonism: Strengths and limitations. NeuroRx 2005;2:495–503. 16. Levine MS, Cepeda C, Hickey MA, Fleming SM, Chesselet MF. Genetic mouse models of Huntington’s and Parkinson’s diseases: Illuminating but imperfect. Trends Neurosci 2004;27(11):691–697. 17. Petters RM, Sommer JP. Transgenic animals as model for human disease. Transgenic Res 2000;9:347–351. 18. Vaitukaitis JL. Animal models of human disease for the 21st century. Lab Anim Sci 1998;48(6):562–564. 19. Chan AWS, Chong KY, Schatten G. Production of transgenic nonhuman primates. In: Pinkert CA, Ed. Transgenic Animal Technology: A Laboratory Handbook, 2nd ed. San Diego, CA: Academic Press, 2002:359–382. 20. Reddy S, Smith DB, Rich MM, Leferovich JM, Reilly P, et al. Mice lacking the myotonic dystrophy protein kinase develop a late onset progressive myopathy. Nat Genet 1996;13(3):325–335. 21. Jansen G, Groenen PJ, Bachner D, Jap PH, Coerwinkel M, et al. Abnormal myotonic dystrophy protein kinase levels produce only mild myopathy in mice. Nat Genet 1996;13(3):316–324. 22. Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, et al. Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science 1997;276(5321):2045–2047. 23. Sango K, Yamanaka S, Hoffmann A, Okuda Y, Grinberg A, et al. Mouse models of Tay-Sachs and Sandhoff diseases differ in neurologic phenotype and ganglioside metabolism. Nat Genet 1995; 11(2):170–176. 24. Yamanaka S, Johnson MD, Grinberg A, Westphal H, Crawley JN, et al. Targeted disruption of the Hexa gene results in mice with biochemical and pathologic features of Tay-Sachs disease. Proc Natl Acad Sci USA 1994;91(21):9975–9979. 25. Adriaan Bouwknecht J, van der Gugten J, Groenink L, Olivier B, Paylor RE. Behavioral and physiological mouse models for anxiety: Effects of flesinoxan in 129S6/SvEvTac and C57BL/6J mice. Eur J Pharmacol 2004;494(1):45–53. 26. Brouillet E, Conde F, Beal MF, Hantraye P. Replicating Huntington’s disease phenotypes in experimental animals. Prog Neurobiol 1999;59:427–468. 27. Yamada M, Mizuno Y, Mochizuki H. Parkin gene therapy for alphasynucleinopathy: A rat model of Parkinson’s disease. Hum Gene Ther 2005;16(2):262–270. 28. Lopez EM, Bell KFS, Ribeiro-da-Silva A, Cuello AC. Early changes in neurons of the hippocampus and neurocortex in transgenic rats expressing intracellular human A–β. J Alzheimers Dis 2004;6(4): 421–431. 29. Echeverria V, Ducatenzeiler A, Alhonen L, Janne J, Grant SM, Wandosell F, Muro A, Baralle F, Li H, Duff K, Szyf M, Cuello AC. Rat transgenic models with a phenotype of intracellular Aβ accumulation in hippocampus and cortex. J Alzheimers Dis 2004;6:209–219.

330

SECTION IV / MODELS FOR SPECIFIC PURPOSES

30. Cao C, Temel Y, Blokland A, Ozen H, Steinbusch HWM, Vlamings R, Nguyen HP, von Horsten S, Schmitz C, Visser-Vandewalle V. Progressive deterioration of reaction time performance and choreiform symptoms in a new Huntington’s disease transgenic rat model. Behav Brain Res 2006;170:257–261. 31. van Horsten S, Schmitt I, Nguyen HP, Holzmann C, Schmidt T, Walter T, Bader M, Pabst R, Kobbe P, Krotova J, Stiller D, Kask A, Vaarmann A, Rathke-Hartlieb S, Schulz JB, Grasshoff U, Bauer I, Vieira-Saecker AMM, Paul M, Jones L, Lindenberg KS, Landwehrmeyer B, Bauer A, Li XJ, Riess O. Transgenic rat model of Huntington’s disease. Hum Mol Genet 2003;12(6):617–624. 32. Chan AW. Transgenic nonhuman primates for neurodegenerative diseases. Reprod Biol Endocrinol 2004;2:39. 33. Kordower JH, Emborg ME, Bloch J, Ma SY, Chu Y, Leventhal L, McBride J, Chen EY, Palfi S, Roitberg BZ, Brown WD, Holden JE, Pyzalski R, Taylor MD, Carvey P, Ling ZD, Trono D, Hantraye P, Deglon N, Aebischer P. Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson’s disease. Science 2000;290:767–773. 34. Yamamoto A, Lucas JJ, Hen R. Reversal of neuropathology and motor dysfunction in a conditional model of Huntington’s disease. Cell 2000;101(1):57–66. 35. Davies SW, Turmaine M, Cozens BA, DiFiglia M, Sharp AH, et al. Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell 1997;90(3):537–548. 36. Bates GP, Mangiarini L, Mahal A, Davies SW. Transgenic models of Huntington’s disease. Hum Mol Genet 1997;6(10):1633– 1637. 37. Schilling G, Becher MW, Sharp AH, Jinnah HA, Duan K, et al. Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin. Hum Mol Genet 1999;8(3):397–407. 38. Chan AWS. Transgenic animals: Current and alternative strategies. Cloning 1999;1(1):25–46. 39. Kordower JH, Bloch J, Ma SY, Chu Y, Palfi S, et al. Lentiviral gene transfer to the nonhuman primate brain. Exp Neurol 1999; 160(1):1–16. 40. Palfi S, Leventhal L, Chu Y, Ma SY, Emborg M, et al. Lentivirally delivered glial cell line-derived neurotrophic factor increases the number of striatal dopaminergic neurons in primate models of nigrostriatal degeneration. J Neurosci 2002;22(12):4942– 4954. 41. de Almeida LP, Rodd CA, Zala D, Aebischer P, Deglon N. Lentiviral-mediated delivery of mutant huntingtin in the striatum of rats induces a selective neuropathology modulated by polyglutamine repeat size, huntingtin expression levels, and protein length. J Neurosci 2002;22(9):3473–3483. 42. Chan AWS, Homan EJ, Ballou LU, Burns JC, Bremel RD. Transgenic cattle produced by reverse-transcribed gene transfer in oocytes. Proc Natl Acad Sci USA 1998;95(24):14028–14033. 43. Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D. Germline transmission and tissue-specific expression of transgenes delivered by lentivirus vectors. Science 2002;295(5556):868–872. 44. Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: Concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci USA 1993;90(17):8033–8037. 45. Yee JK, Miyanohara A, LaPorte P, Bouic K, Burns JC, Friedmann T. A general method for the generation of high-titer, pantropic retroviral vectors: Highly efficient infection of primary hepatocytes. Proc Natl Acad Sci USA 1994;91(20):9564–9568. 46. Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma IM. Development of a self-inactivating lentivirus vector. J Virol 1998;72(10): 8150–8157. 47. Pfeifer A, Ikawa M, Dayn Y, Verma IM. Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem cells and preimplantation. Proc Natl Acad Sci USA 2001;99(4):2140– 2145.

48. Miyoshi H, Takahashi M, Gage FH, Verma IM. Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proc Natl Acad Sci USA 1997;94(19):10319–10323. 49. Naldini L, Blomer U, Gage FH, Trono D, Verma IM. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci USA 1996;93(21):11382–11388. 50. Tiscornia G, Singer O, Ikawa M, Verma IM. A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc Natl Acad Sci USA 2003;100(4):1844– 1848. 51. Davis J, Xu F, Deane R, Romanov G, Previti ML, Zeigler K, Zlokovic BV, Van Nostrand WE. Early-onset and robust cerebral microvascular accumulation of amyloid β-protein in transgenic mice expressing low levels of a vasculotrophic Dutch/Iowa mutant form of amyloid β-protein precursor. J Biol Chem 2004;279(19): 20296–20306. 52. Oddo S, Caccamo A, Kitazawa M, Tseng BP, LaFerla FM. Amyloid deposition precedes tangle formation in a triple transgenic model of Alzheimer’s disease. Neurobiol Aging 2003;24(8):1063–1070. 53. Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE, et al. Triple-transgenic model of Alzheimer’s disease with plaques and tangles: Intracellular Abeta and synaptic dysfunction. Neuron 2003;39(3):409–421. 54. Bales KR, Verina T, Dodel RC, Du Y, Altstiel L, et al. Lack of apolipoprotein E dramatically reduces amyloid beta-peptide deposition. Nat Genet 1997;17(3):263–264. 55. Holtzman DM, Bales KR, Tenkova T, Fagan AM, Parsadanian M, et al. Apolipoprotein E isoform-dependent amyloid deposition and neuritic degeneration in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci USA 2000;97(6):2892–2897. 56. Luo Y, Bolon B, Damore MA, Fitzpatrick D, Liu H, et al. BACE1 (beta-secretase) knockout mice do not acquire compensatory gene expression changes or develop neural lesions over time. Neurobiol Dis 2003;14(1):81–88. 57. Mohajeri MH, Saini KD, Nitsch RM. Transgenic BACE expression in mouse neurons accelerates amyloid plaque pathology. J Neural Transm 2004;111(3):413–425. 58. Rubinsztein DC. Lessons from animal models of Huntington’s disease. Trends Genet 2002;18(4):202–209. 59. Davies S, Ramsden DB. Huntington’s disease. J Clin Pathol Mol Pathol 2001;54:409–413. 60. Cummings CJ, Zoghbi HY. Trinucleotide repeats: Mechanisms and pathophysiology. Annu Rev Genomics Hum Genet 2000;1:281– 328. 61. Mangiarini L, Sathasivam K, Seller M, Cozens B, Harper A, et al. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 1996;87:493–506. 62. Menalled LB, Chesselet MF. Mouse models of Huntington’s disease. Trends Pharmacol Sci 2002;23(1):32–39. 63. Charreau B, Tesson L, Soulillou JP, Pourcel C, Anegon I. Transgenesis in rats: Technical aspects and models. Transgenic Res 1996;5:223–234. 64. Taurog JD, Maika SD, Satumtira N, Dorris ML, McLean IL, et al. Inflammatory disease in HLA-B27 transgenic rats. Immunol Rev 1999;169:209–223. 65. Agca Y, Critser JK. Assisted reproductive technologies and genetic engineering in rats. In: Suckow MA, Weisbroth SH, Franklin CL, Eds. The Laboratory Rats, 2nd ed. San Diego, CA: Academic Press, 2005:165–190. 66. Jacob HJ. Functional genomics and rat models. Genome Res 1999;9(11):1013–1016. 67. Kuramoto T, Yamasaki K, Kondo A, Nakajima K, Yamada M, Serikawa T. Production of WTC.ZI-zi rat congenic strain and its pathological and genetic analyses. Exp Anim 1998;47:75–81. 68. Kose H, Moralejo DH, Ogino T, Mizuno A, Yamada T, Matsumoto K. Examination of OLETF-derived non-insulin-dependent diabetes mellitus QTL by construction of a series of congenic rats. Mamm Genome 2002;13:558–562.

CHAPTER 35 / TRANSGENIC ANIMAL MODELS OF NEURODEGENERATIVE DISEASES

69. Pfeifer A, Ikawa M, Dayn Y, Verma IM. Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc Natl Acad Sci USA 2002;99:2140–2145 70. Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, Kopinja J, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 2003;33:401–406. 71. Kantor O, Temel Y, Holzmann C, Raber K, Nguyen HP, Cao C, Turkoglu HO, Rutten BPF, Visser-Vandewalle V, Steinbusch HWM, Blokland A, Korr H, Riess O, van Horsten S, Schmitz C. Selective striatal neuron loss and alterations in behavior correlate with impaired

331

striatal function in Huntington’s disease transgenic rats. Neurobiol Dis 2006;22:538–547. 72. Takahashi M, Miyoshi H, Verma IM, Gage FH. Rescue from photoreceptor degeneration in the rd mouse by human immunodeficiency virus vector-mediated gene transfer. J Virol 1999;73(9):7812–7816. 73. Gallichan WS, Kafri T, Krahl T, Verma IM, Sarvetnick N. Lentivirus-mediated transduction of islet grafts with interleukin 4 results in sustained gene expression and protection from insulins. Hum Genet Ther 1998;9(18):2717–2726. 74. Armstrong DT, Opavsky MA. Superovulation of immature rats by continuous infusion of follicle-stimulating hormone. Biol Reprod 1988;39(3):511–518.

36

Animal Models of Nociception and Pain JAMES D. ROSE AND C. JEFFREY WOODBURY

ABSTRACT

processes in animals. In spite of great recent progress in deciphering the neurobiological basis of nociception and pain, this knowledge has yet to see large-scale translation into effective pain therapies. A limiting factor has been the often unsuccessful extrapolation from animal models to human clinical practice, as exemplified by recent attempts to develop new pharmacological treatments for migraine headache.3–5 Beneficial applications from animal models could be fostered by rigorous examination of the validity and limits of these models.3 This goal also hinges on a better understanding of the similarities and differences between nociception and pain in humans and in the animal models, an understanding that would make model selection and interpretation more valid for human applications. This chapter’s principal objectives are to clarify distinctions between nociception and pain to improve the interpretation and validity of animal models and to discuss and evaluate commonly used and important models. There are several recent reviews, some highly detailed, on animal models in nociception–pain research that should be consulted by readers wishing further information.3,6–8

Research on the neurobiological bases of nociception and pain and related investigations of potential therapies require great reliance on animal models. There are unique challenges in the development of well-validated models in this field because of the distinction between nociception, the processing and response to potentially pain producing stimuli by lower levels of the nervous system, and pain, the conscious result of nociceptive stimulus processing by the cerebral cortex. The most frequently used models actually represent tests of nociception only and are appropriate for investigating diverse pathophysiological processes that cause nociceptive activity in peripheral tissues, nerves, the spinal cord, or subcortical regions. However, because human pain is a complex end result of nociception and consciousness-dependent processes, models intended to address pain must be validated for this purpose. Models assessing processes related to pain are relatively rare and more difficult to validate and use than those relevant only for nociception. A failure to recognize the pain–nociception distinction has significant practical consequences for successful extrapolation of results from laboratory to clinical practice. Key Words: Nociception–pain dichotomy, Construct validity, Neocortex, Nocifensive behavior, Neural substrate.

DEFINING PAIN IN HUMANS: IMPLICATIONS FOR ANIMAL MODELS

THE NEED FOR ANIMAL MODELS OF NOCICEPTION AND PAIN

A valid working definition of pain is vital for efforts to explain its underlying mechanisms or develop therapeutic interventions. To this end, the International Association for the Study of Pain9 defines human pain as follows: (1) pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage; (2) pain is always subjective; and (3) pain is sometimes reported in the absence of tissue damage and the definition of pain should avoid tying pain to an external eliciting stimulus. One of the most critical conceptual advances in the understanding of pain is the distinction between nociception and pain. As Wall10 emphasized, nociception that is “activity induced in the nociceptor and nociceptive pathways by a noxious stimulus is not pain, which is always a psychological state.” It is also critical to understand that the pain experience requires conscious awareness.11,12 In the usual course of events, tissue-damaging forms of stimuli excite nociceptors and this activity is conducted through peripheral nerves, the spinal cord, and subcortical brain structures to the cerebral cortex. If a person is conscious when nociception-related activity arrives in the cortex, further processing by extensive cortical regions results in pain.11–14

Pain research with human subjects is productive on many fronts, as shown by the large and diverse literature surveyed in the most recent edition of Wall and Melzack’s Textbook of Pain.1 A particularly prominent area of progress is in the use of brain imaging methods such as positron emission tomography and functional magnetic resonance imaging to advance our understanding of the higher brain processes that underlie pain.2 However, there are great limitations on the use of humans in experimental studies of pain and animal subjects continue to be vital. In vivo models are particularly important because pain and its underlying mechanisms are emergent processes of a whole nervous system; these processes cannot be fully simulated in highly reduced cell or tissue systems. In addition to bettering our understanding of nociception and pain, these models are valuable out of welfare concerns for achieving a better understanding of pain–nociception

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

333

334

SECTION IV / MODELS FOR SPECIFIC PURPOSES

The separateness of pain and nociception is seen in diverse ways. First, nociceptive processes do not always lead to pain. People can sustain severe injuries in warfare, sports, or everyday life and either not report pain or report it differently than the extent of an injury would suggest.15–17 Second, people with “functional” pain syndromes experience chronic, disturbing pain without any demonstrable tissue damage or pathology. Third, pain can be reduced by psychological manipulations such as a visual illusion18 or hypnotic suggestion.19 Fourth, pain has a strong social learning component and depends greatly on prior experience with it and interpersonal interactions that accompany this experience.13 THE NEUROLOGY OF NOCICEPTION AND PAIN There has been great progress in identifying the functional neuroanatomy underlying nociception and pain. We will present a brief account of the neural structures and systems implicated in nociception and pain as it pertains to the use and interpretation of animal models. Numerous excellent reviews provide more detailed information.1,2,20–22 Studies of diverse mammals have shown that nociceptive stimuli activate two types of nociceptive receptors in body tissues: those that appear to respond exclusively to noxious mechanical, thermal, or chemical stimuli and those that respond to combinations of these stimuli (polymodal nociceptors). Activity is conducted from these receptors to the spinal cord through both myelinated (Aβ and Aδ) and unmyelinated (C) axons. These axons synapse on dorsal horn spinal neurons, principally in the more superficial laminae, where extensive processing occurs. Ascending projections arise from neurons in diverse dorsal horn laminae, principally lamina I and V, and travel through the contralateral lateral and ventral spinal pathways. The ascending pathways have synaptic terminations in diverse regions of the brainstem, mainly catecholamine neuron groups, parabrachial nuclei of the pons, midbrain periaqueductal gray, and diverse sites through the brainstem reticular formation. The thalamus receives multiple direct spinal projections to topographically organized lateral and nontopographically organized medial nuclei. There are significant differences between mammalian species at this level, particularly in the existence of a posterior ventromedial nucleus that seems to exist only in primates and is greatly enlarged in humans.21 Functional imaging studies in humans have consistently shown a diverse array of cortical structures to be activated specifically in association with perceived pain, including the first and second somatosensory areas, anterior cingulate gyrus, insula, and prefrontal cortex. Diverse evidence indicates that the somatosensory cortical zones are critical for the sensory-discriminative dimension of pain, that pain intensity is related to activation of multiple zones, especially involving both hemispheres, and that the emotional-evaluative (suffering) component depends on the anterior cingulate gyrus, insula, and prefrontal cortex. In addition, it is now well established that in humans, pain experience is absolutely dependent on the functioning of these neocortical and limbic cortical areas.22,23 The dependence of pain on these cortical regions makes sense also when it is considered that pain depends on the concurrent existence of another function: consciousness. Extensive evidence shows that the cortical regions known to be essential for pain greatly overlap with those vital to the existence of consciousness.11,14 In addition to the ascending pathways is a network of descending modulatory controls, centered in the periaqueductal gray and

rostral ventromedial medulla, that exerts both antinociceptive and pronociceptive actions on ascending nociceptive signaling.24 THE ADAPTIVENESS OF NOCIFENSIVE BEHAVIORS Nociceptors form a common underlying thread throughout the evolutionary history of multicellular organisms. Nociceptors have been observed in all bilaterally symmetrical multicellular organisms that have been examined, with the notable exception of elasmobranch fishes.14 Even the leech has nociceptive neurons,25 many of which display close similarity to the polymodal nociceptor population that has been so well characterized in mammals. Although nociceptors are common in the animal kingdom, the existence of nocifensive behaviors, the unconscious protective responses to noxious stimuli, is even more widespread and not specifically tied to possession of nociceptors.26 The single-celled paramecium (absent any possibility of a nervous system) exhibits protective responses to adverse environmental stimuli. Likewise, sponges with no nervous system and jellyfish with simple nerve nets have simple, but functional nocifensive behaviors. In these and more advanced bilaterally symmetrical invertebrate organisms as well as vertebrates, the nociceptive system and nocifensive behaviors constitute an essential component to survival. Importantly, the suite of responses to nociceptive stimuli does not end simply with withdrawal reflexes, but in advanced multicellular organisms also includes complex arrays of endocrine and autonomic responses that help prepare the organism for a defense of disturbed homeostasis. Species with nociceptors showing properties in common with those of mammals could serve as useful models for investigating peripheral nociception. DISTINGUISHING NOCICEPTION FROM PAIN: WHY IT MATTERS As explained above, nociception and pain are distinctly different things, with differing underlying mechanisms. Unless one is studying the processes that specifically mediate the conscious experience of pain or a behavioral response that is specific to such processes, nociception is being studied and the term pain should not be used. Unfortunately, these terms are frequently used in ambiguous or inconsistent ways, with significant practical costs in the use and interpretation of animal models and theoretical costs in understanding of mechanisms. The Nociception–Pain Dichotomy in Clinical Neurology The behavioral separateness of nociception and nocifensive responses from pain is commonly seen in humans with severe neurological injury such as a spinal cord transection. Noxious stimulation of a limb below the level of the transection excites nociceptive sensory receptors and nociceptive pathways within the spinal cord. This spinal activity produces a nocifensive limb withdrawal response, but because nociceptive pathways are interrupted between the spinal cord and the cortex, no pain is felt. The pain–nociception distinction does not stop at this level. Humans with massive damage or dysfunction of the cerebral cortex are unconscious, but can be awake and show grimacing, vocalization, and organized avoidance reactions in response to a nociceptive stimulus.12 The importance of understanding the nociception–pain distinction was shown by the confusion and contentiousness surrounding the recent tragic case of Terri Shaivo, who in 1990 experienced a prolonged period of anoxia. Although examining neurologists agreed that she was unconscious, in an irreversible, persistent vegetative state, Mrs. Shaivo was awake, was quite reactive to noxious stimuli, and exhibited nocifensive and emotion-like behaviors, which led to claims by some that Ms. Schaivo’s behaviors had to be consciously mediated. After a pro-

CHAPTER 36 / ANIMAL MODELS OF NOCICEPTION AND PAIN

tracted legal dispute and interventions by government officials, her feeding tube was removed. Subsequent autopsy confirmed that her cerebral cortex had massively degenerated, a fact consistent with the diagnosis of unconsciousness.28 The important points to be gleaned from this case are that a striking array of nocifensive behaviors can be unconsciously expressed in a human; that these are behaviors mediated by the brainstem and spinal systems rather than conscious, pain-related responses; and there is a widespread public unawareness of such things with associated societal consequences. Valid Selection and Interpretation of Models As stated previously, effective therapies for pain have lagged behind advances in basic research. A practical example will serve to illustrate this point. Substance P was described in 1931 and its role in nociceptive neurotransmission has been known for decades.29 Immense resources have been devoted to development of substance P antagonists, principally NK1 receptor antagonists for pain relief, especially migraine headache. In spite of their effectiveness in animal models of dural inflammation, these drugs were ineffective in relieving human migraine pain. This negative result was not a matter of a receptor affinity mismatch because high level NK1 receptor blockade has been achieved in the brains of humans.30 This costly failure has been attributed to many things, including recognition that there is more to human migraine headache pain than events in the dura mater.4,31 Here, the animal model response measure was at the most peripheral level of the complex hierarchy of nociception–pain process, but the human clinical response measure, the pain report, was at the end stage of all intervening processing. WHAT WOULD THE PAIN OF ANIMALS BE LIKE? A most important question regarding animal models for pain is which species are capable of pain experience, or at least a pain experience that meaningfully resembles that of a human. The foregoing explanation should make it clear that most species in the animal kingdom can detect and respond to noxious stimuli but not all can experience conscious pain. There has been interest in using invertebrates and some nonmammals such as amphibians32 as animal models for nociception on the assumption that they were more humane models than mammals. Examination of the collective neurological and behavioral evidence has led to the conclusion that fishes and amphibians are very unlikely to have a capacity for conscious pain experience, at least anything resembling that of humans.14,32 However, there are differing views.33,34 It is clear, though, that structural organization of the forebrain differs dramatically between mammals and nonmammalian vertebrates. Most notable is the unique presence of a neocortical component of the forebrain pallium in mammals,35,36 with its suite of structures, and especially interconnections between them, essential to pain experience. Nonetheless, the forebrain complexity of reptiles and birds is considerable36,37 and the presence of functional homologs of pain or consciousness-related cerebral structures, especially in birds, remains to be investigated. The majority of animals used in nociception–pain research are mammals and the species used all have, to varying degrees, the cortical regions that correspond to those as essential to human pain experience.14,36 However, large expanses of higher–order nonsensory, nonmotor cortex are part of the pain-related mosaic and in nonprimate mammals, only a small proportion of cerebral cortex, less than 10% in rats and mice,38 fits this structural designation. As mentioned previously, the subcortical pathways that

335

distribute nociception-related information to pain-related cortical zones also differ substantially between primates (especially humans) and nonprimate mammals. Clearly, significant functional differences may exist between species of mammals in general and humans in particular in terms of the how pain processing might occur.

WHEN IS AN ANIMAL MODEL APPROPRIATE FOR THE INVESTIGATION OF PAIN? Much current research employs models specifically addressed at particular types of human pain conditions. Some models are also used to address fundamental mechanistic questions concerning nociceptive processing or actual pain experience. In either case, where an animal model is being used to investigate some aspect of human nociception and pain, it is vital to know that the model system is actually valid for the purpose. In particular, painrelated response measures must be distinguished from purely nociceptive–nocifensive response measures because pain is a psychological process that is not directly observable and must be measured indirectly through behavior. As shown above, clinical neurology provides human examples of the pain–nociception distinction, but clear examples have been in the animal literature for many years. Responses to noxious stimuli have been studied in several mammalian species following decerebration where all of the forebrain above the midbrain is removed. Chronically decerebrate rats39 react strongly to the insertion of a feeding tube, struggling, pushing at it with the forepaws, and vocalizing. When receiving an injection, these rats react indistinguishably from a normal rat: vocalizing, attempting to bite the syringe or the experimenter’s hand and lick the injection site. Since a large body of evidence indicates that consciousness (and accordingly pain experience) depends on the neocortex, it must be concluded that these reactions are nocifensive and unconscious rather than expressions of conscious pain. It can be concluded that nocifensive behaviors can be far more complex than “simple reflexes” and even ostensibly purposive, a fact that makes the behavioral distinction between nociception and pain difficult. In fact, many assumptions about indications of pain have been based on behaviors that are sustained, organized, or directed to the site of nociceptive stimulation,7,8,40 the type of responses fully within the capacity of decerebrate rats. Identification of pain in humans usually depends on a verbal report, but verbal reports and other pain-related behaviors are not always reliably interpretable.19 So, validated rating scales or other tools adopted from cognitive psychology provide sufficiently reliable means of measuring pain in humans. Correspondingly, there is a long history in experimental psychology of using nonverbal behavioral methods to assess the internal “psychological” state of an animal. It is quite possible to assess the aversiveness of a stimulus in terms of whether the animal will learn to avoid the stimulus, escape from it, or perform some behavior to escape that reflects the aversiveness of the nociceptive stimulus. An example of the last case is that a rat will leave a dark chamber and enter a brightly illuminated chamber (normally aversive to a rat) in order to escape a hot plate or electric shock.8 Learned avoidance, or conditioned emotional responses to nociceptive stimuli, however, does not prove the existence of conscious pain, because associative learning of these types is believed to be unconsciously mediated.41

336

SECTION IV / MODELS FOR SPECIFIC PURPOSES

Another effort to establish the existence of pain in an animal, as opposed to measurement of pain intensity, was Bateson’s40 eight criteria for animal pain, which have been of interest to welfare biologists. However, when viewed in terms of what is currently known about pain14 these criteria are flawed because they fail to distinguish nociception and nocifensive behaviors from conscious pain.34

EXAMPLES OF COMMONLY USED ANIMAL MODELS AND AN EVALUATION OF THEIR INTERPRETATION Although overlap occurs in the use of some models, they can be broadly divided into those primarily used for investigation of acute nociception–pain and those for study of chronic nociception–pain. Because of the great clinical importance of chronic pain, more diverse and specialized models are used to represent particular human pathological processes such as neuropathic, bone cancer, or arthritis pain. In using waking animals for studies of nociception–pain, there is the ever-present issue of humane treatment.6,8 Investigators are bound by ethics and statute to minimize the exposure of animals to presumably painful procedures, within the objectives of a study. This requirement has shaped the development of the animal models for nociception–pain research toward the use of relatively low-intensity, brief-duration stimuli. The welfare consideration has been an impetus to develop in vitro models or use species (such as amphibians; see Chapter 37 by Stevens in this volume) viewed as less likely to have a capacity for pain experience. Accordingly, as addressed below, these animal models must be validated for their intended application. CONSTRUCTS AND TERMINOLOGY Nociception (and frequently pain) is a normal consequence of noxious stimulation, of course, but modifications of normal nociception–pain are more commonly of clinical importance. These include allodynia, hyperalgesia, analgesia, dyesthesia, paresthesia, causalgia, and neuropathic pain (Table 36–1). These terms represent human clinical complaints that animal models are intended to represent, but because some of these complaints, like paresthesia or causalgia, are identified by subjective reports, it is impossible to know how well the animal model represents them. There are two main ways to categorize nociception/pain models1: (1) by stimulus duration, such as short duration (acute) stimuli and longer duration (chronic) stimuli, or (2) by level of the nervous system presumed to mediate the responses (Table 36–28). Ideally, a model should meet the following criteria: specificity, sensitivity, construct validity, predictive validity, and reliability (Table 36–1). The Commonly Used Models Contemporary models vary greatly in sophistication and validity but the predominantly used behavioral models employ rats or mice and are based on spinal reflexes such as tail flick and paw withdrawal and entail, variously, measures of response threshold and/or latency. Additional measures associated with paw withdrawal are paw lifting, flinching, guarding, and licking. The most frequently used tests of nociception have been the tail flick, hot plate, paw pressure, writhing, and formalin tests.6,8 The tail flick test entails radiant heat application to a localized tail region or immersion in preheated water. Tail flick is known to be a spinal reflex, but is likely modulated by descending influences from the brainstem.6,8,24 A variation on the tail flick test is the Hargreave’s test in which heat is

Table 36–1 Constructs and terminology Allodynia: pain caused by a normally innocuous stimulus. Analgesia: a consequence of a manipulation, such as drug administration, that causes a previously pain-provoking stimulus to become nonpainful. Causalgia: an abnormal spontaneous or stimulus-evoked burning sensation, generally due to neuropathic conditions. Dyesthesia: an unpleasant abnormal sensation. Hyperalgesia: intensification of the painfulness of a normally painproducing stimulus. Neuropathic pain: diverse pain experiences, including allodynia, hyperalgesia, and spontaneous pain due to a nervous system lesion or disease. Paresthesia: an abnormal spontaneous or stimulus-evoked sensation. Specificity: the stimuli used should be genuinely nociceptive. In practice, test stimuli may activate combinations of nociceptive and nonnociceptive sensory afferents. The behavioral response model should distinguish nonnociceptive from nociceptive stimuli. Sensitivity: it should be possible to detect a range of responsiveness across a range of stimulus magnitudes, from below to above the nocifensive response threshold. Ideally, the model should also be sensitive to pharmacological or other manipulations that might modify responsiveness. Construct validity: a most critical but often overlooked criterion is that the model should actually be an indication of nociception or pain. It is particularly important to know if the model is actually assessing pain as opposed to nociception alone. Put another way, the model should really be assessing the process or variable that it is thought to assess. If bone cancer pain in humans is of fundamental interest, the model must realistically replicate key pathophysiological and psychological attributes of human bone cancer pain. This issue emerges in many contexts, but is particularly salient in consideration of models that do not employ behavioral measures, such as histological or gene expression measures. Predictive validity: effects of pharmacological or other therapies on the animal model should predict the effects of these treatments on human pain. Reliability: The model must give the same results each time it is used, within and between laboratories, to make the results obtained adequately generalizable.

applied to the plantar surface of a foot and withdrawal latency is measured. In the hot plate test the animal is placed on a heated metallic plate and latencies for paw licking and jumping responses are recorded. These behaviors are mediated through a combination of spinal and brainstem processes (Table 36–2). In the plantar pressure test, increasing force is applied on the plantar surface of a foot with a von Frey-type filament and threshold pressure for paw withdrawal is monitored. The latter two responses involve supraspinal, probably brainstem control (Table 36–2). Electrical stimulation of the tail or paw has also been frequently used to elicit a hierarchy of responses, including twitching, escape behavior, vocalization, and biting the electrodes, which are thought to reflect progressively more complex neural mediation. Dental pulp stimulation has also been used for the purpose of simulating trigeminal nociception–pain states and also

CHAPTER 36 / ANIMAL MODELS OF NOCICEPTION AND PAIN

337

Table 36–2 Common nociception testing paradigms,a observed behavioral outcomes, and their presumed neural substrates Stimulus modality

Testing paradigmsb

Quantified behaviors

Minimal central substratesc

Mechanical Thermal

Hargreave’s (von Frey) Tail immersion (hot or cold), tail flick, hot plate, cold plate, Hargreave’s (radiant heat) Formalin, acid, capsaicin, taxol

Withdrawal Withdrawal, jumping, escape, licking, guarding

Segmental (spinal) Segmental and suprasegmental (brainstem)

Licking, biting, guarding, altered posture, writhing, vocalizing

Segmental and suprasegmental

Chemical a

Employing natural stimuli; nonnatural stimuli (e.g., electrical stimulation) are not included; see Le Bars et al.6 Reviewed in Mogil et al.48 c Reviewed in Vierck.8

b

on the (questionable6) assumption that all the associated afferents are nociceptors. The commonly used formalin and writhing tests exemplify tests eliciting long-duration nociceptive stimulation by irritant injection. Intradermal formalin injections are most common, but hypertonic saline, complete Freund’s adjuvant, capsaicin, bee venoms, and other agents have been used. When injected into the dorsal surface of a rat’s forepaw, a 0.5–15% formalin solution evokes a constellation of quantifiable behaviors, including reduced weight bearing on the paw, paw lifting, licking, nibbling, shaking, or biting. Irritant injection into the footpad of rats or mice, in contrast, produces a biphasic response, with an initial phase of about 3 min latency, a subsequent period during which responses dissipate, and a later (20–30 min latency) reappearance of behavioral reactions, which appears to stem from predominant activation of C fiber nociceptors.8 The writhing test involves intraperitoneal injection of any of several irritants, including phenylbenzoquinone, acetylcholine, dilute hydrochloric or acetic acid, and bradykinin. These substances irritate serous membranes of the peritoneal cavity and in rats and mice evoke abdominal contractions, large body movements, including the hind paws, asymmetric contraction of dorsal abdominal muscles, and reduced locomotion. Although intended to be a model for visceral pain, it is likely that somatic afferents are also activated by the algogenic substances. Models using stimulation of hollow organs, particularly gastrointestinal or urogenital structures, have taken various forms: injection of formalin or other algogenic substances into the colon, capsaicin injection into the bladder, distention of organs like the colon and rectum with an inflatable balloon, or solid material introduction into a ureter. The behaviors produced depend to some extent on the specific type and location of the nociceptive stimulus, but include various types of abdominal reflexive contractions, stretching, and abdominal licking. Models specifically designed to investigate chronic nociception–pain often include some of the same behavioral measures and nociceptive treatments previously described for investigations of acute nociception–pain. For example, the threshold nociceptive pressure required for paw withdrawal can be assessed in a normal rat by pressure with a filament (the von Frey test), but this method can test hyperalgesia after injection of an irritant like formalin into a paw. Likewise, this test can be used to assess allodynia associated with neuropathic pain caused by direct nerve injury or chronic nerve constriction. Interpretations of the Commonly Used Behavioral Models The above-described tests are widely used because they

appear to have face validity, that is, they appear to produce states that would be comparable to those that would be associated with pain in humans. In addition, the most widely used of these, like the hot plate and intradermal formalin tests, are relatively easy to perform and score on large numbers of animals, thereby lending themselves to dose–effect studies of potentially analgesic drugs or other antinociceptive manipulations. The seeming simplicity of such tests is illusory. Detailed critiques of the limitations in these models have been presented by Le Bars et al.,6 Blackburn-Munro,3 and Vierck,8 and the interested reader is urged to read these informative and thoughtful reviews. A detailed discussion of the technical and theoretical limitations of the various nociception–pain models is not possible here, but we will outline the major concerns. The first such as noxious temperature paradigms (e.g., tail flick, hot plate, Hargreave’s) or paw withdrawal tests, there are many poorly controlled and variable aspects to nociceptive stimulus application. For example, a rat’s tail is a thermal exchange organ, which places it in a very different functional category from other tissues regarding effects of thermal stimuli.6 Furthermore, in the hot plate test, the animal’s movements make thermal stimulus application highly variable. Although nociceptive stimuli are often administered in ways intended to avoid or minimize tissue trauma, it may result in variable degrees, thereby introducing the dimension of allodynia or hyperalgesia in a test where these effects are not intended. Effects of injected algogenic stimuli will differ depending on the specific agent used (formalin, capsaicin, mustard oil, etc.) and route of administration (intraperitoneal, in the bladder) presenting a complex stimulus, to say the least. In addition, nonnociceptive afferent activation is often a confounding variable in that those afferents may contribute to the elicitation of responses that are presumed to be purely nocifensive. Response measures themselves also present difficulties in the foregoing test paradigms.6,8 Investigator judgments are frequently a major source of variation in quantifying responses. Repeated testing of individual animals in some of the procedures, like the hot plate test, can be associated with learned aversion behaviors that modify stimulus application as well as the character of the responses. Sometimes tests of threshold become confounded with response latency, especially with longer duration stimuli. In addition, activation of nonnociceptive afferents in testing paradigms (above) may elicit flexion reflexes, confounding the interpretation of the nociceptive nature of the stimulus and response. Genetic variability and strain differences can also be a major source of confounding variability in a number of models.6,8

338

SECTION IV / MODELS FOR SPECIFIC PURPOSES

In most cases, the responses examined in the most frequently used tests such as tail flick, hot plate, paw withdrawal, and their variations could be entirely mediated by spinal reflexes or brainstem–spinal motor programs, thus constituting unconscious nocifensive responses (Table 36–28). Nonetheless, some higher brain influence is probably operating in an intact, awake animal, but its presence and nature are hard to separate from subcortical processes and it is also likely to be unconsciously mediated. Consequently, none of these tests can be legitimately viewed as tests of pain, since they do not depend on a consciously mediated response. In some cases, investigators are aware of this constraint and strictly adhere to the term nociception rather than pain in interpreting their results. Unfortunately this is far from a universal practice and erroneous language and inference are common. Frequently “pain processing” or “pain transmission” is used to describe what is clearly nociceptive processing at the receptor, spinal, or subcortical level. Statements that can be found in the literature, such as pain in labor originates in the cervix or that there is inflammatory pain in the bladder, are mechanistically incorrect by implying that the conscious, psychological process of pain is somehow produced and experienced by peripheral tissues. Development of well-validated models for pain, as opposed to nociception, is one of the most significant challenges to this field. To this end, some investigators have utilized diverse paradigms requiring behaviors clearly beyond the realm of reflexes or complex, but probably unconsciously mediated motor programs. Behaviors that are more complex than stimulus-bound flexion reflexes and are directed at the site of nociceptive stimulation, or sustained after stimulus termination, such as the vocalization afterdischarge, are sometimes taken to reflect pain. But recall that even decerebrate rats are capable of such behaviors. Measures involving learned behaviors, tasks where the animal can regulate nociceptive stimulus intensity, and assessments of disrupted feeding, sleep, or social behavior may have more validity as reflecting pain.3,8 Presumably, these types of behaviors reflect more complex, higher-order processing in the brain, but in the absence of rigorous validation, their acceptance as indications of pain should be considered tentative. Interpretation of Nonbehavioral Measures of Nociception/Pain Numerous reduced preparations, even in vitro models, are being used for studies of nociception–pain. Tissue response measures, such as neuronal expression of the early oncogene c-fos42 and other markers of gene expression,43,44 are potentially associated with processes of importance for nociception, but their significance must be qualified because they are quite removed from pain or even the dynamic, functional context of nociception. Expression of c-fos is commonly interpreted as an indication of neuronal response to sensory, including nociceptive stimuli. But unlike c-fos expression, neuronal responses to nociceptive stimuli are not all or none, not always excitatory, and not invariant. Rather, they may entail changes in pattern or inhibition, all of which are mechanistically important, but undetectable with markers such as c-fos. In addition, the proportion of neurons that expresses c-fos is often much smaller than the population that actually responds electrophysiologically42,45 and neurons in some neurophysiologically responsive brain regions do not express cfos.42,45 There are additional problems with false-positive responses and dissociation between behavioral and c-fos responses to nociceptive stimuli.42 Clearly, these indirect measures, like behavioral

measures, require careful validation with respect to what they do or do not mean. Likewise, interpretations from response measures such as reflexes or neurophysiological recordings obtained under anesthesia are also subject to qualification. Measures of brain neuronal activity in animals or measures used in humans, such as functional magnetic resonance imaging, alone are never proof of pain. The relationship of such indicators to pain can be assessed only after pain has independently been shown to coexist with and be essential for putative pain-related properties of such neurophysiological correlates. Furthermore, nothing short of an intact, fully functioning animal, particularly a mammal, is a potentially suitable model for investigations of human-like pain. Highly reduced preparations are primarily valuable for understanding some elements of nociceptive signaling or peripheral pathophysiological processes that might initiate nociceptive signaling.

RECOMMENDATIONS FOR MODEL SELECTION AND INTERPRETATION This chapter has stressed the importance of critically examining the validity of animal models to foster progress in understanding mechanisms of pain and the development of more effective treatments. To this end, we propose that particular consideration be given to the following questions: (1) is the problem or phenomenon of interest expressed at the level of nociceptive processing and nocifensive responses or the higher-order, end stage of pain; (2) is the response measure valid and appropriate for the question under investigation; (3) how valid are assumptions concerning the equivalence between the animal model and the human pain condition; and (4) is peripheral nociception of greatest importance, or is it essential to evaluate pain specifically? The difficulty of knowing how well the putative pain experience of an animal such as a rat or mouse translates to a human-like experience is likely to remain a limiting factor in development of models that validly assess the psychological experience of pain. Future success in development of pain therapies may often hinge on more exacting attention to this issue. There is a good deal of disagreement between investigators of animal psychology on which species might manifest consciousness, a prerequisite to pain experience as we know it,34,46 but there is a different approach to the question of pain. The dependence of the suffering dimension of human pain on cortical functioning, especially the cingulate gyrus, insula, and prefrontal cortex, is now well established.2,13,14,22,23 It is also commonly and probably safely assumed that similar cortical regions, where present, work in at least roughly similar ways across mammalian species. On this basis, it would be possible to provide a preliminary validation of a putative animal model for pain by showing that behaviors allegedly reflecting it depend on the functional integrity of these painmediating cortical zones. There would still be potential for misinterpretation by confusing nocifensive behaviors with paindependent behaviors, but by placing the control of the response measure at the same cortical regions known to be essential to pain experience in humans, the potential for examining common mechanisms would be greatly facilitated. There is currently evidence that investigators of pain are using this approach.8,47

REFERENCES 1. McMahon SB, Koltzenburg M, Eds. Wall and Melzack’s Textbook of Pain, 5th ed. Philadelphia: Elsevier Churchill Livingstone, 2006.

CHAPTER 36 / ANIMAL MODELS OF NOCICEPTION AND PAIN

2. Bushnell CM, Apkarian AV. Representation of pain in the brain. In: McMahon SB, Koltzenburg M, Eds. Wall and Melzack’s Textbook of Pain, 5th ed. Philadelphia: Elsevier Churchill Livingstone, 2006:107–124. 3. Blackburn-Munro G. Pain-like behaviours in animals–how human are they? Trends Pharmacol Sci 2004;25:299–305. 4. Hill R. NK1 (substance P) receptor antagonists–why are they not analgesic in humans? Trends Pharmacol Sci 2000;21:244–246. 5. Villanueva L. Is there a gap between preclinical and clinical studies of analgesia? Trends Pharmacol Sci 2000;21:461–462. 6. Le Bars D, Gozariu M, Cadden SM. Animal models of nociception. Physiol Rev 2001;53:597–652. 7. Hogan Q. Animal pain models. Reg Anesth Pain Med 2002;27: 385–401. 8. Vierck CJ. Animal models of pain. In: McMahon SB, Koltzenburg M, Eds. Wall and Melzack’s Textbook of Pain, 5th ed. Philadelphia: Elsevier Churchill Livingstone, 2006:175–186. 9. Bond MR. Psychiatric disorders and pain. In: McMahon SB, Koltzenburg M, Eds. Wall and Melzack’s Textbook of Pain, 5th ed. Philadelphia: Elsevier Churchill Livingstone, 2006:259–266. 10. Wall PD. Pain: Neurophysiological mechanisms. In: Adelman G, Smith B, Eds. Encyclopedia of Neuroscience, 2nd ed. Amsterdam: Elsevier, 1999:1565–1567. 11. Laureys S, Goldman S, Phillips C, Van Bogaert P, Aerts J, Luxen A, Franck G, Maquet P. Impaired cortical connectivity in vegetative state: Preliminary investigation using PET. Neuroimage 1999;9:377– 382. 12. Young GB, Ropp AH, Bolton CF. Coma and Impaired Consciousness. New York: McGraw-Hill, 1998. 13. Derbyshire SWG. Locating the beginnings of pain. Bioethics 1999;13:1–31. 14. Rose JD. The neurobehavioral nature of fishes and the question of awareness and pain. Rev Fisheries Sci 2002;10:1–38. 15. Beecher HK. Measurement of Subjective Responses. New York: Oxford University Press, 1959. 16. Carlen PL, Wall PD, Nadvrona H, Steinbach T. Phantom limbs and related phenomena in recent traumatic amputations. Neurology 1978;28:211–217. 17. Melzack R, Wall PD, Ty TC. Acute pain in an emergency clinic: Latency of onset and descriptor patterns. Pain 1982;14:33–43. 18. Ramachandran VS, Rogers-Ramachandran D. Synaesthesia in phantom limbs induced with mirrors. Proc Biol Sci 1996;263:377– 386. 19. Price DD, Rainville P. Hypnotic analgesia. In: McMahon SB, Koltzenburg M, Eds. Wall and Melzack’s Textbook of Pain, 5th ed. Philadelphia: Elsevier Churchill Livingstone, 2006:329–338. 20. Craig AD. How do you feel? Interoception: The sense of the physiological condition of the body. Nat Rev Neurosci 2002;3:655–666. 21. Dostrovsky JO, Craig AD. Ascending projection systems. In: McMahon SB, Koltzenburg M, Eds. Wall and Melzack’s Textbook of Pain, 5th ed. Philadelphia: Elsevier Churchill Livingstone, 2006:187–204. 22. Price DD. Psychological Mechanisms of Pain and Analgesia. Seattle: International Association for the Study of Pain, 1999. 23. Treede RD, Kenshalo DR, Jones AKP. The cortical representation of pain. Pain 1999;79:105–111. 24. Fields HL, Basbaum AI, Heinricher HH. Central nervous system mechanisms of pain modulation. In: McMahon SB, Koltzenburg M, Eds. Wall and Melzack’s Textbook of Pain, 5th ed. Philadelphia: Elsevier Churchill Livingstone, 2006:125–142.

339

25. Pastor J, Soria B, Belmonte C. Properties of nociceptive neurons of leech segmental ganglion. J Neurophysiol 1996;75:2268–2279. 26. Dewsbury DA, Rethlingshafer DA. Comparative Psychology, a Modern Survey. New York: McGraw-Hill, 1973. 27. Jouvet M. Coma and other disorders of consciousness. In: Vinken PJ, Bruyn GW, Eds. Handbook of Clinical Neurology. New York: Elsevier, 1969;3:62–79. 28. Thogmartin JR. Report of autopsy #5050439, Theresa Schaivo, Pasco and Pinellas Counties, Florida, 2005. 29. Wahlestedt C. Neuropharmacology: Reward for persistence in substance P research. Science 1998;281:1624–1625. 30. Keller M, Montgomery S, Ball W, Morrison M, Snavely D, Guanghan L, Hargreaves R, Hietala J, Lines C, Beebe K, Reines S. Lack of efficacy of the substance P (neurokinin1 receptor) antagonist aprepitant in the treatment of major depressive disorder. Biol Psychiatry 2006;59:216–223. 31. Ramadan NM. Acute treatments: Some blind alleys. Curr Med Res Opin 2001;17(Suppl. 1):s71–80. 32. Stevens CW. Opioid research in amphibians: An alternative pain model yielding insights on the evolution of opioid receptors. Brain Res Rev 2004;46:204–215. 33. Chandroo KP, Duncan IJH, Moccia RD. Can fish suffer?: Perspectives on sentience, pain, fear and stress. Appl Anim Behav Sci 2004;86:225–250. 34. Rose JD. Anthropomorphism and “mental welfare” of fishes. Dis Aquat Org 2007;75(2):139–154. 35. Northcutt RG, Kaas JH. The emergence and evolution of mammalian neocortex. Trends Neurosci 1995;18:373–379. 36. Nieuwenhuys R, ten Donkelaar HJ, Nicholson C. The Central Nervous System of Vertebrates. Berlin: Springer, 1998. 37. Avian Brain Nomenclature Consortium. Avian brains and a new understanding of vertebrate brain evolution. Nat Rev Neurosci 2005;6:151–159. 38. Mountcastle VB. Perceptual Neuroscience. Cambridge, MA: Harvard University Press, 1998. 39. Woods JW. Behavior of chronically decerebrate rats. J Neurophysiol 1964;27:635–644. 40. Bateson P. Do animals feel pain? New Sci 1992;134:30–33. 41. Macphail EA. The Evolution of Consciousness. New York: Oxford University Press, 1998. 42. Harris J. Using c-fos as a neural marker of pain. Brain Res Bull 1998;45:1–8. 43. Pace MC, Mazzariello L, Passavanti MB, Sansone P, Barabarisi M, Aurilio C. Neurobiology of pain. J Cell Physiol 2006;209:8–12. 44. Lacroix-Fralish ML, Tawfik VL, Tanga FY, Spratt KF, DeLeo JA. Differential spinal cord gene expression in rodent models of radicular and neuropathic pain. Anesthesiology 2006;104:1283–1292. 45. Alexander BM, Rose JD, Stellflug JN, Fitzgerald JA, Moss GE. Foslike immunoreactivity in brain regions of domestic rams following exposure to rams or ewes. Physiol Behav 2001;73:75–80. 46. Wynne CDL. The perils of anthropomorphism. Nature 2004;428: 606. 47. Johansen JP, Fields HL, Manning BH. The affective component of pain in rodents: Direct evidence for a contribution of the anterior cingulate cortex. Proc Natl Acad Sci USA 2001;98:8077– 8082. 48. Mogil JS, Wilson SG, Bon K, Lee SE, Chung K, Raber P, Pieper JO, Hain HS, Belnap JK, Hubert L, Elmer GI, Chung JM, Devor M. Heritability of nociception I: Responses of 11 inbred mouse strains on 12 measures of nociception. Pain 1999;80:67–82.

37

Nonmammalian Models for the Study of Pain CRAIG W. STEVENS

ABSTRACT

Pain research is the study of neural pathways that convey noxious stimuli and the investigation of substances or manipulations that alter the neurotransmission of nociceptive information. As the goal of pain research is to generate safer and more effective treatments for patients in pain, this field of research is better described as “analgesia research,” but this more accurate term is not widely used. Unlike other types of biomedical research employing nonhuman animals, pain and analgesia research has an added ethical component due to the testing of analgesic agents (or manipulations) by first inducing a “painful” state in the animal subject by the application of a noxious stimulus. The effectiveness of the analgesic treatment is determined by the difference of treatment values from baseline values in the duration or intensity of the noxious stimulus at the time the animal exhibits a behavior (a nocifensive behavior) that signals nociception. Though most exposures to the noxious stimuli in the experimental setting are brief (like the hot plate and tail-flick algesiometric tests in rodents), a number of persistent pain models are used to more closely mimic the clinical scenario of chronic pain patients. The core of the ethical concern in pain and analgesia research, like many

ethical issues, comes from a basic lack of knowledge: the capacity of nonhuman animals to experience nociceptive transmission as humans do, i.e., “pain,” is not known. It is unknowable because “pain” is a conscious perception and it is not known if nonhuman animals have the capacity for consciousness that may be needed for the “pain” perception to exist. To think otherwise is not science, but science fiction. But science can guide rational thought and based on the comparative neurology of central nervous system (CNS) pathways and brain structures shown to be important for nociceptive transmission and supraspinal appreciation of pain in mammalian studies, it was proposed that the potential for pain, if it exists in nonhuman animals, is less in earlier-evolved vertebrates than in mammals.1 This idea arose from considerations of an amphibian pain model,2–4 but similar conclusions were made in the discussion of pain and awareness in fish.5 Thus, a continuum of the potential for pain likely exists among vertebrate classes, perhaps correlated with the capacity for consciousness, and the relative difference in this potential for pain between, say, a frog and a rat may justify developing an alternative or adjunct* nonmammalian model for pain and analgesia research on ethical grounds.6 The scientific basis for the use of adjunct models for analgesia research depends on the value of a comparative approach to address the question at hand. For example, the value of investigating mechanisms of opioid analgesia in adjunct models using nonmammalian vertebrates has enriched the understanding of opioid receptors and the selectivity of opioid drugs, as highlighted at the end of this chapter. While there are a great number of pharmacological and nonpharmacological means to modulate nociceptive transmission and therefore many avenues of investigation in pain research, this chapter will focus on nonmammalian models developed primarily to investigate the role of opioids in producing antinociception in behavioral models. An initial review of current knowledge of the nociceptive pathways and endogenous opioid systems in nonmammalian vertebrates is followed by a review of pain and analgesia studies by vertebrate class. A more detailed examination of the methodology and results using an amphibian model follows next. The chapter closes with a “case study” of a comparative approach to investigate the binding of opioid analgesics that leads to an increased understanding of the evolution of vertebrate opioid receptors.

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

*The use of “adjunct model” avoids any connotation present in the term “alternative model.”

Processing nociceptive information is a consistent feature of the nervous system in all vertebrate species. This chapter reviews the nonmammalian models developed for pain and analgesia research, with a special emphasis on models that were used for detecting opioid antinociception. Nociceptive pathways in nonmammalian vertebrates are reviewed and endogenous opioid systems are described. Compared to mammalian models for pain research, there are relatively few models in each vertebrate class. Details are provided for the methods and the results of using an amphibian model for the testing of opioid analgesics. The chapter ends with a case study of the comparative approach in pain and analgesia research applied to the molecular evolution of opioid receptor proteins. Key Words: Opioids, Pain, Analgesia, Animal models, Amphibians, Evolution.

INTRODUCTION: ALTERNATIVES TO THE USE OF MAMMALS FOR PAIN RESEARCH

341

342

SECTION IV / MODELS FOR SPECIFIC PURPOSES

NOCICEPTIVE AND ENDOGENOUS OPIOID SYSTEMS IN NONMAMMALIAN VERTEBRATES This section presents a summary of the existing scientific literature on nociceptive processing and endogenous antinociceptive systems in fish, amphibians, reptiles, and birds. It should be noted that there are relatively few data available on nociception in nonmammalian vertebrates, and as shown in the next section, even fewer data on the action of opioid analgesic agents. However, the basic plan of the nervous system is consistent in all vertebrates, with the major difference being a lack of neocortical brain development in earlier-evolved vertebrate species. Thus, nociceptive transmission at the lower levels in the nervous system (nociceptive primary afferent fibers and spinal cord pathways) is homologous in all vertebrates. In contrast, pain processing at higher, supraspinal centers arising later in vertebrate phylogeny may not be present in fish, amphibians, reptiles, and birds, which do not share homologous supraspinal structures. NOCICEPTIVE PATHWAYS IN EARLIER-EVOLVED VERTEBRATES Noxious information in mammals is parallel processed throughout the nervous system, with separate pathways for the sensory-discriminative and motivational-affective aspects of pain perception.7 To some extent, this duality of pain perception begins with the primary afferent fiber type: thinly myelinated Aδ fibers initiate the sensory-discriminative pathway and the unmyelinated C fibers may signal the motivational-affective component of pain. In mammals, the highest level termination of the sensory pathways is the sensory neocortex and the destination of motivational pathways is the limbic neocortex.8,9 Electrophysiology experiments using dissected nerves and characterizing single nociceptive primary afferent fibers with defined nociceptive receptive fields are not routinely done using nonmammalian species. However, there are a number of studies characterizing nociceptive pathways in the rainbow trout, Oncorhynchus mykiss. Anatomical data and electrophysiological examination of single units recorded from trigeminal ganglia cells identified nociceptors and nociceptive afferents that were not significantly different from those found in other vertebrates.10,11 Notable differences include more Aδ fibers and less C fibers in fish trigeminal nerve than in mammals.12 There are few studies of the central termination of nociceptive afferents in fish spinal cord, although substance P, a marker of nociceptive primary afferent fibers, was localized densely in the superficial aspect of the substantia gelatinosa and scattered throughout the nucleus proprius, intermediate zone, and the ventral horn.13 Investigations of amphibian primary afferent fibers, including nociceptive afferents, were among the first studies characterizing sensory fibers in vertebrates.14–16 In contrast to numerous specialized end organs on mammalian afferent fibers, only three groups of afferent nerve endings were identified in amphibians. These include an “expanded tip” morphology ending at the superficial level of the dermis and two types of free nerve endings: one type terminating in the epidermis and the other ending in the deeper layers of the dermis.17–19 Based on early electrophysiological work, the detection of all noxious sensory stimuli, including chemical, was attributed exclusively to the free nerve endings associated with thermal sensitivity in amphibians.16 Amphibians possess both myelinated and unmyelinated afferent fibers running concurrently in mixed-fiber peripheral sensory nerves. Investigators delineate these fibers into three general classes based on

morphology, conduction velocities, latency of response, and action potential characteristics: large heavily myelinated A fibers, thinly myelinated B fibers, and small unmyelinated C fibers. Their characteristics correlate well with those observed in corresponding classes of somatic sensory afferents in mammals: Aβ, Aδ, and C fibers, respectively. In general, the cutaneous nociceptors in frog skin (those responding to noxious thermal, mechanical, and chemical stimuli) arise from peripheral terminations of the thinly myelinated C fibers. In studies that separated fibers both by conduction velocity and fiber diameter in amphibians, small slowly conducting fibers transmitted the majority of all impulses induced by noxious heat, pinching, pin pricks, and the application of dilute acid to the skin.16,20–22 There is controversy over the central terminations of primary afferent fibers within the amphibian spinal gray,23 although most studies indicate that sensory afferents from the skin terminate in areas of the dorsal spinal cord that correspond to laminae I–IV. The termination of small-caliber fibers associated with nociception was found exclusively in the superficial dorsal laminae (the substantia gelatinosa), while only larger fibers associated with transmission of nonnoxious information penetrate to the deeper laminae.24,25 Electrophysiological evidence supports the existence of primary afferent synapses on motoneuron dendrites within the amphibian dorsal horn. Little research has been done on the nociceptive pathways of reptiles. Reptiles also appear to possess only simple nonencapsulated corpuscular endings and free nerve endings, both of which are distributed throughout the dermis and epidermis.26,27 The separation of receptor structures into categories of noxious and nonnoxious sensory function appears less discreet in the reptile and, thus far, no stimulus-specific receptor structures have been identified.28,29 Likewise, there are remarkably few data available concerning peripheral fiber types in reptiles. In contrast to reports on amphibians, researchers report only a unimodal distribution of sensory fiber sizes, which extends over the sizes of the B and C classes of amphibian fibers.28 Furthermore, all stimulus modalities elicit responses throughout both ranges of conduction velocity and fiber size. Additionally, the dorsal roots containing peripheral sensory afferents entering the spinal cord do not clearly segregate according to large and small fibers as in higher vertebrates.30 Thus it is difficult to classify any specific subgroup of reptilian sensory afferents as primarily nociceptive. However, at least in the turtle, a separation of peripheral fiber types according to their sites of termination in the spinal cord was shown.31 Smaller type A fibers associated with cutaneous sensation and nociception arborized primarily in the superficial laminae (I–III) as compared to muscle spindle and joint proprioceptive fibers that penetrated to the deeper laminae (IV–VII) where they formed monosynaptic reflex pathways with the dendritic endings of motor neurons. Investigation of nociceptive pathways in birds utilized the domestic chicken, Gallus gallus. Electrophysiological recordings of single afferent fibers from teased-apart leg nerves identified both mechanical and thermal nociceptors.32 The majority of responses were classified as C fibers responding to both mechanical and thermal noxious stimuli, but Aδ fibers were also noted. Nociceptive afferents innervating chicken skeletal muscle respond to noxious mechanical stimulation (muscle compression) and also to the injection of acetic acid into the muscle tissue.33 Further studies in chickens showed that nasal trigeminal nociceptors are activated by ammonia vapor,34 that joint nociceptors are activated

CHAPTER 37 / NONMAMMALIAN MODELS FOR THE STUDY OF PAIN

343

Table 37–1 m, d, and k opioid receptor proteins cloned and sequenced in nonmammalian vertebrates Access numbera Class

Common name

Genus species

MOR

DOR

KOR

References

Pisces Amphibia Amphibia

Zebrafish Grass frog Newta

Danio rerio Rana pipiens Taricha granulosa

AF132813 AF530571 AY751784

AJ001596 AF530572 AY751785

AF285173 AF530573 AY725197

138–140 4, 149 146, 147

a

Access numbers for the nucleotide sequences deposited in GenBank at http://www.ncbi.nlm.nih.gov/Genbank/index.html.

by the intraarticular injection of urate crystals,35,36 and that beak amputation stimulates the firing of nociceptive afferents in the trigeminal nerve.37 The spinal cord projections of ankle joint primary afferents were mapped to the upper layers of the chicken spinal cord dorsal horn38 and substance P-containing fibers terminate in laminae I–II.39,40 SUPRASPINAL PATHWAYS OF NOCICEPTION There is scant literature on the supraspinal pathways that may process nociceptive information in nonmammalian vertebrates. Functional studies of evoked responses in supraspinal sites after specific noxious stimulation of peripheral nociceptive fibers are lacking; however, electrical stimulation of the sciatic nerve produced evoked potentials in posterior thalamic nuclei and primordial hippocampal structures in frogs.41 Single unit recordings in trout and goldfish (Carassius auratus) obtained from electrodes placed in the spinal cord, cerebellum, tectum, and telencephalon after timed noxious stimuli revealed supraspinal nociceptive pathways, but more precise mapping by histological confirmation of electrode placement was not done.42 The sensory-discriminative pathway originating from spinal neurons makes direct connections with neuron groups farther toward the front of the brain as we ascend the phylogeny of vertebrates.43 Therefore, fish and amphibians have direct spinal connections to the brainstem, reptiles and birds to the brainstem and the dorsal thalamus in the midbrain, and mammals to the brainstem, thalamus, and primary cortex. Pathways that appear to contribute to the motivational-affective dimension of pain follow a similar evolutionary pattern, except that more medial target sites are contacted in the brain: the medial thalamic nuclei in reptiles and the limbic cortex in mammals. Throughout phylogeny, all the target sites of spinal pathways in the brain increase in complexity, specialization, and number of neurons, suggesting that nociceptive messages to the thalamus in a reptile and mammal may not be comparable.44 ENDOGENOUS OPIOID PEPTIDES Nociceptive information is carried from the peripheral nociceptors to the spinal cord by the primary afferent nerve fibers in all vertebrates. These fibers terminate and synapse on second-order neurons in the dorsal horn, releasing neurotransmitters such as substance P, calcitonin gene-related peptide (CGRP), and glutamate. Using immunohistochemical techniques, all of these substances are readily identified in abundance in the spinal dorsal horn of fish, amphibians, reptiles, and birds.45–48 Substance P and glutamate excite second-order neurons that have their cell bodies in the dorsal horn and send long fibers upward to form the ascending nociceptive pathways. Such second-order neurons within the dorsal horn that receive direct dorsal root input have not yet been identified in nonmammalian vertebrates.49

Also present in the spinal cord are intrinsic neurons that release met-enkephalin, an endogenous opioid peptide. Met-enkephalin inhibits the release of substance P from the central terminations of nociceptive primary afferents and decreases the firing of the second-order pain neurons. Met-enkephalin is present in the spinal cord of all vertebrate species examined.2,39,48,50–57 In amphibians, immobilization stress produces antinociception that is blocked by the pretreatment of animals with the opioid antagonist naloxone.58,59 This finding demonstrates that enkephalinergic neurons modulate nociceptive threshold in earlier-evolved vertebrates as they do in mammals. The molecular evolution of opioid peptides and their precursors is the subject of a number of recent studies.60–64 OPIOID RECEPTORS IN NONMAMMALIAN VERTEBRATES Opioid receptors are membrane proteins belonging to the superfamily of G-protein-coupled receptors (GPCR) and are the products of four distinct genes in mammalian genomes.65 µ, δ, and κ opioid receptors, abbreviated MOR, DOR, and KOR, mediate the analgesic effects of opioids, while the role of the fourth type of opioid receptor, the nociceptin or orphanin FQ receptor (ORL), is less clear.66 Before the advent of the genomic era, binding studies using tissue homogenates demonstrated the presence of opioid-binding sites in fish,67–70 amphibian,71–76 bird,77–80 and reptile81–83 CNS. More definite evidence for the expression of opioid receptor proteins in nonmammalian vertebrates comes from the cloning and sequencing of opioid receptor mRNA. To date, µ, δ, and κ opioid receptors were cloned and sequenced in three nonmammalian vertebrates: the zebrafish, the Northern grass frog, and the rough-skinned newt (see Table 37– 1). These sequences represents two classes of nonmammalian vertebrates (Pisces and Amphibia) with no opioid receptor sequences yet available for species from the Reptilia and Aves classes. As shown at the end of the chapter, an analysis of opioid receptor sequences from nonmammalian and mammalian vertebrates demonstrates the utility of a comparative approach to investigate opioid analgesic action.

PAIN AND ANALGESIA RESEARCH USING NONMAMMALIAN VERTEBRATES Key factors in the development of a nonmammalian model for pain and analgesia research are the identification of a nocifensive behavior that is unique to a noxious stimulus and the demonstration that the same noxious stimulus solely activates nociceptive primary afferents. In an amphibian model called the acetic acid test, the wiping response is the nocifensive behavior and a drop of dilute acetic acid is the noxious stimulus that activates nociceptors and nociceptive primary afferent fibers exclusively.20,22,84 For other nonmammalian models, integration of neurophysiological

344

SECTION IV / MODELS FOR SPECIFIC PURPOSES

data with behavioral data in most cases are still lacking. A review of these adjunct models for analgesia research by vertebrate class is presented next, followed by a detailed examination of an amphibian model for analgesia research. The adjunct models for behavioral assessment of opioid antinociception are listed in Table 37–2. PISCES Fish are not easily amenable to behavioral assays of antinociception. A study aimed primarily at the metabolism of morphine in goldfish (C. auratus) also described a behavioral assay using an electric prod applied caudal to the dorsal fin as the noxious stimulus.85 The nocifensive behavior that was observed was called the agitated swim response. The voltage threshold for eliciting the swim response was consistent for each animal and with repeated testing. Morphine sulfate added to the tank water produced a dose-dependent increase in the electrical voltage

needed to elicit the agitated swim response. It was also shown that goldfish became tolerant to the antinociceptive effects of morphine with repeated administration.85 There was no attempt to attain a measure of morphine’s potency (i.e., no ED50 value of morphine was reported) nor was opioid receptor involvement verified by pretreatment with an opioid antagonist such as naloxone. Further studies using the same model in goldfish showed that morphine administered via the intracranial route (into a space above the optic tectum) produced a dose-dependent increase in the voltage needed to elicit the swim response.86 In this study, opioid receptor involvement was shown by naloxone antagonism of morphine’s effect. The antinociceptive effects of dermorphin, a potent and selective MOR opioid peptide originally isolated from the skin of Phyllomedusa frogs,87 was tested using electrodes implanted in

Table 37–2 Behavioral models for testing opioid antinociception in nonmammalian vertebrate species Class

Animal/(genus species)

Model

Stimulus

Response

Notes

Pisces

Goldfish (Carasius auratus)

Caudal electroshock

Electric current by handheld prod

Agitated swim response

Pisces

Codfish (Gadus orhuamarisalbi)

Caudal electroshock

Electric current by implanted electrode

Agitated swim response

Pisces

Rainbow trout (Oncorhynchus mykiss)

Caudal electroshock

Electric current by implanted electrode

Agitated swim response

Pisces

Carp (Cyprinus carpio)

Caudal electroshock

Electric current by implanted electrode

Agitated swim response

Pisces

Rainbow trout (Oncorhynchus mykiss)

Lip inflammation

Pain-related behaviors

Amphibia

European frog (Rana esculenta)

Electrified grid

Acetic acid/bee venom lip injection Electric current in floor grid

Amphibia

European frog (Rana esculenta)

Hot plate test

Heated surface

Jumping

Amphibia

Northern grass frog (Rana pipiens)

Acetic acid test

Dilute acetic acid

Wiping response

Amphibia

Japanese firebelly newt (Cynops pyrrhogaster)

Tail-flick test

Heat from lamp

Tail-flick

Acute model; measured agitated swim response (ASR); morphine added to tank water or intracranial; blocked by naloxone 85,86 Acute model; ASR quantified by forcetransducer; dermorphin given by intranasal application; βcasomorphin given by IP and IM injection88,90 Acute model; ASR quantified by forcetransducer; dermorphin given by intranasal application89 Acute model; ASR quantified by forcetransducer; tramadol given by IM injection; blocked by naloxone91 Chronic model; pain-related behaviors quantified; morphine effects noted92–95 Acute model; no antinociceptive effect of morphine96 Acute model; no antinociceptive effect of morphine97 Acute model; welldeveloped and validated adjunct model; sensitive to opioid and nonopioid analgesics, stress- and hypothermia-induced analgesia58,59,98,99,121–131,136 Acute model; effects of metenkephalin and RFamide blocked by naloxone100

Jumping

CHAPTER 37 / NONMAMMALIAN MODELS FOR THE STUDY OF PAIN

345

Table 37–2 (continued) Class

Animal/(genus species)

Model

Stimulus

Response

Notes

Reptilia

Green anole lizard (Anolis carolinensis) Nile crocodile (Crocodylus niloticus)

Tail-flick test

Heat from lamp

Tail-flick

Hot plate test

Heated surface

Leg-lift or escape response

Reptilia

Green iguana (Iguana iguana)

Tail-flick test

Heat from thermal electrode

Tail-flick

Aves

Chicken (Gallus gallus)

Toe-pinch

Noxious mechanical

Limb withdrawal

Aves

Chicken (Gallus gallus) Chicken (Gallus gallus)

Wing-shock

Electric current to underside of wing Noxious thermal to feet

Flight response

Aves

Chicken (Gallus gallus)

Formalin test

Noxious chemical in foot

Leg-lifting

Aves

Chicken (Gallus gallus)

Carrageenan inflammation

Aves

Chicken (Gallus gallus)

Monoarthritis model

Inflammationinduced thermal hyperalgesia Urate crystals in joint capsule

Foot withdrawal from thermal stimulus Pain-related behaviors

Acute model; antinociceptive effects of morphine101 Acute model; antinociceptive effects of morphine and meperidine (pethidine)103 Acute model; preliminary investigation of butorphanol104 Acute model; no antinociceptive effect of morphine105 Acute model; morphine antinociception106 Acute model; morphine, codeine hyperalgesia; developmental stage specific106–111 Chronic model; morphine hyperalgesia, strain dependent112,113 Chronic model; morphine produced dose-dependent antinociception114 Chronic model; no effect of morphine, fentanyl, buprenorphine115–117

Reptilia

Aves

Hot plate test

the caudal region of Derjugin codfish, Gadus morhuamarisalbi, as the noxious stimulus.88 The nocifensive response was similar to the agitated swim response mentioned above, but the fish was held in a flow-through chamber and the swim response was quantified by a force transducer. Dermorphin was administered into the intranasal passages (olfactory sacs) of the codfish and produced a dose-dependent antinociceptive effect. Further studies using rainbow trout, O. mykiss, with the same sophisticated behavioral apparatus, mapped the most sensitive areas of noxious stimulation on the trout and confirmed the antinociceptive effect of intranasal dermorphin in this species.89 In codfish, intraperitoneal (IP) and intramuscular (IM) administration of the MOR selective opioid peptide, β-casomorphin, produced antinociceptive effects.90 The opioid analgesic, tramadol, an MOR opioid agonist and norepinephrine/serotonin reuptake inhibitor, produced dose-dependent antinociception in the carp, Cyprinus carpio.91 In this last study, pretreatment with the opioid antagonist naloxone blocked the antinociception produced by tramadol. A chronic pain model was developed in fish by injecting acetic acid or bee venom into the lip region of trout.92,93 These animals develop pain-related behaviors that can be quantified and antinociception determined by the reduction in pain-related behaviors. Morphine had inhibiting effects on nociceptive behaviors per se and in the presence of novel objects.94,95 AMPHIBIA Early studies employing a hot plate test and electrified floor grid in amphibians did not detect an antinocicep-

Jumping

tive effect of morphine.96,97 It is likely that this was due to an inappropriate experimental design, as previously noted.98 A behavioral assay using acetic acid as the noxious stimulus and the wiping response as the nocifensive behavior was developed using the Northern grass frog, Rana pipiens, and is discussed in greater detail in the next section. Other studies using the acetic acid test in amphibians demonstrated that regional hypothermia is antinociceptive and is mediated in part by endogenous opioid peptides.99 A tail-flick test was also used to assess the antinociceptive effects of opioids in the Japanese firebelly newt, Cynops pyrrhogaster.100 Intraperitoneal injection of met-enkephalin and the putative opioid peptide RFamide produced a mild antinociceptive effect that was blocked by the concurrent administration of the opioid antagonist naloxone. REPTILIA To test the antinociceptive effects of opioids in reptiles, a tail-flick apparatus was used in the green anole lizard, Anolis carolinensis.101 Intraperitoneal injection of morphine (5 mg/kg) produced a weak, but significant increase in the latency of the lizard to remove its tail from a noxious heat stimulus. A number of nociceptive assays were characterized in the Nile crocodile, Crocodylus niloticus.102 Crocodiles placed on a hot plate attempt to guard their foot pads by lifting them alternately as well as attempting to escape the heated surface. A dilute capsaicin solution instilled in the eye of the crocodile produces behavioral responses including blinking, rubbing, and head shaking. Injection of formalin in the crocodile forepaw results in

346

SECTION IV / MODELS FOR SPECIFIC PURPOSES

the development of paw guarding behaviors. Morphine or meperidine (pethidine) produced antinociception as measured by the increased latency for crocodiles to lift their leg or escape on the hot plate test, which was the most suitable of three assays.103 However, these studies did not include dose–response analysis and did not assess the opioid nature of the antinociceptive effect by pretreatment with an opioid antagonist, such as naloxone or naltrexone. As mentioned in a clinical review of analgesia for exotic species,104 there are preliminary data that the partial opioid agonist butorphanol produced antinociception in the green iguana, Iguana iguana, using a thermal tail-flick test. AVES An early attempt to measure the antinociceptive effect of opioids in chicks using toe-pinch as the noxious stimulus and limb withdrawal as the nocifensive behavior did not find any antinociceptive effect of morphine up to a dose of 200 mg/kg.105 Using an electroshock to the underside of the wing as the noxious stimulus and an elicited flight response as the nocifensive behavior in chickens, morphine at a single dose of 30 mg/kg produced measurable antinociception in this model.106 Using a heated floor grid, other studies found that morphine produced a decrease in the latency for chickens to jump.107 This hyperalgesic effect was dependent on the age of the chicken; morphine administered to young cockerels less than 14 days old did not produce hyperalgesia.108 The hyperalgesic effect of morphine was dose dependent and exhibited a U-shaped time course curve.109 Using selective opioid antagonists, the hyperalgesic effect of morphine in chickens was shown to be predominantly mediated by µ opioid receptors.110 Codeine produced both hypoalgesia (antinociception) and hyperalgesia in this model.111 A formalin algesiometric assay was developed in chickens, quantified by the pain-related behaviors observed after injection of formalin into the foot.112 There was a strain difference in the hyperalgesic effect of morphine, perhaps due to differences in descending monoamine pathways.113 Carrageenan, a chemical irritant like formalin, provided the noxious stimulus in a chronic inflammatory model using the Hargreaves method.114 In this assay, chickens are measured for the latency to withdrawal of an inflamed foot from a lamp placed under the floor grid. Morphine produced a dose-dependent antinociception but had no effect on the edema produced by carrageenan. A chronic arthritis model was developed in chickens using an intraarticular injection of urate crystals, which models gouty arthritis.115,116 However, intraarticular injection of morphine, fentanyl, or buprenorphine did not have any effect in reducing pain-related behaviors due to the experimentally induced arthritis.117

AN AMPHIBIAN MODEL FOR PAIN RESEARCH THE ACETIC ACID TEST The acetic acid test (AAT) to determine the nociceptive threshold (NT) in frogs consists of 11 concentrations of acetic acid serially diluted from glacial acetic acid (two parts acid : one part water).98 The concentrations are given a code number from 0 to 10 with the lowest code number equal to the lowest concentration of acetic acid. The actual molar concentration of acetic acid per each solution is given by the following equation: Log[M] = 0.1716 × (Code #) − 0.5849 Nociceptive testing is done by placing, with a Pasteur pipette, a single drop of acid on the dorsal surface of the frog’s thigh.

Testing begins with the lowest concentration and proceeds with increasing concentrations until the NT is reached. The NT is defined as the code number of the lowest concentration of acid that causes the frog to vigorously wipe the treated leg with either hindlimb. To prevent tissue damage, the acetic acid is immediately wiped off with a gentle stream of distilled water once the animal responds or after 5 sec if the animal fails to respond. If the animal fails to respond, testing continues on the opposite hindlimb. An animal that fails to respond to the highest concentration (#10) is assigned the cutoff value of 11. The wiping response in frogs, like the tail-flick in rodents, remains intact after a high spinal transection, demonstrating sufficient circuitry in the spinal cord to mediate this behavior.118 The wiping response has not been observed in the laboratory in the absence of noxious stimuli and appears specific for assessing nociception. As noted above, acetic acid was shown to activate nociceptive afferents in amphibians. The wiping response is also the basis for much research on the motor systems of the amphibian spinal cord.119 ADMINISTRATION OF ANALGESICS TO AMPHIBIANS The technique of systemic administration (SC) of drugs in frogs is similar to subcutaneous administration in rodent species. The frog is gently grasped and a tuberculin syringe fitted with a 25-gauge needle is slipped under the skin on the dorsal side of the animal. The initial location of the injection is the mid-back region, which overlies the dorsal lymph sac in amphibians (see Figure 37–1, top). The needle is gently inserted into the lymph sac (felt as a slight resistance as if injecting into a sponge) and the drug is delivered in a volume of 10 µl/g body weight. Each animal is used only once for injections to prevent possible confounding effects of repeated injections in the same animal. A saline-injected control group is run with each experiment. The technique of intraspinal (IS) administration of drugs in frogs is remarkably similar to that described for mice.120 Frogs are gently held with the nondominant hand and using a tapered 26gauge needle fitted to a microsyringe, the needle is introduced into the spinal cord slightly lateral to the neural spine at the articulation between the seventh and eighth vertebrae. This articulation is felt by probing with the needle and is located by triangulation of the dorsal protuberances of the iliac crest of the frog (see Figure 37–1, middle). The region of intraspinal injection overlies the area of the lumbar enlargement. Penetration of the needle sometimes produces a mild hindlimb extension or trembling, which discontinues after a 2–3 sec delivery of the drug. Previous experiments employing radiolabeled morphine demonstrated that over 98% of intraspinal morphine remained within two or three segments of the site of injection throughout the time course of the experiment.121 Using a modification of the methods for intracerebroventricular (ICV) administration in rodents, animals are injected with 3 µl of drug or saline vehicle into the third ventricle. Briefly, animals are administered a local anesthetic (0.5 ml, 2% lidocaine) suffused just under the skin of the skull region. A midline incision is made and the top of the skull is exposed by retraction. A hand-held microsyringe, fitted with an 8 cm length of PE-10 and a 1.5-inch, 29-gauge needle cannula is used to deliver the drugs. Placement of the needle is made through the skull at a depth of 2.5 mm, midline and just anterior to the optic tectum, a site shown in pilot studies to fill the brain ventricles with dye (see Figure 37–1, bottom). Visualization of the brain structures through the thin

CHAPTER 37 / NONMAMMALIAN MODELS FOR THE STUDY OF PAIN

347

enkephalinase inhibitors.59 Other behavioral studies include an investigation of the effects of opioids on noxious and nonnoxious sensory modalities,84,126 and an examination of agents acting on α2-adrenergic receptors after systemic and spinal administration.127,128 Later results of systematic studies examining the antinociception of selective µ, δ, or κ opioid agonists administered by different routes yielded an important finding: the relative antinociceptive potency of µ, δ, or κ opioid agonists after systemic, intraspinal, or intracerebroventricular administration in amphibians was highly correlated to that observed in typical mammalian models and to the relative analgesic potency of opioid analgesics in human clinical studies.129–131 These data established the amphibian model as a robust and predictive adjunct model for the testing of opioid analgesics.1,6,132 Table 37–3 provides a summary of the opioid agonists used in behavioral studies of opioid

Table 37–3 Antinociceptive potency of opioid agonists administered to Rana pipiens

Figure 37–1. Routes of drug administration in frogs. Drugs can be given by the systemic route (top), the intraspinal route (middle), and the intracerebroventricular route (bottom). See text for details.

cartilaginous skull of amphibians also aids in the consistency of injection sites. EFFECTS OF OPIOID ANALGESICS IN AMPHIBIANS Initial studies of the analgesic effects of opioid administration in amphibians were conducted using nonselective opioid agonists, endogenous opioid peptides, and antagonists.121–124 These studies showed that both exogenous opioid agonists and endogenous opioid peptides could raise the nociceptive threshold in amphibians by an action at opioid receptors. Tolerance to the analgesic effects of daily morphine administration was documented125 and stress-induced release of endogenous opioids was shown to produce analgesia, which was potentiated by

Opioid agonist

Receptora

Routeb

Fentanyl CI-977 (enadoline) Remifentanil Levorphanol U50488H Methadone Bremazocine Morphine Buprenorphine Meperidine Codeine Nalorphine Dermorphin DAMGO DSLET DADLE Fentanyl Morphine Remifentanil DPDPE Deltorphin CI-977 (enadoline) Bremazocine U50488H Nalorphine DADLE Morphine DPDPE Fentanyl CI-977 (enadoline) U50488H

MOR KOR MOR MOR KOR MOR KOR MOR MOR MOR MOR KOR MOR MOR DOR MOR MOR MOR MOR DOR DOR KOR KOR KOR KOR MOR MOR DOR MOR KOR KOR

SC SC SC SC SC SC SC SC SC SC SC SC IS IS IS IS IS IS IS IS IS IS IS IS IS ICV ICV ICV ICV ICV ICV

ED50 valuec 1.4 5.8 7.1 7.5 8.5 19.9 44.4 86.3 99.1 128.1 140.3 320.9 0.04 0.13 0.13 0.14 0.94 2.26 3.20 3.29 13.50 13.51 22.89 36.82 43.13 1.3 2.0 8.3 23.0 35.5 61.5

Selectivity of the listed opioid for MOR, DOR, or KOR (µ, δ, or κ opioid receptor). b Route of administration: SC, subcutaneous; IS, intraspinal; ICV, intracerebroventricular. c Dose that gives 50% antinociceptive effects; in nanomoles/gram for SC, and in nanomoles/frog for IS and ICV administration. Data are from references 129, 130, 131, and 148. a

348

SECTION IV / MODELS FOR SPECIFIC PURPOSES

antinociception following systemic, spinal and supraspinal routes of administration. However, emerging results from both behavioral and binding studies in R. pipiens suggested that the selectivity of amphibian opioid receptors was different from the selectivity of mammalian opioid receptors. The MOR, DOR, and KOR type selective opioid antagonists, β-FNA, naltrindole, and nor-BNI,133–135 did not show type selectivity in blocking the antinociceptive effects of selective opioid agonists in amphibians.136 In radioligand binding studies, β-FNA, naltrindole, and nor-BNI competed with [3H]naloxone binding from brain and spinal cord homogenates with equal apparent affinity, but did show type-selective competition against selective opioid agonist radioligands.74–76 Still lacking was the ultimate identification of the types of opioid receptor proteins expressed in R. pipiens from molecular cloning studies.

norvigecus), and human (Homo sapiens). Phylogenetic dendrograms (trees) were constructed using existing alignment (ClustalW) and molecular evolution software. The nearestneighbor joining trees for each vertebrate opioid receptor type gave the accepted evolutionary relationship, with the fourth type of opioid receptors (ORL) used as an outgroup to root the three classical types of opioid receptors (see Figure 37–2). As shown, the three groups of opioid receptor types did not arise from a single, common ancestral sequence but rather the group of κ opioid receptors shared a common ancestral sequence with the branch bifurcating later to the µ and δ opioid receptor groups. As pharmacological selectivity is correlated with similarity of amino acid sequences at the GPCR family level (e.g., opioid receptors vs. muscarinic receptors) it is reasonable to assume that the type selectivity within family members (e.g., MOR, DOR, and

AN EXAMPLE OF THE COMPARATIVE APPROACH: THE EVOLUTION OF VERTEBRATE OPIOID RECEPTORS The number of full-length cDNA sequences for opioid receptors in nonmammalian vertebrates deposited in GenBank is surprisingly small. At present, there are no databank sequences for any type of opioid receptor from any species of Reptilia or Aves. From class Pisces there was an initial entry of µ opioid receptor sequences expressed in the white suckerfish, Castomus commersoni,137 and more recently, the triad of µ, δ, and κ opioid receptor cDNAs was cloned from the zebrafish, Danio rerio.138–140 The cloning of full-length opioid receptors in R. pipiens was particularly needed given that there is a corresponding dataset of opioid behavioral and binding data. Other nonmammalian species do not have a corresponding dataset of opioid binding and behavioral studies to integrate molecular cloning and in vitro results with whole animal, behavioral data on opioid antinociception. CLONING AND SEQUENCING OF AMPHIBIAN OPIOID RECEPTORS Three full-length clones of opioid receptors were obtained by reverse transcriptase polymerase chain reaction (RT-PCR) amplification of R. pipiens brain and spinal cord cDNA. On the basis of high homologies to existing vertebrate sequences, these clones were named rpMOR (GenBank number AF530571), rpDOR (AF530572), and rpKOR (AF530573) as novel orthologs to mammalian µ, δ, and κ opioid receptors. A fourth type of opioid receptor protein, rpORL (AY434690), was recently sequenced and was homologous to existing ORL proteins. The percent identity of amino acids in rpMOR compared to the zebrafish µ opioid receptor was 79%, and ranges from 83 to 84% compared to mammalian µ opioid receptors. The frog δ opioid receptor, rpDOR, had 77% identity with the zebrafish ortholog and 73% identity compared to mammalian δ opioid receptors. Mammalian κ opioid receptors ranged from 70 to 71% identical to rpKOR and the zebrafish κ opioid receptor was 64% identical to rpKOR. BIOINFORMATICS OF VERTEBRATE OPIOID-LIKE RECEPTORS The bioinformatic analysis of the amphibian opioid receptor used existing matched datasets of µ, δ, and κ opioid receptors from other vertebrate species. Somewhat surprising, there are only four vertebrate species, excluding the amphibian data, that have full-length sequences for all three types of opioid receptor cDNA deposited in GenBank. These species are the zebrafish (Danio rerio), mouse (Ms µscularis), rat (Rattus

Figure 37–2. Phylogenetic analysis of the vertebrate OpR proteins. The neighbor-joining algorithm was used (h, human; r, rat; m, mouse; rp, frog; tg, newt; and dr, fish). The corresponding vertebrate ORL proteins were used as the outgroup. Numbers provide the bootstrap value (percent reliability out of 1000 replicates) and the scale bar is the proportional difference (e.g., 0.05 = 5% divergence in sequence) for a given unit of branch length.

CHAPTER 37 / NONMAMMALIAN MODELS FOR THE STUDY OF PAIN

349

Table 37–4 Homology of MOR, DOR, and KOR receptors within different vertebrate speciesa Mean (SEM)b

MOR vs. DOR

MOR vs. KOR

DOR vs. KOR

Identity Zebrafish Frog Rat Mouse Human

68 73 66 61 62

62 65 61 60 60

68 63 61 61 59

66.0 (2.0) 67.0 (3.1) 62.7 (1.7) 60.7 (0.3) 60.3 (0.9)

Similarity Zebrafish Frog Rat Mouse Human

80 85 76 71 73

78 81 75 75 73

82 77 74 73 72

80.0 (1.2)c 81.0 (2.3)c 75.0 (0.6) 73.0 (1.2) 72.7 (0.3)

a Percent identity and similarity determined by BLAST-P. BLAST-P settings: matrix = Blossum62, gap open = 11, gap extension = 1, x-drop-off = 50, expect = 10.00, wordsize = 3, and filter off. b Standard error of the mean. c Denotes significantly different from rat, mouse, and human mean values (p < 0.05, one-way ANOVA followed by post hoc Newman–Kuels test).

KOR) is also correlated with percent identity or similarity. The results of pair-wise BLAST analysis141 for the three types of opioid receptor sequences within each species yielded a rank order of divergence such that in earlier-evolved vertebrates, µ, δ, and κ opioid sequences were more closely related to each other than in humans and other mammals. For the percent similarity comparison, these values reached statistical difference such that the mean values of similarity of µ, δ, and κ opioid receptors in nonmammals were greater than the similarity of µ, δ, and κ opioid receptors in mammals (Table 37–4). Insofar as divergence of molecular sequence is related to the greater type selectivity of opioid receptors, this finding gave rise to the novel hypothesis that opioid receptors are more type selective in mammals than in nonmammalian species. The hypothesis that opioid receptors in earlier-evolved vertebrate are less selective is supported by behavioral and binding studies in amphibians74,136 and by binding studies on zebrafish opioid receptors expressed in Chinese hamster ovary (CHO) cells.138,142 Further bioinformatic analysis of the vertebrate opioid receptor sequence dataset leads to identification of individual amino acids that may be crucial for determining type selectivity of opioid analgesics. Using this comparative approach, novel hypotheses were generated that lead to a greater understanding of the molecular evolution of vertebrate opioid receptors and the mechanism of the opioid analgesic selectivity.4,149

CONCLUSIONS The comparative approach to investigations of pain and analgesia has great promise for contributing to a greater understanding of analgesic mechanisms in humans. Additionally, much research using adjunct nonmammalian models for the assessment of antinociception is needed to make sound clinical decisions in veterinary treatment of exotic animals (i.e., not dogs or cats, but fish, amphibians, reptiles, and birds).143 There has been some transfer of knowledge from basic studies of antinociceptive effects in R. pipiens to the clinical application of analgesics for use during

oocyte harvesting in Xenopus laevis.144 However, for both improved clinical treatment of humans and animals,145 additional studies of nociception in nonmammalian species and the development of adjunct models for analgesia are critically needed.

REFERENCES 1. Stevens CW. An amphibian model for pain research. Lab Anim 1995;24:32–36. 2. Stevens CW. Opioid antinociception in amphibians. Brain Res Bull 1988;21:959–962. 3. Stevens CW. An amphibian model for the assessment of opioid analgesia: Systemic and spinal studies. Analgesia 1995;1:683– 686. 4. Stevens CW. Opioid research in amphibians: An alternative pain model yielding insights on the evolution of opioid receptors. Brain Res Rev 2004;46:204–215. 5. Rose JD. The neurobehavioral nature of fishes and the question of awareness and pain. Rev Fish Sci 2002;10:1–38. 6. Stevens CW. Alternatives to the use of mammals for pain research. Life Sci 1992;50:901–912. 7. Hammond DL. New insights regarding organization of spinal cord pain pathways. News Physiol Sci 1989;4:98–101. 8. Willis WD, Westlund KN. Neuroanatomy of the pain system and of the pathways that modulate pain. J Clin Neurophys 1997; 14:2–31. 9. Wilkinson SV, Neary MT, Jones RO, Sunshein KF. The neuroanatomy of pain. Pain Manag 1994;11:1–13. 10. Sneddon LU. Anatomical and electrophysiological analysis of the trigeminal nerve in a teleost fish, Oncorhynchus mykiss. Neurosci Lett 2002;319(3):167–171. 11. Sneddon LU. Trigeminal somatosensory innervation of the head of a teleost fish with particular reference to nociception. Brain Res 2003;972(1–2):44–52. 12. Sasaki Y, Matsui M, Taguchi M, et al. D-ARG2-dermorphin tetrapeptide analogs: A potent and long-lasting analgesic activity after subcutaneous administration. Biochem Biophys Res Comµn 1984;120:214–218. 13. Ritchie TC, Leonard RB. Immunohistochemical studies on the distribution and origin of candidate peptidergic primary afferent neurotransmitters in the spinal cord of an elasmobranch fish, the atlantic stingray (Dasyatis sabina). J Comp Neurol 1983;213:414– 425.

350

SECTION IV / MODELS FOR SPECIFIC PURPOSES

14. Adrian ED. The impulses produced by sensory nerve endings. Part 4: Impulses from pain receptors. J Physiol 1926;62:33–51. 15. Adrian ED. Impulses in sympathetic fibres and in slow afferent fibres. Proc Physiol Soc 1930;20–21. 16. Adrian ED, Cattell M, Hoagland H. Sensory discharges in single cutaneous nerve fibres. J Physiol 1931;72:377–391. 17. Roberts A, Hayes BP. The anatomy and function of free nerve endings in an amphibian skin sensory system. Proc Biol Sci 1977;196:415–420. 18. Fox H, Whitear M. Observations of Merkel cells in amphibians. Biol Cell 1978;32:223–232. 19. Spray DC. Cutaneous receptors: Pain and temperature receptors of anurans. In: Llinas R, Precht W, Eds. Frog Neurobiology: A Handbook. Berlin: Springer-Verlag, 1976:607–628. 20. Hamamoto DT, Forkey MW, Davis WL, Kajander KC. The role of pH and osmolarity in evoking the acetic acid-induced wiping response in a model of nociception in frogs. Brain Res 2000; 862:217–229. 21. Maruhashi J, Mizuguchi K, Tasaki I. Action currents in single afferent nerve fibres elicited by stimulation of the skin of the toad and the cat. J Physiol 1952;117:129–151. 22. Hamamoto DT, Simone DA. Characterization of cutaneous primary afferent fibers excited by acetic acid in a model of nociception in frogs. J Neurophysiol 2003;90:566–577. 23. Nikundiwe AM, De Boer-van Huizen R, Ten Donkelaar HJ. Dorsal root projections in the clawed toad (Xenopus laevis) as demonstrated by anterograde labeling with horseradish peroxidase. Neuroscience 1982;7:2089–2103. 24. Szekely G. The morphology of motoneurons and dorsal root fibers in the frog’s spinal cord. Brain Res 1976;103:275–290. 25. Szekely G, Matesz K, Baker RE, Antal M. The termination of cutaneous nerves in the dorsal horn of the spinal cord in normal and in skin-rotated frogs. Exp Brain Res 1982;45:19–28. 26. Landmann L, Halata Z. Merkel cells and nerve endings in the labial epidermis of a lizard. Cell Tissue Res 1980;210:353–357. 27. Pac L. Nerve endings in the lizard skin (Lacerta viridis). Z Mikrosk Anat Forsch 1984;98:939–950. 28. Kenton B, Kruger L, Woo M. Two classes of slowly adapting mechanoreceptor fibres in reptile cutaneous nerve. J Physiol 1971;212:21–44. 29. Kenton B, Kruger L. Information transmission in slowly adapting mechanoreceptor fibers. Exp Neurol 1971;31:114–139. 30. Kusuma A, Ten Donkelaar HJ. Dorsal root projections in various types of reptiles. Brain Behav Evol 1980;17:291–309. 31. Ruigrok TJ, Crowe A, Ten Donkelaar HJ. Morphology of primary afferents to the spinal cord of the turtle Pseudemys scripta elegans. Anat Embryol (Berl) 1985;171:75–81. 32. Gentle MJ, Tilston V, McKeegan DE. Mechanothermal nociceptors in the scaly skin of the chicken leg. Neuroscience 2001;106:643– 652. 33. Sandercock DA. Putative nociceptor responses to mechanical and chemical stimulation in skeletal muscles of the chicken leg. Brain Res Brain Res Rev 2004;46:155–162. 34. McKeegan DE, Demmers TG, Wathes CM, Jones RB, Gentle MJ. Response characteristics of nasal trigeminal nociceptors in Gallus domesticus. NeuroReport 2002;13:1033–1035. 35. Gentle MJ, Thorp BH. Sensory properties of ankle joint capsule mechanoreceptors in acute monoarthritic chickens. Pain 1994; 57:361–374. 36. Gentle MJ. Sodium urate arthritis: Effects on the sensory properties of articular afferents in the chicken. Pain 1997;70:245–251. 37. Gentle MJ. The acute effects of amputation on peripheral trigeminal afferents in Gallus gallus var domesticus. Pain 1991;46:97–103. 38. Gentle MJ, Hunter LN, Sterling RJ. Projections of ankle joint afferents to the spinal cord and brainstem of the chicken (Gallus g. domesticus). J Comp Neurol 1995;361:669–680. 39. Atsumi S, Sakamoto H, Kawate T, Zhai XY. Substance P-containing primary afferent receives inhibitory modulation directly from enkephalin- and GABA-containing interneurons in the dorsal horn of the chicken. Regul Pept 1993;46:410–412.

40. Zhai XY, Atsumi S. Large dorsal horn neurons which receive inputs from numerous substance P-like immunoreactive axon terminals in the laminae I and II of the chicken spinal cord. Neurosci Res 1997;28:147–154. 41. Vesselkin NP, Agayan AL, Nomokonova LM. A study of thalamotelencephalic afferent systems in frogs. Brain Behav Evol 1971; 4:295–306. 42. Dunlop R, Laming P. Mechanoreceptive and nociceptive responses in the central nervous system of goldfish (Carassius auratus) and trout (Oncorhynchus mykiss). J Pain 2005;6:561–568. 43. Sarnat HB, Netsky MG. Evolution of the Nervous System, 2nd ed. New York: Oxford University Press, 1981. 44. Northcutt RG. Evolution of the vertebrate central nervous system: Patterns and processes. Am Zool 1984;24:701–716. 45. Inagaki S, Senba E, Shiosaka S. Regional distribution of substance P-like immunoreactivity in the frog brain and spinal cord: Immunohistochemical analysis. J Comp Neurol 1981;201:243–254. 46. Lorez HP, Kemali M. Substance P, met-enkephalin and somatostatin-like immunoreactivity distribution in the frog spinal cord. Neurosci Lett 1981;26:119–124. 47. Wouters W, Ermes-Busio MCE, Van Den Bercken J. Potentiation by naloxone of reflex activity in the isolated spinal cord of Xenopus. Eur J Pharmacol 1979;55:431–433. 48. Luthman J, Fernandez A, Radmilovich M, Trujillo-Cenoz O. Immunohistochemical studies on the spinal dorsal horn of the turtle Chrysemys d’orbigny. Tissue Cell 1991;23:515–523. 49. Simpson JI. Functional Synaptology of the Spinal Cord. In: Llinas R, Precht W, Eds. Frog Neurobiology: A Handbook. Berlin: Springer-Verlag, 1976:728–749. 50. Naik DR, Sar M, Stumpf WE. Immunohistochemical localization of enkephalin in the central nervous system and pituitary of the lizard, Anolis carolinensis. J Comp Neurol 1981;198:583–601. 51. Snow PJ, Renshaw GM, Hamlin KE. Localization of enkephalin immunoreactivity in the spinal cord of the long-tailed ray Himantura fai. J Comp Neurol 1996;367:264–273. 52. Vecino E, Perez MT, Ekstrom P. Localization of enkephalinergic neurons in the central nervous system of the salmon (Salmo salar L.) by in situ hybridization and immunocytochemistry. J Chem Neuroanat 1995;9:81–97. 53. Vecino E, Pinuela C, Arevalo R, Lara J, Alonso JR, Aijon J. Distribution of enkephalin-like immunoreactivity in the central nervous system of the rainbow trout: An immunocytochemical study. J Anat 1992;180:435–453. 54. Cameron AA, Plenderleith MB, Snow PJ. Organization of the spinal cord in four species of elasmobranch fish: Cytoarchitecture and distribution of serotonin and selected neuropeptides. J Comp Neurol 1990;297:201–218. 55. Kawate T, Sakamoto H, Yang C, Li Y, Shimada O, Atsumi S. Immunohistochemical study of delta and mu opioid receptors on synaptic glomeruli with substance P-positive central terminals in chicken dorsal horn. Neurosci Res 2005;53:279–287. 56. Hamada S, Ogawa M, Okado N. Immunohistochemical examination of intraspinal serotonin neurons and fibers in the chicken lumbar spinal cord and coexistence with leu-enkephalin. Cell Tissue Res 1995;282:387–397. 57. Du F, Dubois PM. Development and distribution of enkephalinimmunoreactive elements in the chicken spinal cord. Neuroscience 1988;27:251–266. 58. Pezalla PD, Dicig M. Stress-induced analgesia in frogs: Evidence for the involvement of an opioid system. Brain Res 1984; 296:356–360. 59. Stevens CW, Sangha S, Ogg BG. Analgesia produced by immobilization stress and an enkephalinase-inhibitor in amphibians. Pharmacol Biochem Behav 1995;51:675–680. 60. Lecaude S, Alrubaian J, Sollars C, Propper C, Danielson P, Dores RM. Organization of proenkephalin in amphibians: Cloning of a proenkephalin cDNA from the brain of the anuran amphibian, Spea µltiplicatus. Peptides 2000;21:339–344. 61. Danielson PB, Hoversten MT, Fitzpatrick M, Schreck C, Akil H, Dores RM. Sturgeon orphanin, a molecular “fossil” that bridges the

CHAPTER 37 / NONMAMMALIAN MODELS FOR THE STUDY OF PAIN

62.

63. 64.

65.

66.

67.

68.

69.

70. 71. 72.

73.

74.

75.

76.

77.

78.

79.

80.

81. 82.

83.

gap between the opioids and orphanin FQ/nociceptin. J Biol Chem 2001;276:22114–22119. Dores RM, Lecaude S, Bauer D, Danielson PB. Analyzing the evolution of the opioid/orphanin gene family. Mass Spectrom Rev 2002;21(4):220–243. Dores RM, Lecaude S. Trends in the evolution of the proopiomelanocortin gene. Gen Comp Endocrinol 2005;142:81–93. Alrubaian J, Lecaude S, Barba J, Szynskie L, Jacobs N, Bauer D, Brown C, Kaminer I, Bagrosky B, Dores RM. Trends in the evolution of the preprodynorphin gene in teleosts: Cloning of eel and tilapia prodynorphin cDNAs. Peptides 2006;27:797–804. Fredriksson R, Lagerstrom MC, Lundin LG, Schioth HB. The G-protein-coupled receptors in the human genome form five main families: Phylogenetic analysis, paralogon groups, and fingerprints. Mol Pharmacol 2003;63:1256–1272. Mogil JS, Pasternak G. The molecular and behavioral pharmacology of the orphanin FQ/nociceptin peptide and receptor family. Pharmacol Rev 2001;53:381–415. Gonzalez-Nunez V, Barrallo A, Traynor JR, Rodriguez RE. Characterization of opioid binding sites in zebrafish brain. J Pharmacol Exp Ther 2006;316:900–904. Brooks AI, Standifer KM, Cheng J, Ciszewska GR, Pasternak GW. Opioid binding in giant toad and goldfish brain. Receptor 1994;4:55–62. Bird DJ, Jackson M, Baker BI, Buckingham JC. Opioid binding sites in the fish brain: An autoradiographic study. Gen Comp Endocrinol 1988;70:49–62. Buatti MC, Pasternak GW. Multiple opiate receptors: Phylogenetic differences. Brain Res 1981;218:400–405. Simon EJ, Hiller JM, Groth J, Itzhak Y, Holland MJ, Beck SG. The nature of opiate receptors in toad brain. Life Sci 1982;31:1367–1370. Benyhe S, Varga E, Hepp J, Magyar A, Borsodi A, Wollemann M. Characterization of kappa1 and kappa2 opioid binding sites in frog (Rana esculenta) brain membrane. Neurochem Res 1990;15:899– 904. Simon J, Szucs M, Benyhe S, Borsodi A, Zemlan FP, Wollemann M. Solubilization and characterization of opioid binding sites from frog (Rana esculenta) brain. J Neurochem 1984;43:957–963. Newman LC, Wallace DR, Stevens CW. Selective opioid agonist and antagonist displacement of [3H]-naloxone binding in amphibian brain. Eur J Pharmacol 2000;397:255–262. Newman LC, Wallace DR, Stevens CW. Selective opioid receptor agonist and antagonist displacement of [3H]-naloxone binding in amphibian spinal cord. Brain Res 2000;884:184–191. Newman LC, Sands SS, Wallace DR, Stevens CW. Characterization of µ, κ, and δ opioid binding in amphibian whole brain tissue homogenates. J Pharmacol Exp Ther 2002;301:364–370. Kawashima M, Imai S, Takahashi T, Kamiyoshi M, Tanaka K. An opiate receptor in the neurohypophysis of laying hens. Poult Sci 1995;74:716–722. Csillag A, Stewart MG, Szekely AD, Magloczky Z, Bourne RC, Steele RJ. Quantitative autoradiographic demonstration of changes in binding to delta opioid, but not mu or kappa receptors, in chick forebrain 30 minutes after passive avoidance training. Brain Res 1993;613:96–105. Martin R, McGregor GP, Halbinger G, Falke N, Voigt KH. Methionine5-enkephalin and opiate binding sites in the neurohypophysis of the bird, Gallus domesticus. Regul Pept 1992;38:33–44. Csillag A, Bourne RC, Stewart MG. Distribution of mu, delta, and kappa opioid receptor binding sites in the brain of the one-day-old domestic chick (Gallus domesticus): An in vitro quantitative autoradiographic study. J Comp Neurol 1990;302:543–551. Pert CB, Aposhian D, Snyder SH. Phylogenetic distribution of opiate binding. Brain Res 1974;75:356–361. Xia Y, Haddad GG. Major difference in the expression of δ- and µ-opioid receptors between turtle and rat brain. J Comp Neurol 2001;436:202–210. Bakalkin GY, Pivovarov AS, Kobylyansky AG, Nesterenko PN, Yarygin KN. Lateralization of opioid receptors in turtle visual cortex. Brain Res 1989;480:268–276.

351

84. Willenbring S, Stevens CW. Thermal, mechanical and chemical peripheral sensation in amphibians: Opioid and adrenergic effects. Life Sci 1996;58:125–133. 85. Jansen GA, Greene NM. Morphine metabolism and morphine tolerance in goldfish. Anesthesiology 1970;32:231–235. 86. Ehrensing RH, Michell GF, Kastin AJ. Similar antagonism of morphine analgesia by MIF-1 and naloxone in Carassius auratus. Pharmacol Biochem Behav 1982;17:757–761. 87. Stevens CW, Yaksh TL. Spinal action of dermorphin, an extremely potent opioid peptide from frog skin. Brain Res 1986;385: 300–304. 88. Chervova LS, Kamenskii AA, Malyukhina GA, Baturina EY, Deigin VI, Yaroav EP. Study of dermorphin’s intranasal action mechanism in fish and mammal species. Zh Evolyutsionnoi Biokhim Fiziol 1992;28:45–48. 89. Chervova LS, Lapshin DN, Kamenskii AA. Pain sensitivity of trout and analgesia induced by intranasal administration of dermorphin. Doklady Biol Sci 1994;338:424–425. 90. Chervova LS. Pain sensitivity and behavior of fishes. J Ichthyol 1997;37:98–102. 91. Chervova LS, Lapshin DN. Opioid modulation of pain threshold in fish. Doklady Biol Sci 2000;375:703–704. 92. Sneddon LU, Braithwaite VA, Gentle MJ. Do fishes have nociceptors? Evidence for the evolution of a vertebrate sensory system. Proc Biol Sci 2003;270:1115–1121. 93. Sneddon LU. Evolution of nociception in vertebrates: Comparative analysis of lower vertebrates. Brain Res Brain Res Rev 2004;46:123– 130. 94. Sneddon LU, Braithwaite VA, Gentle MJ. Novel object test: Examining nociception and fear in the rainbow trout. J Pain 2003; 4:431–440. 95. Sneddon LU. The evidence for pain in fish: The use of morphine as an analgesic. Appl Anim Behav Sci 2003;83:153–162. 96. Nistri A, Pepeu G, Cammelli E, Spina L, De Bellis AM. Effects of morphine on brain and spinal acetylcholine levels and nociceptive threshold in the frog. Brain Res 1974;80:199–209. 97. Nistri A, Pepeu G. Increase in brain and spinal acetylcholine levels without antinociceptive actions following morphine administration in the frog. Br J Pharmacol 1973;47:650P-651P. 98. Pezalla PD. Morphine-induced analgesia and explosive motor behavior in an amphibian. Brain Res 1983;273:297–305. 99. Suckow M, Terril L, Grigdesby C, March P. Evaluation of hypothermia-induced analgesia and influence of opioid antagonists in leopard frogs (Rana pipiens). Pharmacol Biochem Behav 1999;63: 39–43. 100. Kanetoh T, Sugikawa T, Sasaki I, et al. Identification of a novel frog RFamide and its effect on the latency of the tail-flick response of the newt. Comp Biochem Physiol C Toxicol Pharmacol 2003;134:259–266. 101. Mauk MD, Olson RD, LaHoste GJ, Olson GA. Tonic immobility produces hyperalgesia and antagonizes morphine analgesia. Science 1981;213:353–354. 102. Kanui TI, Hole K, Miaron JO. Nociception in crocodiles: Capsaicin instillation, formalin and hot plate tests. Zool Sci 1990;7:537–540. 103. Kanui TI, Hole K. Morphine and pethidine antinociception in the crocodile. J Vet Pharmacol Ther 1992;15:101–103. 104. Hawkins MG. The use of analgesics in birds, reptiles, and small exotic mammals. J Exot Pet Med 2006;15:177–192. 105. Schneider C. Effects of morphine-like drugs in chicks. Nature 1961;191:607–608. 106. Bardo MT, Hughes RA. Shock-elicited flight response in chickens as an index of morphine analgesia. Pharmacol Biochem Behav 1978;9:147–149. 107. Hughes RA, Sufka KJ. The ontogeny of thermal nociception in domestic fowl: Thermal stimulus intensity and isolation effects. Dev Psychobiol 1990;23:129–140. 108. Hughes RA, Bowes M, Sufka KJ. Morphine hyperalgesic effects on developmental changes in thermal nociception and respiration in domestic fowl (Gallus gallus).Pharmacol Biochem Behav1992;42:535– 539.

352

SECTION IV / MODELS FOR SPECIFIC PURPOSES

109. Sufka KJ, Hughes RA. Dose and temporal parameters of morphineinduced hyperalgesia in domestic fowl. Physiol Behav 1990;47: 385–387. 110. Sufka KJ, Hughes RA, Giordano J. Effects of selective opiate antagonists on morphine-induced hyperalgesia in domestic fowl. Pharmacol Biochem Behav 1991;38:49–54. 111. Sufka KJ, Hughes RA. Time-dependent codeine hypoalgesia and hyperalgesia in domestic fowl. Pharmacol Biochem Behav 1992;41:349–353. 112. Hughes RA, Sufka KJ. Morphine hyperalgesic effects on the formalin test in domestic fowl (Gallus gallus). Pharmacol Biochem Behav 1991;38:247–251. 113. Sufka KJ, Hoganson DA, Hughes RA. Central monoaminergic changes induced by morphine in hypoalgesic and hyperalgesic strains of domestic fowl. Pharmacol Biochem Behav 1992;42:781– 785. 114. Roach JT, Sufka KJ. Characterization of the chick carrageenan response. Brain Res 2003;994:216–225. 115. Gentle MJ, Corr SA. Endogenous analgesia in the chicken. Neurosci Lett 1995;201:211–214. 116. Gentle MJ. Pain-related behaviour following sodium urate arthritis is expressed in decerebrate chickens. Physiol Behav 1997;62:581– 584. 117. Gentle MJ, Hocking PM, Bernard R, Dunn LN. Evaluation of intraarticular opioid analgesia for the relief of articular pain in the domestic fowl. Pharmacol Biochem Behav 1999;63:339– 343. 118. Fukson OI, Berkinblit MB, Feldman AG. The spinal frog takes into account the scheme of its body during the wiping reflex. Science 1980;209:1261–1263. 119. Giszter SF, McIntyre J, Bizzi E. Kinematic strategies and sensorimotor transformations in the wiping movements of frogs. J Neurophysiol 1989;62:750–767. 120. Hylden JLK, Wilcox GL. Intrathecal morphine in mice: A new technique. Eur J Pharmacol 1980;67:313–316. 121. Stevens CW, Pezalla PD. A spinal site mediates opiate analgesia in frogs. Life Sci 1983;33:2097–2103. 122. Stevens CW, Pezalla PD. Naloxone blocks the analgesic action of levorphanol but not of dextrorphan in the leopard frog. Brain Res 1984;301:171–174. 123. Pezalla PD, Stevens CW. Behavioral effects of morphine, levorphanol, dextrorphan and naloxone in the frog Rana pipiens. Pharmacol Biochem Behav 1984;21:213–217. 124. Stevens CW, Pezalla PD, Yaksh TL. Spinal antinociceptive action of three representative opioid peptides in frogs. Brain Res 1987; 402:201–203. 125. Stevens CW, Kirkendall K. Time course and magnitude of tolerance to the analgesic effects of systemic morphine in amphibians. Life Sci 1993;52:PL111–116. 126. Willenbring S, Stevens CW. Spinal µ, δ and κ opioids alter chemical, mechanical and thermal sensitivities in amphibians. Life Sci 1997;61:2167–2176. 127. Brenner GM, Klopp AJ, Deason LL, Stevens CW. Analgesic potency of alpha adrenergic agents after systemic administration in amphibians. J Pharmacol Exp Ther 1994;270:540–545. 128. Stevens CW, Brenner GM. Spinal administration of adrenergic agents produces analgesia in amphibians. Eur J Pharmacol 1996;316:205–210. 129. Stevens CW, Klopp AJ, Facello JA. Analgesic potency of µ and κ opioids after systemic administration in amphibians. J Pharmacol Exp Ther 1994;269:1086–1093. 130. Stevens CW. Relative analgesic potency of µ, δ and κ opioids after spinal administration in amphibians. J Pharmacol Exp Ther 1996;276:440–448.

131. Stevens CW, Rothe KS. Supraspinal administration of opioids with selectivity for µ-, δ-, and κ-opioid receptors produces analgesia in amphibians. Eur J Pharmacol 1997;331:15–21. 132. Stevens CW. Opioid research in amphibians: A unique perspective on mechanisms of opioid analgesia and the evolution of opioid receptors. Rev Analgesia 2003;7:122–136. 133. Portoghese PS, Sultana M, Takemori AE. Naltrindole, a highly selective and potent non-peptide δ opioid receptor antagonist. Eur J Pharm 1988;146:185–186. 134. Takemori AE, Ho BY, Naeseth JS, Portoghese PS. Norbinaltorphimine, a highly selective κ-opioid antagonist in analgesic and receptor binding assays. J Pharmacol Exp Ther 1988;246: 255–258. 135. Ward SJ, Portoghese PS, Takemori AE. Pharmacological characterization in vivo of the novel opiate, beta-funaltrexamine. J Pharmacol Exp Ther 1982;220:494–498. 136. Stevens CW, Newman LC. Spinal administration of selective opioid antagonists in amphibians: Evidence for an opioid unireceptor. Life Sci 1999;64:PL125–PL130. 137. Darlison MG, Greten FR, Harvey RJ, et al. Opioid receptors from a lower vertebrate (Castomus commersoni): Sequence, pharmacology, coupling to a G-protein-gated inward-rectifying potassium channel (GIRK1), and evolution. Proc Natl Acad Sci USA 1997;94:8214–8219. 138. Barrallo A, González-Sarmiento R, Alvar F, Rodríguez RE. ZFOR2, a new opioid receptor-like gene from the teleost zebrafish (Danio rerio). Mol Brain Res 2000;84:1–6. 139. Rodríguez RE, Barrallo A, Garcia-Malvar F, McFadyen IJ, González-Sarmiento R, Traynor JR. Characterization of ZFOR1, a putative δ-opioid receptor from the teleost zebrafish. Neurosci Lett 2000;288:207–210. 140. Alvarez FA, Rodríguez-Martin I, Gonzalez-Nunez V, de Velasco EM, Gonzalez SR, Rodríguez RE. New κ opioid receptor from zebrafish Danio rerio. Neurosci Lett 2006;405:94–99. 141. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403–410. 142. McFadyen IJ, Metzger TG, Paterlini MG, Ferguson DM. Exploring the unique pharmacology of a novel opioid receptor, ZFOR1, using molecular modeling and the ‘message-address’ concept. Protein Eng 2001;14:953–960. 143. Read MR. Evaluation of the use of anesthesia and analgesia in reptiles. J Am Vet Med Assoc 2004;224:547–552. 144. Green SL. Postoperative analgesics in South African clawed frogs (Xenopus laevis) after surgical harvest of oocytes. Comp Med 2003;53:244–247. 145. Paul-Murphy J, Ludders JW, Robertson SA, Gaynor JS, Hellyer PW, Wong PL. The need for a cross-species approach to the study of pain in animals. J Am Vet Med Assoc 2004;224:692–697. 146. Bradford CS, Walthers EA, Searcy BT, Moore FL. Cloning, heterologous expression and pharmacological characterization of a kappa opioid receptor from the brain of the rough-skinned newt, Taricha granulosa. J Mol Endocrinol 2005;34:809–823. 147. Bradford CS, Walthers EA, Stanley DJ, Baugh MM, Moore FL. ∆ and µ opioid receptors from the brain of a urodele amphibian, the rough-skinned newt, Taricha granulosa: Cloning, heterologous expression, and pharmacological characterization. Gen Comp Endocrinol 2006;146:275–290. 148. Mohan SK, Stevens CW. Systemic and spinal administration of the mu opioid, remifentanil, produces antinociception in amphibians. Eur J Pharmacol 2006;534:89–94. 149. Stevens CW, Brasel CM, Mohan SK. Cloning and bioinformatics of amphibian mu, delta, kappa, and nocieptin opioid receptors expressed in brain tissue: evidence for opioid receptor divergence in mammals. Neurosci Lett 2007;419:189–194.

MODELS OF BEHAVIOR

V

Cardiovascular

A

38

Animal Models of Vascular Development and Endothelial Cell Biology VICTORIA L. BAUTCH

ABSTRACT

tube system to maximize efficient function. Moreover, it was recently shown that the formation of lumens in developing vessels proceeds via vesicle fusion within the endothelial cells, in a manner very similar to lumen formation in the fly tracheal system.3 This chapter will focus on four major vertebrate models of blood vessel formation: the frog (Xenopus laevis and tropicalis), the zebrafish (Danio rerio), the avian chick (Gallus gallus) and the Japanese quail (Coturnix japonica), and the rodent mouse (Mus musculus) and a stem cell model derived from mouse cells. Historically the frog and avian embryos were the tools of choice, for reasons outlined below. However, in recent years the relatively new field of zebrafish development has blossomed, and this vertebrate is particularly useful for aspects of vessel development. The mouse has also gained in popularity in recent years, with the advent of genetic tools and more sophisticated technology for analysis of mammalian embryos. Finally, we and others have utilized mouse embryonic stem cells, which can differentiate to produce endothelial cells and primitive blood vessels, as a parallel model to address questions of mammalian vascular development.

There have been great advances in the past 15 years in our understanding of how blood vessels form and function. Many of these advances result from the availability of several animal models that allow for embryological, molecular, and genetic manipulations of the developing vasculature. Here I describe four vertebrate animal models used in studies of vascular development. The frog, zebrafish, avian, and mouse embryos are compared historically and currently. Recent advances using each model are highlighted. Finally, I describe the use of animal derivatives, the chick chorioallantoic membrane and mouse embryonic stem cells, for studies of blood vessel formation. Key Words: Vascular development, Angiogenesis, Animal models, Xenopus embryos, Zebrafish embryos, Avian embryos, Mouse embryos, Transgenesis, Genetic analysis.

OVERVIEW OF ANIMAL MODELS OF VESSEL DEVELOPMENT Numerous animal models, both historically and currently, have helped us understand blood vessel formation. Important steps include the differentiation of angioblasts to endothelial cells, the initial assembly of endothelial cells to form vessels in the embryo, the expansion of the vessel network, and the remodeling of vessels to accommodate the physiological needs of the organism. These processes were first described, then perturbed using physical agents, pharmacological compounds, and genetic tools. The first known description of embryonic blood vessels was provided by Aristotle in 350 bc, when he cracked open chicken eggs and followed the stages of avian development!1 The animal models of vascular development revolve around vertebrate organisms, since invertebrates do not have a closed circulatory system. However, it might be argued that the fruitfly Drosophila melanogaster has provided a model of blood vessel formation, because a tracheal system develops that opens to the environment and allows diffusion of oxygen into the organism.2 Elegant genetic dissection of tracheal development in the fly has led to a model of tube formation that seems to hold for vertebrate vessels in its essential points. Specifically, both systems appear to develop according to a “hard-wired” genetic program that starts the process, while later physiological inputs from the organism are translated into molecular cues that modify and modulate the

XENOPUS AS A MODEL OF VASCULAR DEVELOPMENT The historical advantages of the frog X. laevis are that the embryos are large and development occurs rapidly outside the mother, allowing for manipulation and visualization. Thus fate mapping studies showed that the endothelial cells that form blood vessels arise from mesoderm in the ventral part of the embryo.4,5 The frog was also used by Cleaver and Krieg in ground-breaking studies showing that migration of precursor cells (angioblasts) from lateral areas to the midline in response to a vascular endothelial growth factor (VEGF) signal initiates formation of the dorsal aorta, a large and important vessel for all vertebrates.6,7 More recently, the injection of a compound that binds endothelial cells, DiI-Ac-LDL, was used to image the patent vasculature at successive stages of frog development.8 Despite these and other important studies, until recently the use of Xenopus has been limited because much of the frog vascular development is not amenable to perturbation by morpholinos, the method of choice to manipulate gene expression in Xenopus embryos. This is because morpholinos, which block gene expression by blocking splicing or translation, are injected early and diluted out with cell division. Thus they are ineffective for

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

355

356

SECTION V / MODELS OF BEHAVIOR

probing many aspects of vascular development in the frog that occur at later stages. However, in recent years a second species, X. tropicalis, has gained in popularity because it is possible to do forward genetics in this model. That is to say, the generation time is such that it is feasible to mutagenize a male, and derive offspring that carry mutations that can be screened and analyzed.9 This is not possible in the original X. laevis. Thus we can look forward to genetic dissection of aspects of vascular development in the frog to complement forward genetics in zebrafish. This will be useful since the frog eventually develops lungs and breathes air, while the fish has gills and gets oxygen from the water. Moreover, it was recently shown that the frog develops a lymphatic vasculature that resembles that of mammals,10 so the development of this parallel vascular system to move lymph fluid can be dissected using the frog model.

ZEBRAFISH AS A MODEL OF VASCULAR DEVELOPMENT HISTORY OF ZEBRAFISH AS A MODEL ANIMAL As mentioned above, the zebrafish has only recently gained popularity as an animal model for development and vascular development. In the 1970s, George Streisinger discovered that zebrafish embryos could be physically manipulated to develop as homozygous diploids, thus allowing for easy genetic analysis.11 This technology proved difficult—more developmental defects were introduced by the process than by the mutagen used to generate mutations! However, he and others realized that the zebrafish had a generation time that allowed for forward genetic screens, with many of the physical advantages of the frog embryo. The zebrafish was firmly established when several major investigators conducted genetic screens for developmental defects and began to analyze these mutations.12,13 ADVANTAGES OF ZEBRAFISH FOR VASCULAR DEVELOPMENT The zebrafish embryo presents several advantages for the study of vascular development. Similar to the frog embryo, it is fairly large, development occurs outside the mother, and the embryos are clear, allowing for easy visualization of developmental processes. Once blood flow commences, patent vessels can be easily seen through the movement of the blood cells. Additionally, it is possible to use microangiography, and several years ago Weinstein and colleagues used this technology to produce a complete atlas of vascular development in the zebrafish.14 Even more recently, several groups have generated transgenic zebrafish that express green fluorescent protein (GFP) in the developing vasculature,15,16 thus allowing for identification of subtle vascular defects. Additionally, it is possible to perturb cardiac or vascular development in the zebrafish embryo without these perturbations inducing immediate lethality, because the embryo obtains oxygen from the water. Thus cardiovascular mutations and perturbations can be dissected in vivo, and the effects of cardiac function on vessel development can be assessed. For example, a mutation named silent heart fails to produce a beating heart, yet the vasculature continues to develop for some time, indicating that a heartbeat or blood flow is not essential for these stages of vascular development.17 These advantages have led several groups to invest in the development of highly sophisticated imaging technologies that allow for live image analysis of vascular development in zebrafish, and lineage tracing of progenitor cells.18,19

The major advantage of the zebrafish model is that it is a vertebrate embryo with a developing vasculature that is amenable to forward genetic screens. Thus a series of mutations can be isolated, characterized, and cloned to further our understanding of novel pathways that may be involved in vascular development, as well as novel relationships among players and pathways already identified.20 Investigators have combined tools for visualization of vascular development described above with forward genetic screens.21 It is also possible to do chemical screens to identify compounds that perturb wild-type vascular development or rescue vascular mutations, since embryos will develop for a time in 96-well formats.22 This aspect of zebrafish biology is especially attractive to pharmaceutical companies that have access to chemical libraries and would like to uncover new therapeutic targets or drugs. RECENT ADVANCES USING ZEBRAFISH TO STUDY VASCULAR DEVELOPMENT Among the recent outpouring of studies using the zebrafish to study blood vessel formation, the studies that elucidated a genetic pathway for specification of arteries vs. veins are particularly compelling. It was known that both genetic cues23 and blood flow24 influenced the decision of vessels to become arteries or veins, but the mechanism was not known. Weinstein and colleagues used a combination of available mutants, morpholino or RNA injection, and chemical inhibitors to define a molecular pathway that determines artery fate in zebrafish. In genetic terms, hedgehog signaling is upstream of vegf signaling, which in turn is upstream of Notch signaling, to determine arteries.25,26 A mutant screen uncovered an additional mutation that proved to be a lesion in the phospholipase C γ gene, and this gene is downstream of vegf signaling in arterial specification.27

AVIAN MODELS OF VASCULAR DEVELOPMENT HISTORY OF AVIAN MODELS OF VASCULAR DEVELOPMENT As mentioned above, the chick embryo was used to visualize vascular development historically. The chick, along with its cousin the quail, has continued to be used in recent times, primarily because the embryos are accessible to manipulation in ovo, and further development is possible. The identification of an epitope on quail angioblasts and blood vessels via production of a specific monoclonal antibody called QH128 led to the first complete description of blood vessel formation in a model animal.29 Moreover, the QH1 antibody has allowed for distinction of quail vessels (QH1 is not found on chick vessels) in transplants of quail tissue into chick hosts. These quail–chick chimeras were used to show that most mesoderm contains cells that can differentiate into endothelial cells and form vessels,30 and that angioblasts from different mesoderm sources have different potentials. Specifically, mesoderm near endoderm is thought to produce angioblasts that can give rise to “hemogenic endothelium,” endothelium that can differentiate into blood cells, while mesoderm near ectoderm does not have this capacity.31,32 The avian models have been overshadowed in recent times because they are not amenable to genetic analysis, but as we shall see below, new technologies can overcome that deficit to some extent. ADVANTAGES OF AVIANS AS A MODEL OF VASCULAR DEVELOPMENT The accessibility of the avian embryo has been combined with gene delivery techniques to successfully manipulate gene expression in some areas of the embryo. Some studies have used viral vectors that encode genes for overexpression studies.33,34 However, recently embryo electroporation has

CHAPTER 38 / ANIMAL MODELS OF VASCULAR DEVELOPMENT AND ENDOTHELIAL CELL BIOLOGY

been used to deliver gene expression to embryonic sites. The most popular site is the neural tube, since it is possible to deliver the DNA to the central cavity prior to electroporation.35,36 This has so far limited the use of this technology for widespread studies of vascular development in the avian, but technically any tissue can be targeted if DNA can be provided to it. The DNA usually encodes proteins that lead to overexpression of the gene of interest, or dominant negative constructs that block function. However, recently modified vectors to produce small hairpin RNAs that lead to local loss of function have been reported, and it is likely that viral delivery of interfering RNA will become a popular method to produce loss-of-function phenotypes in the avian vasculature.37 We and others have adapted the protocol to deliver genes to quail embryos, thus allowing for subsequent analysis of vessel pattern with the QH1 antibody (J. James and V.L. Bautch, unpublished results). RECENT ADVANCES USING AVIAN MODELS OF VASCULAR DEVELOPMENT How Does Blood Flow Influence Vessel Identity? Eichmann and colleagues24 used the avian yolk sac to manipulate flow parameters in an elegant study showing that blood flow is required to maintain arterial identity once it is established. They took advantage of the accessibility of the embryo and yolk sac and ligated the right vitelline artery in the yolk sac. The arterial marker neuropilin 1 was immediately downregulated, and with time venous markers such as neuropilin 2 and Tie-2 were upregulated. Moreover, reperfusion led to upregulation of the arterial markers, showing that in the avian yolk sac blood flow is a determinant of vessel identity. Imaging Vascular Development Little and colleagues have produced an elegant analysis of vascular development in the avian embryo using dynamic imaging techniques.38,39 They used the avian embryo because the early stages of development are relatively flat and accessible compared to the mouse. They labeled the QH1 antibody with a fluoror, and injected it into living embryos. The labeled antibody binds the developing vessels and allows for imaging of early events. Using this live imaging technology they have described migration of endothelial cells over preexisting vessels. How Does the Neural Tube Pattern Blood Vessels? We took advantage of the avian model to develop mouse–quail chimeras that carried grafts of mouse presomitic mesoderm, a source of angioblasts. These grafts and embryonic stem (ES) cell-derived embryoid body grafts were shown to contribute mouse endothelial cells to the vascular plexus that forms around the developing avian neural tube (perineural vascular plexus, PNVP).40,41 Further embryo manipulations placed a mouse neural tube ectopically into the avian embryo, and showed that the neural tube was the source of a signal leading to PNVP formation.42 Studies using explanted tissues placed in collagen showed that VEGF-A is a critical component of the signal.42 Recent work in our laboratory has utilized electroporation technology to manipulate gene expression locally in the developing neural tube, with a goal of further defining the role of VEGF-A in vessel patterning around the neural tube (J. James and V.L. Bautch, unpublished results). AVIAN DERIVATIVES AS MODELS OF VASCULAR DEVELOPMENT It is noteworthy that alongside the avian embryo, the developing vasculature of an extraembryonic tissue called the chorioallantoic membrane (CAM) has been used extensively to study aspects of angiogenesis. The CAM was first used

357

to show that tumor tissue could induce new blood vessel formation,43 and many cancer researchers have tested both pro- and antiangiogenic compounds using the CAM. Briefly, a day 3 chick embryo is opened, and a paper or bead containing a compound is placed on top of the CAM. After 3–4 days of incubation the CAM is harvested and the amount of vasculature determined by quantitative measures.

RODENT MODELS OF VASCULAR DEVELOPMENT HISTORY OF RODENT MODELS OF VASCULAR DEVELOPMENT Mouse and rat are the primary experimental models for mammalian development because of their size, ability to reproduce, and a rich history of genetics for the mouse. However, their utility for studies of vascular development was hampered by the fact that development occurs in utero and is difficult to visualize. Moreover, most perturbations to the developing vasculature quickly lead to embryonic death, and historically there was not a good in situ marker for immature mammalian vessels. However, the finding that PECAM (platelet endothelial cell adhesion molecule) is expressed on mouse angioblasts and early vessels44–46 and the development of a high-quality monoclonal antibody to mouse PECAM47 have provided a powerful tool for visualization of mouse embryonic vessels. This and advances in genetic manipulation discussed below have made the mouse the premier model for studies of mammalian vascular development in recent years. Of the models discussed here, the processes and mechanisms of vascular development in the mouse most closely mimic those of human vascular development, so there is great interest in using and refining this model of vascular development. ADVANTAGES OF THE MOUSE AS A MODEL OF VASCULAR DEVELOPMENT The primary advantage of the mouse is that it is possible to manipulate the genome by introducing mutations into specific genes.48 This results from several technologies developed in the 1980s and 1990s. First, ES cells that can produce descendants contributing to the germline of the animal have been isolated. These ES cells can be manipulated in vitro prior to reintroduction to a mouse embryo. Second, it is possible to target a specific gene locus in ES cells via homologous recombination. Thus it is possible to mutate or delete one of the two alleles of a gene in ES cells, select for the rare homologous recombination (“targeting”) events, and then reintroduce the cells to embryos, eventually leading to animals with the mutation in both alleles. In this way numerous genes have been deleted, and the effects on mammalian vascular development analyzed. Examples are the two high-affinity receptors for VEGF-A, flk-1 (VEGFR-2) and flt-1 (VEGFR-1), that when deleted lead to perturbed vascular development and death.49,50 However, the phenotypes of the mutant embryos were very different; deletion of flk-1 led to reduced vessel development, while deletion of flt-1 led to vessel overgrowth. This was among the first evidence that the receptors did not function in the same way in vascular development. In recent years it has become possible to selectively delete genes in the developing vasculature.51,52 This is done by introducing recombination sites (loxP) into one allele of a gene in ES cells, and then generating an animal with sites in both alleles. These mice are then mated with mice that express the relevant recombinase (Cre) in only the developing blood vessels.53–56 This modification has allowed investigators to analyze the function of genes

358

SECTION V / MODELS OF BEHAVIOR

in vessels that may have required roles in other places of the embryo earlier in development. Even more recently, an inducible form of the recombinase has been used, and in this scenario excision of the allele occurs in vascular tissues only when the inducer is present.57,58 Along with these techniques to manipulate gene function, the ability to analyze vascular phenotypes in the mouse embryo has grown quite sophisticated. There are now numerous markers of endothelial cells: markers that distinguish arteries from veins and markers that distinguish blood-carrying vessels from lymphatic vessels.59 These markers are of three types: antibodies that recognize proteins or epitopes that are vessel specific (see PECAM description above), antisense RNA in situ hybridization probes that recognize vessel-specific mRNAs, and reporter genes that are placed into a gene locus, then are expressed when gene expression is activated. The most popular of the reporter genes is bacterial lacZ. The lacZ gene encodes the β-galactosidase protein that turns a substrate blue only in the cells that express lacZ, so vessels or vessel subsets are stained blue after the chemical reaction. Recently GFP has also been used as a reporter gene because it allows for live imaging. RECENT ADVANCES USING MOUSE MODELS OF VASCULAR DEVELOPMENT What Regulates Blood Vessel Branching? Analysis of the pattern of blood vessels in mid-gestation mouse embryos carrying mutations has implicated several molecular signaling pathways in the regulation of blood vessel branching. Among these branching regulators are pathways that regulate axon guidance in the developing nervous system, including the semaphorin (Sema) pathway and the netrin pathway. The Sema pathway is complicated, because some Semas interact with neuropilins, and the neuropilin coreceptors can complex with either vascular-expressed flk-1 or neuronal or vascular-expressed plexins. However, recently it was shown that plexin D1 is expressed primarily in blood vessels, and a knockout mutation led to vascular defects.60 The netrin pathway was shown to be involved in branching by analysis of a mutation in a netrin receptor, UNC5B.61 It is not clear whether this pathway promotes or decreases branching,62,63 but it is clearly involved in the processes. Does Vasculogenesis Occur after Birth? Until the mid1990s the dogma in the field was that vasculogenesis, the process whereby endothelial progenitor cells form a vessel while differentiating, occurred only during the early stages of development. Subsequent to those stages, all vessel formation and expansion were thought to occur via angiogenesis—the migration, division, and fusion of endothelial cells already in vessels. However, evidence suggested that perhaps endothelial cells could be formed de novo in some situations, and evidence for this model was provided through the use of the mouse. Mice were irradiated so their bone marrow was destroyed, and then bone marrow from a “marked” donor was grafted into the host. Once recovered, the chimeric mouse now had a “marked” bone marrow, and it was subjected to various situations in which new vessels form. In ischemic injury recovery and tumor angiogenesis, marked cells were seen in the perivascular area, suggesting that bone marrowderived cells could contribute to neoangiogenesis.64 While there is controversy about the lineage and ultimate fate of the bone marrow cells, most investigators think that an endothelial progenitor (EPC) resides in the bone marrow, and it can be mobilized to sites of new vessel formation and perhaps differentiate into an

endothelial cell. Thus vasculogenesis may not be confined to embryonic stages, but may occur alongside angiogenesis in the adult as well. MOUSE DERIVATIVES AS MODELS OF VASCULAR DEVELOPMENT The major mouse derivative used for studies of vascular development is the ES cell that was discussed above in the context of targeted mutations. ES cells can be aggregated and differentiate in vitro to form embryoid bodies, which can then cavitate to become cystic.65,66 This programmed differentiation leads to the formation of endoderm and mesoderm, and subsequently derivatives of these lineages.67,68 Amazingly, endodermderived factors such as VEGF-A provide appropriate signals for the differentiation of endothelial precursors, vasculogenesis leads to the formation of primitive vessels, and then angiogenesis results in the expansion of a primitive vascular plexus in this model.69,70 We and others realized that if the embryoid bodies are reattached prior to cavitation, vascular development proceeds as described above.67,69 Thus embryoid bodies or ES cell cultures can be manipulated both genetically and pharmacologically to address questions of vascular development. Simon and colleagues have shown a requirement for hypoxia-driven gene expression in vascular development using the ES cell model.71 We recently developed ES cell lines that express vascular-restricted GFP, and this allowed us to image the dynamic processes of vascular development.68 Using this tool we showed that the VEGF-A receptor flt-1 (VEGFR-1) positively modulates branching morphogenesis of developing vessels, probably via modulation of the amount and/or spatial context of signaling through the flk-1 (VEGFR-2) receptor.72 Keller and colleagues73,74 used the ES cell model to elucidate lineage relationships. Specifically, evidence for a bipotential common precursor of both blood and endothelial cells, called the hemangioblast, was presented. Mouse ES cells were partially differentiated as described above, then dissociated to single cells and plated in conditions conducive to differentiation of both blood cells and endothelial cells. The finding that single cells could give rise to both endothelial and blood cells was consistent with the presence of a hemangioblast among the cells. Subsequent studies in vivo corroborated this finding,75 but these studies did not distinguish between a true bipotential cell and a multipotential progenitor that could also differentiate into other mesodermal lineages. Recently an elegant study using zebrafish provided evidence that a bipotential hemangioblast does exist, although many endothelial cells do not seem to go through this intermediate.19 The latter study points out the utility of having multiple models of vascular development available for analysis. The cells were tracked by injecting a compound that was then uncaged in a single progenitor cell using a laser. This was technically challenging in the zebrafish embryo and is not currently feasible in the other models.

CONCLUSIONS Our understanding of blood vessel formation has benefited enormously from the availability of several animals models of vascular development, and there have been great strides in our understanding of vascular development at the cell and molecular level in the past several years. I predict that in the near future much will be added to this knowledge base through the creative use of animal models of vascular development such as those described here. More to the point, even more advances will likely

CHAPTER 38 / ANIMAL MODELS OF VASCULAR DEVELOPMENT AND ENDOTHELIAL CELL BIOLOGY

come from the integrated use of multiple animal models of vascular development. For example, it is possible to envision a forward genetic screen in zebrafish to uncover a novel gene important in vascular development, then manipulation of the gene through RNA and morpholinos in both the fish and frog, followed by vascular-specific inducible deletion in the mouse. In this way a comprehensive analysis of the role of a particular gene in vascular development should be elucidated. The sequencing of the relevant genomes and reagents for microarray analysis will no doubt augment future efforts to further understand the mechanisms regulating the development of blood vessels.

ACKNOWLEDGMENTS This work was supported by grants from the NIH (R01HL43174 and R01HL83262) to V.L.B.

REFERENCES 1. Gilbert S. Developmental Biology. Sunderland, MA: Sinauer Associates, Inc., 2003:838. 2. Ghabrial A, Luschnig S, Metzstein MM, Krasnow MA. Branching morphogenesis of the Drosophila tracheal system. Annu Rev Cell Dev Biol 2003;19:623–647. 3. Kamei M, Brian Saunders W, Bayless KJ, Dye L, Davis GE, Weinstein BM. Endothelial tubes assemble from intracellular vacuoles in vivo. Nature 2006;442:453–456. 4. Rovainen C. Labeling of developing vascular endothelium after injections of rhodamine-dextran into blastomeres of Xenopus laevis. J Exp Zool 1991;259:209–221. 5. Mills KR, Kruep D, Saha MS. Elucidating the origins of the vascular system: A fate map of the vascular endothelial and red blood cell lineages in Xenopus laevis. Dev Biol 1999;209:352–368. 6. Cleaver O, Tonissen KF, Saha MS, Krieg PA. Neovascularization of the Xenopus embryo. Dev Dyn 1997;210:66–77. 7. Cleaver O, Krieg PA. Molecular mechanisms of vascular development. In: Harvey RP, Rosenthal N, Eds. Heart Development. New York: Academic Press, 1999:221–252. 8. Levine AJ, Munoz-Sanjuan I, Bell E, North AJ, Brivanlou AH. Fluorescent labeling of endothelial cells allows in vivo, continuous characterization of the vascular development of Xenopus laevis. Dev Biol 2003;254:50–67. 9. Carruthers S, Stemple DL. Genetic and genomic prospects for Xenopus tropicalis research. Sem Cell Dev Biol 2006;17:146–153. 10. Ny A, Koch M, Schneider M, Neven E, Tong RT, Maity S, Fischer C, Plaisance S, Lambrechts D, Heligon C, Terclavers S, Ciesiolka M, Kalin R, Man WY, Senn I, Wyns S, Lupu F, Brandli A, Vleminckx K, Collen D, Dewerchin M, Conway EM, Moons L, Jain RK, Carmeliet P. A genetic Xenopus laevis tadpole model to study lymphangiogenesis. Nat Med 2005;11:998–1004. 11. Streisinger G, Walker C, Dower N, Knauber D, Singer F. Production of clones of homozygous diploid zebra fish (Brachydanio rerio). Nature 1981;291:293–296. 12. Mullins MC, Hammerschmidt M, Haffter P, Nusslein-Volhard C. Large-scale mutagenesis in the zebrafish: In search of genes controlling development in a vertebrate. Curr Biol 1994;4:189–202. 13. Amsterdam A, Burgess S, Golling G, Chen W, Sun Z, Townsend K, Farrington S, Haldi M, Hopkins N. A large-scale insertional mutagenesis screen in zebrafish. Genes Dev 1999;13:2713–2724. 14. Isogai S, Horiguchi M, Weinstein BM. The vascular anatomy of the developing zebrafish: An atlas of embryonic and early larval development. Dev Biol 2001;230:278–301. 15. Lawson ND, Weinstein BM. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev Biol 2002;248:307– 318. 16. Jin S-W, Beis D, Mitchell T, Chen J-N, Stainier DYR. Cellular and molecular analyses of vascular tube and lumen formation in zebrafish. Development 2005;132:5199–5209.

359

17. Isogai S, Lawson ND, Torrealday S, Horiguchi M, Weinstein BM. Angiogenic network formation in the developing vertebrate trunk. Development 2003;130:5281–5290. 18. Weinstein BM. Vascular cell biology in vivo: A new piscine paradigm? Trends Cell Biol 2002;12:439–445. 19. Vogeli KM, Jin S-W, Martin GR, Stainier DYR. A common progenitor for haematopoietic and endothelial lineages in the zebrafish gastrula. Nature 2006;443:337–339. 20. Stainier DYR, Fouquet B, Chen J, Warren K, Weinstein BM, Meiler S, Mohi M, Neuhauss S, Solnica-Krezel L, Schier A, Zwartkruis F, Stemple DL, Driever W, Fishman MC. Mutations affecting the formation and function of the cardiovascular system in the zebrafish embryo. Development 1996;123:285–292. 21. Weinstein BM. Plumbing the mysteries of vascular development using the zebrafish. Semin Cell Dev Biol 2002;13:515–522. 22. Peterson RT, Shaw SY, Peterson TA, Milan DJ, Zhong TP, Schreiber SL, MacRae CA, Fishman MC. Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation. Nat Biotechnol 2004;22:595–599. 23. Mukouyama YS, Shin D, Britsch S, Taniguchi M, Anderson DJ. Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin. Cell 2002;109:693–705. 24. le Noble F, Moyon D, Pardanaud L, Yuan L, Djonov V, Matthijsen R, Breant C, Fleury V, Eichmann A. Flow regulates arterial-venous differentiation in the chick embryo yolk sac. Development 2004; 131:361–375. 25. Lawson ND, Scheer N, Pham VN, Kim CH, Chitnis AB, CamposOrtega JA, Weinstein BM. Notch signaling is required for arterialvenous differentiation during embryonic vascular development. Development 2001;128:3675–3683. 26. Lawson ND, Vogel AM, Weinstein BM. Sonic hedgehog and vascular endothelial growth factor act upstream of the notch pathway during arterial endothelial differentiation. Dev Cell 2002;3:127–136. 27. Lawson ND, Mugford JW, Diamond BA, Weinstein BM. Phospholipase C gamma-1 is required downstream of vascular endothelial growth factor during arterial development. Genes Dev 2003;17: 1346–1351. 28. Pardanaud L, Altmann C, Kitos P, Dieterlen-Lievre F, Buck CA. Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells. Development 1987;100: 339–349. 29. Coffin JD, Poole TJ. Embryonic vascular development: Immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia in quail embryos. Development 1988;102:735–748. 30. Noden DM. Embryonic origins and assembly of blood vessels. Am Rev Respir Dis 1989;140:1097–1103. 31. Pardanaud L, Yassine F, Dieterlen-Lievre F. Relationship between vasculogenesis, angiogenesis and haemopoiesis during avian ontogeny. Development 1989;105:473–485. 32. Pardanaud L, Luton D, Prigent M, Bourcheix LM, Catala M, Dieterlen-Lievre F. Two distinct endothelial lineages in ontogeny, one of them related to hemopoiesis. Development 1996;122: 1363–1371. 33. Iba H. Gene transfer into chicken embryos by retrovirus vectors. Dev Growth Differ 2000;42:213–218. 34. Ishii Y, Reese D, Mikawa T. Somatic transgenesis using retroviral vectors in the chicken embryo. Dev Dyn 2004;229:630–642. 35. Ogura T. In vivo electroporation: A new frontier for gene delivery and embryology. Differentiation 2002;70:163–171. 36. Swartz M, Eberhart J, Mastick GS, Krull CE. Sparking new frontiers: Using in vivo electroporation for genetic manipulations. Dev Biol 2001;233:13–21. 37. Bomberg-White J, Webb C, Patacsil V, Miranti C, Williams B, Holmen S. Delivery of short hairpin RNA sequences by using a replication-competent avian retroviral vector. J Virol 2004;78: 4914–4916. 38. Rupp PA, Czirok A, Little CD. Novel approaches for the study of vascular assembly and morphogenesis in avian embryos. Trends Cardiovasc Med 2003;13:283–288.

360

SECTION V / MODELS OF BEHAVIOR

39. Rupp PA, Czirok A, Little CD. αvβ3 integrin-dependent endothelial cell dynamics in vivo. Development 2004;131:2887–2897. 40. Ambler CA, Nowicki JL, Burke AC, Bautch VL. Assembly of trunk and limb blood vessels involves extensive migration and vasculogenesis of somite-derived angioblasts. Dev Biol 2001;234:352–364. 41. Ambler CA, Schmunk GA, Bautch VL. Stem cell-derived endothelial cells/progenitors migrate and pattern in the embryo using the VEGF signaling pathway. Dev Biol 2003;257:205–219. 42. Hogan KA, Ambler CA, Chapman DL, Bautch VL. The neural tube patterns vessels developmentally using the VEGF signaling pathway. Development 2004;131:1503–1513. 43. Norrby K. In vivo models of angiogenesis. J Cell Mol Med 2006;10:588–612. 44. Baldwin H, Shen H, Yan H, DeLisser H, Chung A, Mickanin C, Trask T, Kirschbaum N, Newman P, Albelda S. Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): Alternatively spliced, functionally distinct isoforms expressed during mammalian cardiovascular development. Development 1994;120:2539–2553. 45. DeLisser H, Christofidou-Solomidou M, Strieter R, Burdick M, Robinsion C, Wexler R, Kerr J, Garlanda C, Merwin J, Madri J, Albelda S. Involvement of endothelial PECAM-1/CD31 in angiogenesis. Am J Pathol 1997;151:671–677. 46. Redick SD, Bautch VL. Developmental platelet endothelial cell adhesion molecule expression suggests multiple roles for a vascular adhesion molecule. Am J Pathol 1999;154:1137–1147. 47. Nourshargh S, Krombach F, Dejana E. The role of JAM-A and PECAM-1 in modulating leukocyte infiltration in inflamed and ischemic tissues. J Leukoc Biol 2006;80:714–718. 48. Downing GJ, Battey JF Jr. Technical assessment of the first 20 years of research using mouse embryonic stem cell lines. Stem Cells 2004;22:1168–1180. 49. Shalaby F, Ho J, Stanford WL, Fischer KD, Schuh AC, Schwartz L, Bernstein A, Rossant J. A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis. Cell 1997;89:981– 990. 50. Fong GH, Rossant J, Gertsenstein M, Breitman M. Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 1995;376:66–70. 51. Metzger D, Chambon P. Site- and time-specific gene targeting in the mouse. Methods 2001;24:71–80. 52. Sauer B. Inducible gene targeting in mice using the cre/lox system. Methods 1998;14:381–392. 53. Gustafsson E, Brakebusch C, Hietanen K, Fassler R. Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice. J Cell Sci 2001;114:671–676. 54. Kisanuki YY, Hammer RE, Miyazaki J-i, Williams SC, Richardson JA, Yanagisawa M. Tie2-Cre transgenic mice: A new model for endothelial cell-lineage analysis in vivo. Dev Biol 2001;230: 230–242. 55. Motoike T, Markham D, Rossant J, Sato T. Evidence for novel fate of Flk1 progenitor: Contribution to muscle lineage. Genesis 2003;35:153–159. 56. Alva JA, Zovein A, Monvoisin A, Murphy T, Salazar A, Harvey N, Carmeliet P, Iruela-Arispe M. VE-Cadherin-Cre-recombinase transgenic mouse: A tool for lineage analysis and gene deletion in endothelial cells. Dev Dyn 2006;235:759–767. 57. Forde A, Constien R, Grone H, Hammerling G, Arnold B. Temporal Cre-mediated recombination exclusively in endothelial cells using Tie2 regulatory elements. Genesis 2002;33:191–197. 58. Gothert JR, Gustin SE, Hall MA, Green AR, Gottgens B, Izon DJ, Begley CG. In vivo fate-tracing studies using the Scl stem cell

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74. 75.

enhancer: Embryonic hematopoietic stem cells significantly contribute to adult hematopoiesis. Blood 2005;105:2724–2732. Eichmann A, Yuan L, Moyon D, le Noble F, Pardanaud L, Breant C. Vascular development: From precursor cells to branched arterial and venous networks. Int J Dev Biol 2005;49:259–267. Gitler AD, Lu MM, Epstein JA. PlexinD1 and semaphorin signaling are required in endothelial cells for cardiovascular development. Dev Cell 2004;7:107–116. Lu X, le Noble F, Yuan L, Jiang Q, de Lafarge B, Sugiyama D, Breant C, Claes F, De Smet F, Thomas J-L, Autiero M, Carmeliet P, Tessier-Lavigne M, Eichmann A. The netrin receptor UNC5B mediates guidance events controlling morphogenesis of the vascular system. Nature 2004;432:179–186. Park KW, Crouse D, Lee M, Karnik SK, Sorensen LK, Murphy KJ, Kuo CJ, Li DY. The axonal attractant Netrin-1 is an angiogenic factor. Proc Natl Acad Sci USA 2004;101:16210–16215. Wilson BD, Ii M, Park KW, Suli A, Sorensen LK, Larrieu-Lahargue F, Urness LD, Suh W, Asai J, Kock GAH, Thorne T, Silver M, Thomas KR, Chien C-B, Losordo DW, Li DY. Netrins promote developmental and therapeutic angiogenesis. Science 2006;313: 640–644. Barber CL, Iruela-Arispe ML. The ever-elusive endothelial progenitor cell: Identities, functions and clinical implications. Pediatr Res 2006;59:26R-32R. Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R. The in vitro development of blastocyst-derived embryonic stem cell lines: Formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol 1985;87:27–45. Risau W, Sariola H, Zerwes HG, Sasse J, Ekblom P, Kemler R, Doetschman T. Vasculogenesis and angiogenesis in embryonicstem-cell-derived embryoid bodies. Development 1988;102:471– 478. Bautch VL. Embryonic stem cell differentiation and the vascular lineage. In: Turksen K, Ed. Methods in Molecular Biology, Vol. 185, Embryonic Stem Cells: Methods and Protocols. Totowa, NJ: Humana Press, 2001:117–125. Kearney JB, Bautch VL. In vitro differentiation of mouse ES cells: Hematopoietic and vascular development. Methods Enzymol 2003;365:83–98. Bautch VL, Stanford WL, Rapoport R, Russell S, Byrum RS, Futch TA. Blood island formation in attached cultures of murine embryonic stem cells. Dev Dyn 1996;205:1–12. Bautch VL, Redick SD, Scalia A, Harmaty M, Carmeliet P, Rapoport R. Characterization of the vasculogenic block in the absence of vascular endothelial growth factor-A. Blood 2000;95:1979–1987. Ramirez-Bergeron DL, Runge A, Dahl KDC, Fehling HJ, Keller G, Simon MC. Hypoxia affects mesoderm and enhances hemangioblast specification during early development. Development 2004;131: 4623–4634. Kearney JB, Kappas NC, Ellerstrom C, DiPaola FW, Bautch VL. The VEGF receptor flt-1 (VEGFR-1) is a positive modulator of vascular sprout formation and branching morphogenesis. Blood 2004;103: 4527–4535. Choi K, Kennedy M, Kazarov A, Papadimitriou J, Keller G. A common precursor for hematopoietic and endothelial cells. Development 1998;125:725–732. Park C, Ma YD, Choi K. Evidence for the hemangioblast. Exp Hematol 2005;33:965–970. Huber TL, Kouskoff V, Joerg Fehling H, Palis J, Keller G. Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature 2004;432:625–630.

39

Models of Behavior Cardiovascular LINA BADIMON, LAURA CASANÍ,

ABSTRACT

AND

GEMMA VILAHUR

The study of thrombosis and its inhibition is based on in vitro, ex vivo, and in vivo approaches that combined have helped to widen the knowledge of the thrombotic process itself. Indeed, the availability of transgenic and knockout animals makes it possible to pinpoint the relative functional importance of single changes in specific gene products and hence facilitates the formulation of new strategies for cardiovascular protection and the prevention and treatment of thrombosis. However, on the other hand, domestic swines have been widely used as biomedical research models for human diseases due to their similarities in cardiovascular anatomy and physiology as well their ability to develop spontaneous or diet-induced atherosclerosis. Nevertheless, the objectives of a study should mandate the type of animal model to use in order to avoid repetitive and unneeded experiments. For instance, although swine are up to 90% similar to humans, there are some molecular differences in the mechanisms involved in arterial thrombosis that must be taken into account in order to properly evaluate the data.

Experimental models of thrombosis are important research resources that have helped to increase our understanding and knowledge of the pathogenesis of the atherothrombotic process as well as to develop novel therapeutic approaches. In fact, different animal models and procedures have been implemented in thrombosis research in order to provide new insights into the mechanisms that have already been outlined in isolated cells and protein studies. Moreover, animal preclinical studies have rendered a solid rationale for conducting prospective randomized trials in patients with coronary artery disease.

INTRODUCTION This chapter reviews the more common animal models used in the study of thrombotic disease, focusing primarily on the management, particularities, and topics related to the porcine animal model because of its human resemblance, particularly in the cardiovascular system.1–7

ANIMAL SIZE MATTERS? THE USE OF ANIMAL MODELS TO STUDY ARTERIAL THROMBOSIS

Some of the reasons for the frequent use of small animal models in research include (1) low cost, (2) ready availability, (3) reduced ethical concern compared to large animals, especially primates, and (4) small size that limits the quantities of new agents required for in vivo screening. These characteristics have permitted rapid evaluation of new agents in sufficiently large populations to perform meaningful statistical analyses. In addition to these practical indications for their use, small animal models have the added advantage of well-defined genetic characterization, and, in the case of mice, the availability of transgenic and gene knockout animals. Furthermore, small animals have figured prominently in the study of the contributions of thrombosis to luminal narrowing following arterial injury.12 In contrast, large animal models such as pigs have the advantage of being more comparable to humans, especially because of their susceptibility to develop atherosclerosis.1,7,8,13 Furthermore, their large size has permitted the evaluation of their coronary arteries, rather than the aortas studied in small animals because of its relatively larger size. In addition, the ability to induce atheromatous disease, which simulates the lesions observed in humans, is a strong attribute of the pig as a large animal model. Conversely, the disadvantages of large animal models are primarily the reverse of the advantages of small animal

Autopsy and angiographic studies have pointed out the importance of plaque rupture with subsequent exposure of atherosclerotic components as a key step of thrombus formation and the subsequent clinical events. However, “the paradigm of plaque rupture” presents several contradictions that must be considered. As such, autopsies have shown that a substantial minority of victims of sudden coronary death shows superficial plaque erosion without plaque rupture,8,9 and although plaque disruption accounts for up to two-thirds of patients in whom unstable angina or acute myocardial infarction develops, in most cases plaque disruptions are asymptomatic, although they may contribute to the growth of the atherosclerotic plaque.10,11 Despite this, there is no doubt that a defect in the surface of the atherosclerotic plaque is almost always associated with mural thrombosis, which, at least, leads to plaque progression.

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

361

362

SECTION V / MODELS OF BEHAVIOR

models. These include greatly increased cost, heightened ethical concerns, less precise genetic characterization, and a scarcity of transgenic models and antibodies.

THE PIG AS A COMMONLY ACCEPTED RESEARCH ANIMAL MODEL Previously published articles mainly present three types of atherothrombotic-related experiments, sometimes combined, performed in pigs: (1) feeding animals with fat-rich diets, (2) intravascular interventions to induce damage (balloon, grafting, etc.), and (3) extracorporeal arteriovenous shunts into which flow chambers of varying patterns are coupled. However, before thoroughly reviewing these topics several pig-related features deserve to be acknowledged to better understand the swine model of atherothrombosis. SWINES NUTRITIVE REQUIREMENTS The majority of swines used in research weight between 10 and 80 kg and require approximately 600–2800 g of food per day.14 Experimental animals can be fed with the commercial chow used for feeding farm animals, although it has been specially formulated in order to obtain a rapid growth rate. However, if there is a research requirement to use animals between 9 and 18 kg (40–60 days old) they should be fed with a nutrient-enriched diet (i.e., postweaning chow). Diet transition should be performed by mixing increasing parts of growing chow with the postweaning chow in order to reduce the presence of food-related diarrheas. For long time period studies, it is desirable to feed the animal with a restricted diet (3.5% of their total weight) to avoid handling difficulties related to animal overweight. This food restriction (also used in farm animals) covers all the animal nutritive and metabolic requirements as published by the National Research Council (NRC).15 Commercial diets for farm pigs usually contain antibiotics, promoters, and other supplements to increase weight gain. If these components can somehow interfere with the study objectives, free-additive diets can be especially formulated and obtained from local venders. Finally, oral drug administration in swines is easy to perform, especially after overnight fasting. The suggestion is to mix or dissolve the drug within a small amount of chow before offering them the calculated ration. Swine approximately require 2.5 liters of water per kg of consumed food per day. Water should be provided by an automated watering system to avoid water restriction-related neurological syndromes such as “salt poisoning” or “sodium toxicosis.”16,17 HEART ANATOMY AND VASCULARIZATION The heart of the pig is anatomically similar to humans except for the presence of the left azygous (hemiazygous) vein, which drains the intercostal system into the coronary sinus,18 and for the size, which tends to be a bit smaller. The heart blood supply is mostly right side dominant since it originates from the posterior septal artery19 and both anatomy and function of the pig coronary system as well as the histological anatomy of the aorta are comparable to humans. However, on the other hand, pig blood vessels are more friable and prone to vasospasm during manipulation, and thus require careful handling during blood withdrawals. Although animal models generally have the advantage of a shorter time to lesion development, pig atherosclerosis is quite slow and occurs both spontaneously and by experimental induction (i.e., intake of

a high atherogenic diet). Moreover, if allowed to develop over time, mild atherosclerotic lesions first appear in coronary arteries and both atherosclerotic plaque distribution and composition (lipid, fibrinogen, smooth muscle cells, and macrophage content)20 follow a pattern comparable to that of humans.21–23 Human-like pig lipoprotein metabolisms may help to explain in part the above mentioned similarities. In relation to pig hemodynamics, either physiological cardiac function or mechanically induced myocardial infarction and the subsequent arrhythmogenic activity in reperfusion are also analogous to humans as well as the woundhealing process.24 Actually, although wound healing in the myocardium has typically used both swine and sheep models, ruminants’ healing somehow differs from humans since it is mainly characterized by the formation of collagenous scars.25 Finally, unlike other animal models, after gradual occlusion of the coronary vessels induced by both balloon injury and an atherogenic diet, swines may develop coronary restenosis syndrome.19,21,22,24,26–28 HUSBANDRY AND MANAGEMENT Recommendations for housing laboratory swine are regulated by the European Directive 2003/65/CE, by local governmental animal care laws, and in the United States by the National Institutse of Health, Guide for the Care and Use of Laboratory Animals.29 Plastic slats (PVC, vinyl) are preferred in biomedical research facilities because they are easy to clean and better isolate animals from humidity. If possible, heating plates should be installed in all pens (Figure 39–1). Swine reared for meat received standard treatments for endoparasites and ectoparasites and vaccinations against common swine diseases at appropriate ages and intervals. However, according to our previous experience, animals that are going to be part of a long-term project should undergo preventive treatment with antibiotics and antiparasites for 3 days as soon as they arrive at the animal facilities to avoid the presence of infectious diseases that may modify physiological pig parameters. Restraint methods should be discouraged in biomedical research because of the stress they induce.30 Subsequently, shortterm chemical restraint agents and anesthetics may be used in order to obtain blood samples. ANESTHESIA, ANALGESIA, AND SURGERY Animal pain and suffering are clinically important conditions that adversely affect the quality of life of the animals and as a result may influence the experimental data. Drugs, techniques, or husbandry methods used to prevent and control pain must be tailored to individual animals and should be based, in part, on the species, breed, age, procedure performed, degree of tissue trauma, individual behavioral characteristics, degree of pain, and health status. Preventive analgesia before major surgery must be performed with agents that do not interfere with platelets or the coagulation system. Accordingly, the use of opioids, particularly buprenorphine and fentanyl, is highly recommended. In our group, we use different agents to induce swine anesthesia depending on the amount of sample required and on the nature of the objectives we want to accomplish. For instance, tranquilization with 2.2 mg/kg of azaperone is more than enough to obtain a small blood sample (less than 10 ml) from the auricular vein or to cannulate a superficial vein (auricular or mammary vein). Once tranquilized, animals can be induced to anesthesia with an intravenous bolus of pentobarbital (5 mg/kg) to either obtain larger

CHAPTER 39 / MODELS OF BEHAVIOR

363

A'

A

B

E

C

D

Figure 39–1. Swine PVC pens in a biomedical research facility. (A, A′) General view of pens. The walls in contact with the animals are covered with stainless steel. These are 1-m height plastic separations between pens and slip-free fiberglass slat flooring for greater hygiene. (A) The size of the pens allows a resting and a feeding area;

toys can be as simple as balls. (C–E) Details of integrated aluminum heating plates, stainless-steel feeders (easily fixed to the floor with a mobile screw), and nipple drinkers placed at a standard height of 30 cm.

amounts of blood via the femoral vein/artery or to perform small surgical procedures. An intramuscular combination of tiletamine/ zolazepam and medetomidine induces a fast anesthetic status sufficient to perform small surgery such as central vessel catheterization. To achieve general deep anesthesia a continuous infusion of pentobarbital-saline (infusion rate of 2 mg/min) should be used, rather than repeated pentobarbital bolus injections. Deep anesthesia can also be induced by pentobarbital and maintained with volatile anesthesia (i.e., isofluorane). Within this context, it is important to emphasize that higher doses of pentobarbital (>30 mg/ kg) have previously been described to reduce platelet reactivity31 and an overdose can cause fatal respiratory and cardiac depression.

with a standard hypercholesterolemic diet, we have previously observed that almost all pigs developed early atherosclerotic lesions (fatty streaks) localized in the abdominal aorta and to a lesser extent in the coronary arteries. In such cases, lesion composition was similar to early stage human atherosclerosis.20 As expected, increasing the diet induction period to 100 and 150 days was associated with a higher degree of lesion severity made evident when suitable fat stain was applied (Sudan IV). Notwithstanding, animals employed in these long period studies tend to be within the prepuberty period and as a consequence their lesions are not as severe as those developed in adult humans over the years. Older animals (over 6 months) can be used to obtain atherosclerotic models of greater human resemblance,32 but management will be even more difficult because of their considerable weight (up to 200 kg). To overcome size-related problems, minipigs are becoming increasingly used. Indeed, miniature swine are preferable to commercial swine as animal models because of their small size and small growth rate, which allow them to maintain weight and size throughout adulthood. They are docile and easily handled, and there are several miniature strains available to better fit the scientific purpose. Miniature pigs have been used in several fields of biomedical

THE PIG AS A TOOL FOR EVALUATING ATHEROTHROMBOTIC DISEASE DIET-INDUCED ATHEROSCLEROSIS Swine can develop hypercholesterolemia and atherosclerotic lesions by diet induction (high cholesterol content diets), reaching plasma cholesterol levels similar to those in humans. Indeed, after a 50-day period

364

SECTION V / MODELS OF BEHAVIOR

research studies such as cardiovascular disease (models of thrombosis and restenosis), diabetes, transplantation, and dermal applications. Miniature pigs can be allocated in the same pens as larger pigs (increasing the number of animals per pen) and feeders and drinkers can be adapted to their size. However, the principal disadvantages of using miniature pigs as a substitute for common pigs are the maintenance requirements and the high cost of the animals. As a consequence, miniature pig breeders are not extensively distributed in all countries. EX VIVO PORCINE MODEL OF THROMBOSIS The ex vivo porcine model of thrombus formation (e.g., flow chambers coupled to extracorporeal shunts) has become essential for testing the effect of blood elements and rheology as well as atherosclerotic vessel components in thrombus formation in a controlled manner.33–40 At the same time it is reproducible and easy to manipulate and thus allows the evaluation of the thrombogenic effect of different plaque constituents (e.g., collagen, fatty streaks, smooth muscle cells),33–35 the different degrees of shear stress (to mimic different degrees of stenosis),36,37 and the antiplatelet effects of new antithrombotic compounds.20,35,37,40–43 Shear stress measures the difference in blood velocity between the center of the vessel and along the vessel wall. Atherosclerosis preferentially occurs in areas of turbulent blood flow and low fluid shear stress, whereas laminar blood flow and high shear stress are atheroprotective. The Badimon perfusion chamber allows control of the blood flow as well as the degree of stenosis and thus evaluates moderate to high-risk thrombotic situations. A positive correlation between vessel stenosis and platelet deposition has previously been described.44 Furthermore, platelet axial distribution analysis indicates that the apex (not the flow recirculation zone distal to the apex) is the segment with the greatest platelet accumulation, suggesting the crucial role of sudden changes in the degree of stenosis after plaque rupture, that is, shear stress changes in acute platelet response.36 The Badimon perfusion chamber also makes it possible to analyze the thrombogenicity of different degrees of vascular injury as occurs during atherothrombogenesis. As such, in situations in which a relatively mild injury to the vessel wall may occur, the thrombogenic stimulus is relatively small, and the resultant thrombus is labile and may then be incorporated into the plaque, as may occur in the episodic asymptomatic progression of atherosclerotic plaques as well as in patients with unstable angina and transient ischemia. In contrast, deep vessel injury, particularly in the presence of severe stenosis, leads to relatively persistent thrombotic occlusion as may occur in myocardial infarction.

INSIGHTS FROM VON WILLEBRAND DISEASE ANIMAL MODELS von Willebrand disease (vWD) is a genetic bleeding disorder that arises from the abnormalities in the vW factor (vWF). It is the most common inherited bleeding disorder in humans, and over the past years several animal species have also been described as suffering from this disease, whether through spontaneous mutation (pigs and dogs) or through a genetically engineered mutation (mice).45 All these different animal models have been extremely useful in exploring the characteristics of vWD and in testing new treatments. Indeed, the interaction of platelets with vWF is crucial in the initiation and development of any

thrombotic process since it enables platelets, via its surface glycoprotein receptors, to adhere to exposed subendothelium and to respond to blood shear stress.46–48 In fact, Fuster and Badimon1,49 were among the first to show a striking difference in the atherosclerotic lesions in the aorta between normal pigs and vWD pigs, both in spontaneous and in diet-induced atherosclerosis, demonstrating protection against atherosclerosis in V vWD pigs. The pig is a good model for studying vWD since vWF localization in endothelial cells and platelets mimics that of humans as do clotting and platelet characteristics. Additionally, in normal pigs the level of vWF is close to the human level whereas in cow, sheep, or goat it is 6- to 12-fold higher.45 Canine vWD is also similar to humans. Over the years, many dog breeds have been identified as suffering from this disease, making vWD the most common inherited bleeding disorder in dogs. However, under the term “canine vWD” there seems to be a very heterogeneous group of bleeding disorders with different subtypes and mode of inheritance.50 Actually, canine vWD can vary in genetic transmission, clinical severity, and diagnostic laboratory findings. Thus, in contrast to pigs, vWD dogs have not been used extensively for research purposes.51 Finally, it deserves to be mentioned that vWD has also been reported in other animal species such as murine,52,53 rabbits,54 and cats.55 However, extrapolation of the results obtained from these animals to humans remains to be completely elucidated.

ANIMAL MODELS OF PLAQUE RUPTURE By its very nature, rupture of an atherosclerotic plaque is difficult to study in humans. Moreover, since rupture of an atherosclerotic plaque occurs in a stochastic fashion, it is also difficult to identify triggering factors and equally hard to investigate treatments addressed toward plaque stabilization. Thus, a good animal model not only may help to better understand the mechanisms behind its rupture but also to test treatments to prevent it from happening. Spontaneous hemorrhage and rupture are considered extremely rare events in large animal models and thus have been found only in the coronary arteries of pigs with inherited hyperlow-density lipoprotein cholesterolemia or in cholesterol-fed pigs with streptozotocin-induced diabetes.56,57 In contrast, many interesting rodent studies related to plaque rupture have been published in recent years. Some authors have described the presence of blood-filled channels within the advanced coronary lesions as well as plaque ruptures and thrombi in the aortic origin of old apolipoprotein (apo) E−/− mice58 whereas other studies have reported the presence of luminal thrombi in ruptured plaques of spontaneously dead apoE−/− mice.59 On the other hand, other investigators have evaluated the harmful effect of combining several risk factors. As such, it has been reported that either in double knockout mice with homozygous null mutations in the apoE and the high-density lipoprotein (HDL) receptor, scavenger receptor class B, or a combination of hypertension and dyslipemia (hypertensive rats transgenic from human cholesteryl ester transfer protein) increases plaque vulnerability and thrombi formation. Despite all these, the most simplistic approach to induce plaque rupture and subsequent thrombus formation is by direct mechanical injury of the vessel of interest. Table 39–1 describes different approaches already established in order to induce plaque rupture and the subsequent thrombi formation in different animal models.60–70

CHAPTER 39 / MODELS OF BEHAVIOR

365

Table 39–1 Previously applied methods to induce plaque rupture and/or thrombus formation Reference

Method applied to induce plaque rupture

Reddick et al.60 Gertz et al.,61 Rekhter et al.62

Forceps squeezing of the aorta Combination of double balloon injury and hypercholesterolemia and further angioplasty-induced plaque rupture Photochemical reaction to previously formed atherosclerotic plaques Intraperitoneal injection of Russell’s viper venom followed by intravenous injection of histamine (vasopressor), hypercholesterolemic serotonin, or angiotensin II Intracerebroventricular injection of corticotropin-releasing factors (“stress hormone”) Carotid angioplasty Thrombogenic effect of shear stress and atherosclerotic vessel components on the extracorporeal perfusion system von Willebrand factor deficiency

Eitzman et al.63 Constantinides et al.,64 Abela et al.,65 Nakamura et al.66

Rekhter et al.67

Heras et al.68 Badimon et al.,34 Lassila et al.44

Fuster et al.,49,69 Badimon et al.1

RABBIT MODELS OF THROMBOSIS The use of rabbits has been widely extended because with the appropriate precaution, they are easy to handle and cost-effective. There are three breeds of rabbits commonly used in biomedical research: New Zealand White, Dutch Belted, and Flemish Giant. All of them are mainly used as animal models for the study of atherogenesis,2,61 plaque instability and rupture,62,65 and myocardial infarction,71 and to evaluate the efficacy of new antithrombotic and antiatherosclerotic drugs.72 Rabbits do not develop spontaneous atherosclerosis as they are vegetarian. However, rabbits can rapidly induce vascular lesions after the administration of a rich atherosclerotic diet (0.5–4% cholesterol content) during 8–16 weeks.2 Moreover, transgenic rabbits have also been developed to obtain models of hereditary hyperlipidemia (WHHL-rabbit) Yet, rabbit atherosclerotic lesions differ from human atheroma since their lipid and macrophage content is much higher10 as is their hypercholesterolemic index. Accelerated atherosclerotic lesions can also be developed in rabbits by a combination of balloon injury and hypercholesterolemia feeding.61

MICE MODELS OF THROMBOSIS Mice do not develop atherosclerosis without genetic manipulation since they have a lipid physiology that is radically different from that in humans, most of the cholesterol being transported in HDL-like particles. On the other hand, the mouse has not been as widely used as the pig as a model to test the possible therapeutic usefulness of antithrombotic agents.8 The reasons for this may be related to the small size of the murine arteries, which limits the manipulation of isolated vessel segments, the lack of standardization of the model, and mostly the differences from the human model, which decidedly limit the extrapolation of mouse findings

Animal model and/or thrombus formation ApoE−/−mice Rabbits ApoE−/− mice Rabbits

ApoE−/− mice

Pig

Pigs with von Willebrand disease

to clinical relevance in humans. Thus, an accurate determination of the mouse hemostatic system with regard to coagulation and fibrinolytic systems, platelet structure, and platelet receptor/ enzyme system is required and differences between the two species must be acknowledged and appreciated in the interpretation of the data. As an example, platelet counts in mice on average are four times those of humans and platelets are only approximately one-half the volume of human platelets. An excellent and comprehensive review of the murine hemostatic system has been provided by Tsakiris et al.,72 and important aspects of murine platelet receptor function have recently been reviewed by Ware.73 However, despite these species-related differences, the use of murine models is particularly advantageous. On one hand, it makes it possible to easily perform several techniques of vascular injury as described in Table 39–2. On the other hand, the murine model not only offers numerous immunological approaches (e.g., monoclonal antibodies), but also provides genetically modified animal strains with defined defects in platelet function that open new ways to identify the individual roles and the interplay of platelet proteins in thrombus formation.74–81 Finally, the usefulness of intravital microscopy in the mice model of thrombosis deserves to be mentioned since it allows testing the in vivo relevance of the in vitro observations. Indeed, the study of the microcirculation by intravital microscopy represents a sophisticated research tool to examine complex biological interactions and disease mechanisms as well as to develop and test novel approaches aimed at the prevention or attenuation of disease-associated microvascular disorders and cellular dysfunction such as the ones that occur in atherosclerosis and thrombosis pathogenesis. In addition, using the intravital microscopic technique, circulatory and cellular disorders in surgical diseases and procedures, such as ischemia/reperfusion, trauma, sepsis, and inflammation, as well as wound healing, may be analyzed.

366

SECTION V / MODELS OF BEHAVIOR

Table 39–2 Different models to induce vascular injury Vessel wall injury models Photochemical injury

Mechanical injury

Electrical injury

Chemical injury

Laser injury

Method Vessel irradiation in the presence of rose Bengal or sodium fluorescein77 Pinching arteries with needle forceps (Folts model)78 Denuding the endothelium via balloon catheters Infusion of a portion of a vessel with air or saline Application of an electrical current through the vessel with an electromicrocoagulator Ex vivo application of ferric chloride; applicable only in guinea pigs, rats, and mice79–81 Argon laser: irradiation followed by visualization of the injury with intravital microscopy82

ANIMAL MODELS OF VENOUS THROMBOSIS Thrombogenesis in veins is mainly attributed to some combination of hypercoagulability, stasis, and vascular injury. All these triggering factors induce tissue thromboplastin (tissue factor) release to the flowing blood forming thrombin and fibrin that trap red blood cells. Thus, in contrast to arterial thrombotic lesions (platelet-rich thrombus, “white thrombus”), venous thrombosis is predominantly “red” and fibrin-rich. Animal models for venous thrombosis appear to be particularly useful for studying the pathophysiology of blood coagulation in vivo and the pathogenesis of venous thrombosis. For instance, this model has helped to define the role of activated protein C, the interplay between inflammatory and procoagulant mediators, and the regulatory role of PAI-1 in thrombolysis in addition to therapeutic approaches.82 In fact, Dorffler-Melly83 and Levi82 have previously published interesting literature searches in MEDLINE and EMBASE databases offering an in-depth review of small and large animal models of venous thrombosis, pointing out their advantages, usefulness, and limitations.

CONCLUSIONS Clinical observations provide the substrate to build up pathophysiological hypotheses, but for obvious ethical reasons our ability to test these hypotheses in humans is very limited. Cell biology-related studies have helped to answer mechanistic questions, but lack the complexity of a real disease, thus limiting the scope of testable hypotheses. On the other hand, studies using rodent or large animal models have proved to be essential for proof of concept since they yield in vivo approaches to confirm critical hypotheses previously evaluated in relevant in vitro models. Moreover, DNA technology made it possible to create a number of knockout and transgenic rodent models that have enhanced our understanding of cardiovascular disease and expedited the development of new gene therapies. However, while small animal models provide experimental convenience and ease

in manipulation, more clinically relevant models are necessary to study the mechanisms surrounding human atherothrombogenesis. Large animal models, although associated with higher cost and handling-related difficulties, have shown an atherothrombotic pattern more comparable to that of humans. To date, considering all models, the porcine model is one of the most useful currently available atherothrombotic models. Indeed, pig animal models have been shown to address specific questions related to blood and atherosclerotic vessel mechanisms involved in thrombus formation that have been eventually translated to clinical situations. Despite all of this, efforts in developing the “ideal” animal model for atherothrombotic evaluation must continue. Furthermore, for this purpose, it may be critical to construct animal preclinical trials that resemble human trials in order to make clear what data and important conclusions can be justifiably extracted from animal models.

REFERENCES 1. Badimon L, Steele P, Badimon JJ, Bowie EJ, Fuster V. Aortic atherosclerosis in pigs with heterozygous von Willebrand disease. Comparison with homozygous von Willebrand and normal pigs. Arteriosclerosis 1985;5:366–370. 2. Badimon L, Fuster V, Chesebro JH, Dewanjee MK. New “ex vivo” radioisotopic method of quantitation of platelet deposition—studies in four animal species. Thromb Haemost 1983;50:639–644. 3. Steele PM, Chesebro JH, Stanson AW, et al. Balloon angioplasty. Natural history of the pathophysiological response to injury in a pig model. Circ Res 1985;57:105–112. 4. Fuster V, Badimon L, Badimon JJT, V, Lie J, Bowie E. Experimental approach to vascular disease in swine with von Willebrand’s disease: In: Tumbleson ME, Ed. Swine in Biomedical Research. New York: Plenum Publishing Corp., 1986:1527–1542. 5. Galvez A, Badimon L, Badimon JJ, Fuster V. Electrical aggregometry in whole blood from human, pig and rabbit. Thromb Haemost 1986;56:128–132. 6. Badimon L, Badimon JJ, Turitto VT, Fuster V. Thrombosis: Studies under flow conditions. Ann NY Acad Sci 1987;516:527–540. 7. Fuster V, Lie JT, Badimon L, Rosemark JA, Badimon JJ, Bowie EJ. Spontaneous and diet-induced coronary atherosclerosis in normal swine and swine with von Willebrand disease. Arteriosclerosis 1985;5:67–73. 8. Farb A, Burke AP, Tang AL, et al. Coronary plaque erosion without rupture into a lipid core. A frequent cause of coronary thrombosis in sudden coronary death. Circulation 1996;93:1354–1363. 9. van der Wal AC, Becker AE, van der Loos CM, Das PK. Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory process irrespective of the dominant plaque morphology. Circulation 1994;89:36–44. 10. Badimon L. Atherosclerosis and thrombosis: Lessons from animal models. Thromb Haemost 2001;86:356–365. 11. Burke AP, Kolodgie FD, Farb A, et al. Healed plaque ruptures and sudden coronary death: Evidence that subclinical rupture has a role in plaque progression. Circulation 2001;103:934–940. 12. Handley DA. Experimental therapeutics and clinical studies in (re)stenosis. Micron 1995;26:51–68. 13. Johnson GJ, Griggs TR, Badimon L. The utility of animal models in the preclinical study of interventions to prevent human coronary artery restenosis: Analysis and recommendations. On behalf of the Subcommittee on Animal, Cellular and Molecular Models of Thrombosis and Haemostasis of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost 1999;81:835–843. 14. Lewis A, Southern L. Swine Nutrition, 2nd ed. Boca Raton, FL: CRC Press Inc., 2000. 15. Committee on Animal Nutrition, National Research Council. Nutrient Requirements of Swine, 10th revised ed. Washington, DC: National Academies Press Publications, 1998.

CHAPTER 39 / MODELS OF BEHAVIOR

16. Swindle M. Defining appropriate health status and management programs for specific-pathogen-free swine for xenotransplantation. Ann NY Acad Sci 1998;862:111–120. 17. Laber K, Whary M, Bingel S, et al. Biology diseases of swine in laboratory Medicine In: Fox JG, Anderson LC, Loew FM, Quimby FW, Eds. Laboratory Animal Medicine, 2nd ed. San Diego, CA: Academic Press, 2002. 18. Swindle MM, Horneffer PJ, Gardner TJ, et al. Anatomic and anesthetic considerations in experimental cardiopulmonary surgery in swine. Lab Anim Sci 1986;36:357–361. 19. Gardner T, Johnson D. Cardiovascular system. In: Swindle MM, Adams RJ, Eds. Experimental Surgery and Physiology: Induced Animal Models of Human Disease. Philadelphia, PA: Lippincott, Williams & Wilkins, 1988:74–124. 20. Casani L, Sanchez-Gomez S, Vilahur G, Badimon L. Pravastatin reduces thrombogenicity by mechanisms beyond plasma cholesterol lowering. Thromb Haemost 2005;94:1035–1041. 21. Gal D, Chokshi SK, Mosseri M, Clarke RH, Isner JM. Percutaneous delivery of low-level laser energy reverses histamine-induced spasm in atherosclerotic Yucatan microswine. Circulation 1992;85: 756–768. 22. White FC, Carroll SM, Magnet A, Bloor CM. Coronary collateral development in swine after coronary artery occlusion. Circ Res 1992;71:1490–1500. 23. White FC, Bloor CM. Coronary vascular remodeling and coronary resistance during chronic ischemia. Am J Cardiovasc Pathol 1992;4:193–202. 24. White C, Ramee S, Banks A, Wiktor D, Price H. The Yucatan miniature swine: An atherogenic model to assess the early potency rates of an endovascular stent. In: Swindle MM, Ed. Swine as Models in Biomedical Research. Ames, IA: Iowa State University Press, 1992:156–162. 25. Mehran R, Ricci M, Graham A, Carter K, Smyes J. Porcine model for vascular graft studies. J Invest Surg 1991;4(l):37–44. 26. Swindle MM. Defining appropriate health status and management programs for specific-pathogen-free swine for xenotransplantation. Ann NY Acad Sci 1998;862:111–120. 27. Bloor C, White F, Roth D. The pig as a model of myocardial ischemia and gradual coronary artery occlusion. In: Swindle MM, Ed. Swine as Models in Biomedical Research. Ames, IA: Iowa State University Press, 1992:163–175. 28. Stanton H, Mersmann H. Swine in Cardiovascular Research, Vols. 1 and 2, Boca Raton, FL: CRC Press, Inc., 1986. 29. Institute of Laboratory Animal Resources Commission on Life Sciences, National Research Council. Guide for the Care and Use of Laboratory Animals. Washington, DC: National Academies Press Publications, 1996. 30. Swindle M, Smith A, Laber-Laird K, Dungan L. Swine in biomedical research: Management and models. ILAR News 1994;36(l):1–5. 31. O’Rourke S, Folts J, Albrecht R. Studies on the inhibition of canine platelet aggregation by barbiturates. J Lab Clin Med 1986; 108:206–212. 32. Jorgensen L. The role of platelets in the initial stages of atherosclerosis. J Thromb Haemost 2006;4:1443–1449. 33. Fernandez-Ortiz A, Badimon JJ, Falk E, et al. Characterization of the relative thrombogenicity of atherosclerotic plaque components: Implications for consequences of plaque rupture. J Am Coll Cardiol 1994;23:1562–1569. 34. Badimon L, Badimon JJ, Galvez A, Chesebro JH, Fuster V. Influence of arterial damage and wall shear rate on platelet deposition. Ex vivo study in a swine model. Arteriosclerosis 1986;6:312–320. 35. Vilahur G, Duran X, Juan-Babot O, Casani L, Badimon L. Antithrombotic effects of saratin on human atherosclerotic plaques. Thromb Haemost 2004;92:191–200. 36. Badimon L, Badimon JJ. Mechanisms of arterial thrombosis in nonparallel streamlines: Platelet thrombi grow on the apex of stenotic severely injured vessel wall. Experimental study in the pig model. J Clin Invest 1989;84:1134–1144. 37. Vilahur G, Segales E, Salas E, Badimon L. Effects of a novel platelet nitric oxide donor (LA816), aspirin, clopidogrel, and combined

38.

39.

40.

41.

42.

43.

44.

45. 46.

47.

48.

49.

50.

51.

52.

53.

54. 55.

56.

57.

367

therapy in inhibiting flow- and lesion-dependent thrombosis in the porcine ex vivo model. Circulation 2004;110:1686–1693. Roussi J, Berge N, Bal dit Sollier C, et al. Clopidogrel-induced qualitative changes in thrombus formation correlate with stent patency in injured pig cervical arteries. Thromb Res 2002;105: 209–216. Mailhac A, Badimon JJ, Fallon JT, et al. Effect of an eccentric severe stenosis on fibrin(ogen) deposition on severely damaged vessel wall in arterial thrombosis. Relative contribution of fibrin(ogen) and platelets. Circulation 1994;90:988–996. Roque M, Rauch U, Reis ED, Chesebro JH, Fuster V, Badimon JJ. Comparative study of antithrombotic effect of a low molecular weight heparin and unfractionated heparin in an ex vivo model of deep arterial injury. Thromb Res 2000;98:499–505. Badimon JJ, Lettino M, Toschi V, et al. Local inhibition of tissue factor reduces the thrombogenicity of disrupted human atherosclerotic plaques: Effects of tissue factor pathway inhibitor on plaque thrombogenicity under flow conditions. Circulation 1999;99: 1780–1787. Vilahur G, Segales E, Casani L, Badimon L. A novel anti-ischemic nitric oxide donor inhibits thrombosis without modifying haemodynamic parameters. Thromb Haemost 2004;91:1035–1043. Osende JI, Shimbo D, Fuster V, Dubar M, Badimon JJ. Antithrombotic effects of S 18886, a novel orally active thromboxane A2 receptor antagonist. J Thromb Haemost 2004;2:492–498. Lassila R, Badimon JJ, Vallabhajosula S, Badimon L. Dynamic monitoring of platelet deposition on severely damaged vessel wall in flowing blood. Effects of different stenoses on thrombus growth. Arteriosclerosis 1990;10:306–315. Denis CV, Wagner DD. Insights from von Willebrand disease animal models. Cell Mol Life Sci 1999;56:977–990. Ruggeri ZM, Orje JN, Habermann R, Federici AB, Reininger AJ. Activation-independent platelet adhesion and aggregation under elevated shear stress. Blood 2006;108:1903–1910. Reininger AJ, Heijnen HF, Schumann H, Specht HM, Schramm W, Ruggeri ZM. Mechanism of platelet adhesion to von Willebrand factor and microparticle formation under high shear stress. Blood 2006;107:3537–3545. Donadelli R, Orje JN, Capoferri C, Remuzzi G, Ruggeri ZM. Size regulation of von Willebrand factor-mediated platelet thrombi by ADAMTS13 in flowing blood. Blood 2006;107:1943–1950. Fuster W, Bowie EJ, Lewis JC, Fass DN, Owen CA Jr, Brown AL. Resistance to arteriosclerosis in pigs with von Willebrand’s disease. Spontaneous and high cholesterol diet-induced arteriosclerosis. J Clin Invest 1978;61:722–730. Johnson GS, Turrentine MA, Kraus KH. Canine von Willebrand’s disease. A heterogeneous group of bleeding disorders. Vet Clin North Am Small Anim Pract 1988;18:195–229. Nichols TC, Bellinger DA, Reddick RL, et al. The roles of von Willebrand factor and factor VIII in arterial thrombosis: Studies in canine von Willebrand disease and hemophilia A. Blood 1993;81: 2644–2651. Sweeney JD, Novak EK, Reddington M, Takeuchi KH, Swank RT. The RIIIS/J inbred mouse strain as a model for von Willebrand disease. Blood 1990;76:2258–2265. Denis C, Methia N, Frenette PS, et al. A mouse model of severe von Willebrand disease: Defects in hemostasis and thrombosis. Proc Natl Acad Sci USA 1998;95:9524–9529. Benson R, Dodds W. Autosomal factor VIII deficiency in rabbits: Size variations of rabbit factor VIII. Thromb Haemost 1977;38:380. French TW, Fox LE, Randolph JF, Dodds WJ. A bleeding disorder (von Willebrand’s disease) in a Himalayan cat. J Am Vet Med Assoc 1987;190:437–439. Prescott MF, McBride CH, Hasler-Rapacz J, Von Linden J, Rapacz J. Development of complex atherosclerotic lesions in pigs with inherited hyper-LDL cholesterolemia bearing mutant alleles for apolipoprotein B. Am J Pathol 1991;139:139–147. Gerrity RG, Natarajan R, Nadler JL, Kimsey T. Diabetes-induced accelerated atherosclerosis in swine. Diabetes 2001;50:1654– 1665.

368

SECTION V / MODELS OF BEHAVIOR

58. Rosenfeld ME, Polinsky P, Virmani R, Kauser K, Rubanyi G, Schwartz SM. Advanced atherosclerotic lesions in the innominate artery of the apoE knockout mouse. Arterioscler Thromb Vasc Biol 2000;20:2587–2592. 59. Johnson JL, Jackson CL. Atherosclerotic plaque rupture in the apolipoprotein E knockout mouse. Atherosclerosis 2001;154:399–406. 60. Reddick RL, Zhang SH, Maeda N. Aortic atherosclerotic plaque injury in apolipoprotein E deficient mice. Atherosclerosis 1998; 140:297–305. 61. Gertz SD, Fallon JT, Gallo R, et al. Hirudin reduces tissue factor expression in neointima after balloon injury in rabbit femoral and porcine coronary arteries. Circulation 1998;98:580–587. 62. Rekhter MD, Hicks GW, Brammer DW, et al. Animal model that mimics atherosclerotic plaque rupture. Circ Res 1998;83:705–713. 63. Eitzman DT, Westrick RJ, Xu Z, Tyson J, Ginsburg D. Hyperlipidemia promotes thrombosis after injury to atherosclerotic vessels in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 2000;20:1831–1834. 64. Constantinides P, Chakravarti RN. Rabbit arterial thrombosis production by systemic procedures. Arch Pathol 1961;72:197–208. 65. Abela GS, Picon PD, Friedl SE, et al. Triggering of plaque disruption and arterial thrombosis in an atherosclerotic rabbit model. Circulation 1995;91:776–784. 66. Nakamura M, Abe S, Kinukawa N. Aortic medial necrosis with or without thrombosis in rabbits treated with Russell’s viper venom and angiotensin II. Atherosclerosis 1997;128:149–156. 67. Rekhter MD, Hicks GW, Brammer DW, et al. Hypercholesterolemia causes mechanical weakening of rabbit atheroma: Local collagen loss as a prerequisite of plaque rupture. Circ Res 2000;86:101–108. 68. Heras M, Chesebro JH, Penny WJ, Bailey KR, Badimon L, Fuster V. Effects of thrombin inhibition on the development of acute platelet-thrombus deposition during angioplasty in pigs. Heparin versus recombinant hirudin, a specific thrombin inhibitor. Circulation 1989;79:657–665. 69. Fuster V, Fass DN, Kaye MP, Josa M, Zinsmeister AR, Bowie EJ. Arteriosclerosis in normal and von Willebrand pigs: Long-term prospective study and aortic transplantation study. Circ Res 1982;51: 587–593. 70. Shiomi M, Ito T, Yamada S, Kawashima S, Fan J. Correlation of vulnerable coronary plaques to sudden cardiac events. Lessons from

71.

72.

73. 74.

75.

76.

77.

78.

79. 80.

81.

82.

83.

a myocardial infarction-prone animal model (the WHHLMI rabbit). J Atheroscler Thromb 2004;11:184–189. Alfon J, Pueyo Palazon C, Royo T, Badimon L. Effects of statins in thrombosis and aortic lesion development in a dyslipemic rabbit model. Thromb Haemost 1999;81:822–827. Tsakiris DA, Scudder L, Hodivala-Dilke K, Hynes RO, Coller BS. Hemostasis in the mouse (Mus musculus): A review. Thromb Haemost 1999;81:177–188. Ware J. Dysfunctional platelet membrane receptors: From humans to mice. Thromb Haemost 2004;92:478–485. Nieswandt B, Aktas B, Moers A, Sachs UJ. Platelets in atherothrombosis: Lessons from mouse models. J Thromb Haemost 2005;3: 1725–1736. Carmeliet P, Moons L, Collen D. Mouse models of angiogenesis, arterial stenosis, atherosclerosis and hemostasis. Cardiovasc Res 1998;39:8–33. Rosenblum WI, El-Sabban F. Platelet aggregation in the cerebral microcirculation: Effect of aspirin and other agents. Circ Res 1977;40:320–328. Gallagher KP, Osakada G, Kemper WS, Ross J Jr. Cyclical coronary flow reductions in conscious dogs equipped with ameroid constrictors to produce severe coronary narrowing. Basic Res Cardiol 1985;80: 100–106. Aoki T, Cox D, Senzaki K, et al. Comparison of the antithrombotic effects of FK633, GPIIb/IIIa antagonist, and aspirin in a guinea pig thrombosis model. Thromb Res 1998;89:129–136. Kurz KD, Main BW, Sandusky GE. Rat model of arterial thrombosis induced by ferric chloride. Thromb Res 1990;60:269–280. Fay WP, Parker AC, Ansari MN, Zheng X, Ginsburg D. Vitronectin inhibits the thrombotic response to arterial injury in mice. Blood 1999;93:1825–1830. Rosen ED, Raymond S, Zollman A, et al. Laser-induced noninvasive vascular injury models in mice generate platelet- and coagulationdependent thrombi. Am J Pathol 2001;158:1613–1622. Levi M, Dorffle-Melly J, Johnson GJ, Drouet L, Badimon L. Usefulness and limitations of animal models of venous thrombosis. Thromb Haemost 2001;86:1331–1333. Dorffler-Melly J, Schwarte LA, Ince C, Levi M. Mouse models of focal arterial and venous thrombosis. Basic Res Cardiol 2000;95: 503–509.

40

Animal Models for Atherosclerosis, Restenosis, and Endovascular Aneurysm Repair ANDRÁS KÓNYA, KENNETH C. WRIGHT, MATTHEW GOUNIS, KRISHNA KANDARPA

ABSTRACT

AND

play an important role in the selection of the laboratory animal model to be used. The validity of any information derived from the animal is dependent on the appropriateness of the model. Thus, a detailed consideration of species and model features that are favorable and applicable to the specific purpose of the research is important. Since new animal models are continually being identified and characterized, a thorough literature search should always be conducted to determine what models are available and which are the most relevant.

Animal models have significantly advanced the understanding of mechanisms involved in atherosclerosis and restenosis and have allowed the evaluation of therapeutic options. The current focus of research is to develop strategies to prevent restenosis. These include pharmacological and biological interventions directed primarily against smooth muscle cell proliferation, endovascular devices for recanalization and/or drug delivery, and an integrated approach using both devices and pharmacobiological agents. Devices aimed at the percutaneous endoluminal exclusion of aortic aneurysms have also generated a great deal of interest. Experience over many decades with animal models for vascular research has established that a single, ideal model for atherosclerosis, restenosis, or aneurysm formation does not exist, although recent development of transgenic variants of currently available models has widened the available options. Nevertheless, an appreciation of the individual features of natural or stimulated disease in each species is of the utmost importance for the proper design and execution of relevant experiments. Key Words: Animal models, Atherosclerosis, Arteriosclerosis, Restenosis, Angioplasty, Stenting, Drug-eluting stents, Endovascular aneurysm repair, Endovascular grafts, Transcatheter embolization.

ATHEROSCLEROSIS BACKGROUND Atherosclerosis is a focal disease that localizes mainly in areas of disturbed blood flow1 and bears many features of chronic inflammation.2 The lesion of plaque consists of endothelial cells, monocytes/macrophages, smooth muscle cells (SMC), and a matrix of collagen, proteoglycans, and calcification. Lesion formation begins with the influx of monocytes/ macrophages, which become loaded with cholesteryl ester to create the so-called “fatty streak.” These lesions may progress by the influx of additional monocytes and T cells, whose secretory products, along with those of endothelial cells, result in migration of SMC, where they proliferate and synthesize matrix components. Some cells in the plaque die, leaving a necrotic core and extracellular cholesterol clefts, which are hallmarks of advanced lesions. The luminal face of the lesion often has a fibrous cap, generated by SMC stabilizing the plaque.3 Previously, atherosclerosis has been viewed as a disease of uncontrolled plaque growth eventually leading to total occlusion.4 New clinical findings suggest that acute coronary events are triggered by rupture and hemorrhage of vulnerable atherosclerotic plaques.5 The plaque consists of a highly thrombogenic core and a slender and fragile fibrous cap that separates the core from blood coagulation factors, thereby precluding formation of thrombus on the surface of the intact plaque.4 Disturbance of cap integrity leads to exposure of the plaque’s thrombogenic interior and subsequent thrombosis. Such ruptures of the fibrous cap may recur over many years without causing complete occlusion of the vessel, and thereby may often remain clinically silent. The reasons for the thinning and rupture of the fibrous cap are not known. One theory hypothesizes that the rupture is a result of

INTRODUCTION Almost all medical knowledge and treatment, especially in the last century, has involved work with laboratory animal models. Since the early part of the twentieth century, research using animal models has contributed to more than half of the significant medical discoveries and during some periods, accounted for more than 75% of the major advances. Two-thirds of the Nobel prizes awarded since 1901 have been for discoveries involving the use of laboratory animal models. The selection of the laboratory species, breed, and strain to be used as a model for human disease is one of the most important decisions to be made by an investigator. The goals of the research

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

369

370

SECTION V / MODELS OF BEHAVIOR

Table 40–1 Characteristics of an ideal animal model of arteriosclerosis11 1. 2. 3. 4. 5. 6. 7.

Inexpensive Easy to maintain and handle Proper size Reproducible in a laboratory setting Well-defined genetic characteristics Sharing important aspects of human disease Lesion development: Develop naturally on a normal diet Progress slowly over the animals’ lifetime Cause clinical sequelae at middle to old age 8. Lesion pathogenesis: Complete range from fatty streak to atheromatous plaques 9. Lesion complications: Calcification Ulceration Hemorrhage Stenosis Thrombosis

loss of SMC, thought to be the main producers of cap-stabilizing collagen. This is caused by the loss of cells from apoptosis that may be mediated by the interaction between SMC and monocytes/ macrophages.6,7 According to a second hypothesis, plaque rupture is the result of an imbalance between the production of plaquestabilizing collagen and the action of corrosive enzymes.8 An immunohistochemical study has shown that macrophages accumulated in the plaque express high levels of matrix metalloproteinases and tissue factors9 that are thought to make the fibrous plaque fragile by digesting the extracellular matrix. Therefore, the search for an animal model of atherosclerotic plaque rupture should be seen against this background. Arteriosclerosis is a disease that really affects only humans and one or two other species, such as the pig and certain nonhuman primates, and because of this researchers must resort to genetically modified models to even partially reproduce the condition.10 It has never been easy to find a good animal model of the atherosclerotic process itself. The ideal animal model of human arteriosclerosis should posses several important characteristics11 (Table 40–1). ANIMAL MODELS OF ATHEROGENESIS Diet-Induced Nonmurine Models Historically, relatively large animals, such as nonhuman primates, swine, and rabbits, have been predominantly used for arteriosclerosis-related research. Rats and dogs are not good models because they do not develop spontaneous lesions and require heavy modifications of diet to produce a vascular lesion. In recent years, however, some transgenic rat models have been produced that develop lesions resembling human atherosclerosis.12,13 Rabbits are useful because they are highly responsive to cholesterol manipulation and develop lesions in a fairly short time.14 The lesions, however, are much more fatty and macrophage-rich (i.e., more inflammatory) than human lesions and plasma cholesterol levels are extraordinarily high.15 The Watanabe heritable hyperlipidemic rabbits (WHHL) are genetic variants that mimic familial hypercholesterolemia.16 This strain of rabbit was produced by inbreeding from a mutant discovered in 1973. The rabbits consistently showed spontaneous development of aortic

arteriosclerosis over 5 months of age. The existence of rabbit strains with congenital abnormalities in lipid metabolism has contributed to the feasibility of creating particularly informative transgenic rabbits. The guinea pig provides valuable models for studying early arteriosclerosis development.17 Guinea pigs carry the majority of their plasma cholesterol in low-density lipoprotein (LDL), and present other striking similarities to humans in terms of cholesterol and lipoprotein metabolism making them a unique animal model. Many of the mechanisms by which guinea pigs regulate cholesterol and lipoprotein metabolism as a response to diet or drug treatment are analogous to those reported in clinical human studies. Nonhuman primates including chimpanzees and various monkeys fed a high-cholesterol diet develop a form of atherosclerosis that is very similar to that of humans. However, the cost of nonhuman primates is prohibitive and many of these species are protected. Therefore, arteriosclerosis research in primates is today generally confined to the study of complex issues such as the effects of psychological stress.10 The domestic pig is one of the most useful currently available large animal models of arteriosclerosis. Over time, pigs develop atherosclerosis even on a normal porcine diet.18 When fed a highcholesterol diet, they develop elevated plasma cholesterol levels and atherosclerotic lesions that are similar to those seen in humans. However, the maintenance of pigs can be expensive and they require special facilities and handling. Miniature pigs (e.g., Yucatan miniswine) have been used in several studies to gain insight into early-stage arteriosclerotic disease. One study concluded that early pathological changes (i.e., increased intima-media thickness and foam cell accumulation) induced by a high fat cholesterol diet precede decreases in endothelium-dependent relaxation (endothelial dysfunction) in femoral arteries.19 This model may be an important tool to evaluate the effectiveness of interventions in modifying or blocking the development of diet-induced vascular disease and the role of the vascular endothelium in these processes. Yucatan minipigs have been used to study material property alterations in femoral and carotid arteries with atheroma induced by endothelial denudation and a high cholesterol diet.20,21 The data demonstrated a striking difference between the two arteries. While lesion progression resulted in stiffening vascular wall elastic modulus in carotid arteries, the elastic modulus in the femoral arteries decreased first with early stages of atheroma development and increased as lesions progressed. The use of pig models of atherosclerosis initially revealed that monocyte infiltration was one of the primary cellular events in the atherogenic process.22 Studies in monkeys and rabbits were pivotal in defining the cellular events in the initiation and development of atherosclerotic lesions.23,24 Murine Models In recent years there has been an explosion in the number of in vivo studies that is largely attributable to the use of murine models to study atherogenic mechanisms.25 Mice are highly resistant to atherosclerosis. The only exception is the C57BL/6 strain. A special diet (30% fat, 5% cholesterol, 2% cholic acid) led to arteriosclerosis in C57BL/6 mice, but the lesions differed from the human condition in their histological nature and location. Additionally, this diet was toxic and resulted in weight loss and respiratory infections. Paigen et al. modified the diet (15% fat, 1.25% cholesterol, 0.5% cholic acid), but the

CHAPTER 40 / ANIMAL MODELS FOR ATHEROSCLEROSIS, RESTENOSIS, AND ENDOVASCULAR ANEURYSM REPAIR

lesions were very small (range of 200–1000 µm2) in the aortic root and usually did not develop beyond the early foam-cell, fatty streak stage.26 It is now possible to add exogenous transgenes into mice as well as many other species. However, uniquely in mice, it is also possible to knock out or replace endogenous genes, which is one of the main advantages of working with the mouse.25 Knockout murine models can be used to identify arteriosclerosis susceptibility modifying genes, the role of various cell types in atherogenesis, and environmental factors affecting atherogenesis and to assess therapies that block atherogenesis or lesion progression.25 Although genetic modifications render mice susceptible to atherogenesis, lipid-enriched diets are indispensable to induce or accelerate the rate of development of atherosclerotic lesions. It appears that the induction of persistent hypercholesterolemia to levels >300 mg/dl is required for the development of experimental atherosclerosis in the mouse where dietary cholesterol is the major proatherogenic component.27 Apolipoprotein E-Deficient Mice Apolipoprotein E (apoE) is a glycoprotein that is a structural component of all lipoprotein particles other than low-density lipoprotein (LDL). One of its most important functions is to serve as a high-affinity ligand for the apoB- and apoE-LDL receptor and for the chylomicronremnant receptor, thereby allowing the specific uptake of apoEcontaining particles by the liver.28 In 1992, apoE-deficient mice were generated by selectively inactivating the apoE gene.29 The apoE-knockout mice exhibited increased total plasma cholesterol levels, which were five times those of normal littermates. Mice naturally have high levels of high-density lipoprotein (HDL) and low levels of LDL, in contrast to humans who have high LDL and low HDL. The mice lack the cholesteryl ester transferase enzyme that converts HDL to very low-density lipoprotein (VLDL) to LDL. The sequential events involved in the formation of atherosclerotic lesions in apoEdeficient mice are strikingly similar to those in humans. Initial fatty streak lesions rapidly progress to advanced lesions with a necrotic core surrounded by proliferating SMC and varying amounts of extracellular matrix. These lesions have well-formed fibrous caps. It is not uncommon for the inflammatory lesion to erode deep into the medial wall of the aorta, sometimes resulting in the formation of aortic aneurysms. When apoE-deficient mice are fed a “Western type” diet (21% fat, 0.15% cholesterol, no cholic acid),30 they exhibit over a 3-fold elevation in plasma cholesterol (to 2000 mg/dl), while wild-type mice show a 2-fold elevation. On this diet, formation of atherosclerotic lesions is greatly accelerated and lesion size is increased. Lesions in the apoE-deficient mouse, as in humans, tend to develop at vascular branch points and progress from a foam cell stage to a fibroproliferative stage with well-defined fibrous caps and necrotic lipid cores. ApoE-deficient mice are considered to be one of the most relevant models for arteriosclerosis since they are hypercholesterolemic and develop spontaneous arterial lesions.31 The apoE-deficient mouse contains the entire spectrum of lesions observed during atherogenesis and was the first mouse model to develop lesions similar to those in humans. In 1995, Kashyap et al. described the successful correction of apoE deficiency in apoE-deficient mice by using an approach involving systemic delivery of recombinant adenoviral vectors expressing human apoE to the liver. Thus the single genetic

371

change causing the absence of apoE and severe hypercholesterolemia is sufficient to convert the mouse from a species that is highly resistant to atherosclerosis to one that is highly susceptible.32 Low-Density Lipoprotein Receptor-Deficient Mice Gene targeting in embryonic stem cells has recently been used to create LDL receptor-knockout mice, a model of familial hypercholesterolemia.33 The lipoprotein abnormality in these mice is more modest than that in the apoE-deficient mice, featuring increases in LDL and VLDL with plasma cholesterol of about 250 mg/dl on a normal chow diet without developing arteriosclerosis. However, after being fed the Paigen diet, their plasma cholesterol level soars to 1500 mg/dl and large arteriosclerotic lesions form.34 The less toxic Western type diet also leads to the development of large lesions in these mice with a plasma cholesterol level of 400 mg/dl. Other Murine Models Overexpression of human apoA-I in apoE-deficient mice increased HDL cholesterol levels 2-fold and substantially decreased fatty streaks and advanced fibroproliferative lesion formation.35,36 All but 3–5% of apoE-deficient mice had detectable fatty streaks by 4 months of age, and had lesions that were highly organized and that occluded on an average 25% of the aortic lumen by 8 months of age. In contrast, more than 50% of apoE-deficient mice that overexpressed human apoA-I had no lesions by 4 months of age and by 8 months of age the lesions that occurred occluded on average only 5% of the aortic lumen. These data suggest that overexpression of apoA-I can diminish lesion size and slow the initiation of fatty streak formation. A human apoB transgenic murine model has also been created. These mice have mildly increased LDL and total cholesterol levels with a lipoprotein profile showing a distinct LDL peak, which differs from the wild-type mice that have only a distinct HDL peak.37,38 This model also shows a strong diet responsiveness; while a normal chow diet is not atherogenic, arteriosclerotic lesions develop upon feeding the Paigen and/or the Western diet. ApoE/LDL receptor (LDLR) double knockout mice have recently been created.34 These animals develop severe hyperlipidemia and arteriosclerosis39 and have been reported to develop more marked arteriosclerotic lesions with accelerated progression than the apoE-deficient mice even when fed a regular chow diet.40 Thus, the double knockout model is suitable for studying antiarteriosclerotic compounds without having to feed the animals an atherogenic diet. More recently, Veniant et al. successfully elevated blood cholesterol levels in chow-fed apoE-deficient and LDLR-deficient mice by making both mouse models homozygous for the apolipoprotein B-100 allele.41 That change resulted in alleviating the hypercholesterolemia in the setting of apoE deficiency, but worsening it in the setting of LDLR deficiency. Furthermore, the LDLR-deficient apoB100/100 mice developed extensive arteriosclerosis even on a normal chow diet. Conclusions In summary, the apoE-deficient mice are the most widely used experimental model of atherosclerosis because they spontaneously produce lesions that simulate complex human atherosclerotic lesions.42 It is generally believed that apoEdeficient animals, even when maintained on regular chow, exhibit more severe or complex lesions than do LDLR-deficient mice fed a Western type diet despite higher plasma cholesterol levels. The

372

SECTION V / MODELS OF BEHAVIOR

apoE-deficient mouse model, however, is not an ideal model for most forms of human arteriosclerosis, which often involve an elevation in LDL. In that regard, the LDLR-deficient mouse coupled with apoB-editing deficiency (double knockout mice) seems to be more promising as an LDL model that develops arteriosclerosis on a low-fat diet.41 Transgenic Rabbit Models By filling the gap between the laboratory mouse and larger domesticated mammals, transgenic rabbits expressing human genes have become a relatively large mammalian model to explore the pathophysiological processes of arteriosclerosis. To date, a wide variety of human transgenes [e.g., apo(a), apoA-I, apoB-100, apoE-2, and apoE-3] have been expressed in rabbits. These models have helped to elucidate the metabolic roles of selected proteins in atherogenesis. Elevated plasma levels of lipoprotein(a) [Lp(a)] constitute an independent risk factor for coronary heart disease, stroke, and restenosis.43 However, apo(a), a unique component of Lp(a), is naturally present exclusively in Old World monkeys, humans, and hedgehogs. Therefore, there are no convenient experimental animal models for studying Lp(a). Using the apo(a) transgenic rabbit model, no arteriosclerotic lesions were found on a regular chow diet, suggesting that lower plasma apo(a) is not atherogenic.44 When transgenic rabbits were fed a 0.3% cholesterol diet for 16 weeks they exhibited more extensive atherosclerotic lesions than nontransgenic rabbits, although cholesterol levels were similarly elevated. Compared to the lesions in nontransgenic control animals, the areas of atherosclerotic lesions in the transgenic rabbits were increased in the aorta as well as the iliac, carotid, and coronary arteries. Transgenic Rabbits versus Transgenic Mice Rabbit strains have even more diverse genetic backgrounds than inbred and outbred mouse strains. On the one hand, this might be advantageous for studying complex disease models such as arteriosclerosis, but it is obviously a disadvantage for defining the effects of gain or loss of target gene function. Rabbits like humans are LDL mammals and have features of lipoprotein metabolism that differ from mice that are HDL mammals. Therefore, the two species show different phenotypes even when the same gene is introduced. For instance, overexpression of apoE in mice caused inhibition of atherosclerosis,45 but led to increased plasma LDL and spontaneous arteriosclerosis in transgenic rabbits.46 ANIMAL MODELS OF PLAQUE VULNERABILITY Prevention of heart attack and stroke depends on the detection of vulnerable plaques and the development of plaque-stabilizing therapies.47 Vulnerable plaque is one of the toughest cases in animal model design. Plaque rupture is a complication of an already complex atherosclerotic process, and precise mechanisms of this complication remain hypothetical.48 The current paradigm of plaque rupture may be described as a combination of four overlapping components: mechanical, thrombogenic, inflammatory, and lipid.4 Plaque rupture occurs as a result of interactions between external and internal mechanical triggers and vulnerable (i.e., weak) regions of the plaque when forces acting on the plaque exceed its tensile strength. Disruption of the protective fibrous cap allows the thrombogenic (lipid/necrotic) core to come into contact with the blood coagulation system, which leads to the formation of thrombus. Plaques with a thin fibrous cap and large core are considered vulnerable. Hence, the cap thickness, core size, and cap/

core ratio are the most basic established “vulnerability endpoints.” Models of Plaque Rupture and Plaque-Associated Thrombosis Spontaneous Plaque Rupture/Thrombosis Spontaneous hemorrhage and rupture were observed in the coronary arteries of 39- to 54-month-old pigs with inherited hyper-LDL cholesterolemia bearing mutant alleles for apolipoprotein B.49 In apoE-deficient mice aged 42–54 weeks, a high frequency (up to 75%) of intraplaque hemorrhage and fibrotic conversion of necrotic zones was found with the loss of the fibrous cap in the innominate (brachiocephalic) artery.42 Surprisingly, intraplaque hemorrhages were not associated with mural thrombosis. These lesions had been previously overlooked because the majority of studies concentrated on the aortic root and the aortic arch. These observations indicate that the character of atherosclerotic lesions is site dependent. In another study, 37- to 59-week-old apoE-deficient mice fed the Western type diet died spontaneously. Necropsy revealed luminal thrombi associated with ruptured plaques in the brachiocephalic artery.50 Recently, these observations were further extended. Of 98 mice, 51 had acutely ruptured plaque in the same locations. The major finding was a significant increase in the number of buried caps in ruptured versus intact lesions, probably indicating previous ruptures in the same plaque.51 Induced Plaque Rupture/Thrombosis The use of direct mechanical injury to induce plaque rupture and thrombosis ignores the complexity of clinically relevant trigger mechanisms. Platelet and fibrin-rich thrombi have been induced in association with atherosclerotic plaques in apoE-deficient mice after squeezing the aorta between forceps.52 Similarly, accelerated atherosclerotic lesions were developed in rabbits by combining balloon injury and hypercholesterolemia, and subsequently mechanically disrupted by inflation of an angioplasty balloon in the arterial lumen.53 In another labor-intensive rabbit model, arteriosclerotic plaque could be ruptured at will after an inflatable balloon was embedded into the plaque.54 This model could also be used for induction of thrombi associated with plaque rupture where the pressure needed to inflate the plaque-covered balloon is an indicator of the overall mechanical strength of the plaque. A more recent study described a pharmacological model of plaque rupture in which plaque was first made vulnerable and then challenged by a systemic stressor.55 First, accelerated arteriosclerosis was initiated in apoE-deficient mice by placement of a perivascular collar. The resulting plaques were transfected with the p53 gene, which led to an increase in cap cell apoptosis, and in return resulted in a marked decrease in the cellular and extracellular content of the cap. Whereas spontaneous plaque rupture was rare, it was found in 40% of cases after treatment with the vasopressor compound phenylephrine. Myocardial Infarction-Prone Rabbit Model Originally, the Watanabe heritable hyperlipidemic (WHHL) rabbit was established as a model of human familial hypercholesteremia due to an LDL receptor deficiency.16 Based on the WHHL rabbit strain, a special rabbit model was developed, designated as WHHL-MI, in which myocardial infarction (MI) occurred spontaneously.56 This model possesses several types of coronary plaques that are possibly correlated to sudden cardiac events. Although many of the coronary plaques in WHHL-MI rabbits appeared histologically to be rupture prone in nature, no occlusive thrombus was detected

CHAPTER 40 / ANIMAL MODELS FOR ATHEROSCLEROSIS, RESTENOSIS, AND ENDOVASCULAR ANEURYSM REPAIR

in any rabbits, suggesting that some additional stimuli play a definitive role in causing disruption of the rupture-prone plaques and thrombosis. The most common feature of WHHL-MI rabbits is plaques that occlude most of the arterial lumen due to macrophage accumulation, suggesting that they are responsible for sudden cardiac events. This model could be useful for studying the mechanisms of plaque rupture and thrombogenesis if plaque rupture/thrombosis could be induced by additional triggering factors. Mouse Models Simulating Plaque Vulnerability The currently used mouse models are plaque-centric and do not take into consideration the processes that lead to occlusive thrombus formation after plaque rupture. The current modeling of vulnerable plaques emphasizes the acute nature of plaque rupture. In reality, several layers of spontaneous plaque fissures are frequently observed in coronary arteries and aortas indicating a series of disruptions of the protective cap in which only the last one becomes a clinical event.48 Even more disturbingly, emerging clinical data suggest that coronary thrombi can arise without plaque rupture and without signs of severe local inflammation.57 Any model is based upon a limited set of assumptions and therefore by default has its own limitations. A model often exaggerates only one feature of a complex process to make analysis more convenient and more accurate.48 Researchers have largely focused on mouse models for the study of arteriosclerosis because of their obvious advantages (e.g., easy handling, extensive knowledge of mouse physiology, large amount of available genetic information). The mouse models, however, have fundamental limitations.10 Mice have a lipid physiology that is radically different from that in humans (HDL vs. LDL dominance) and therefore they do not develop arteriosclerosis without genetic manipulation. Furthermore, mice weigh about 25–30 g, some 3000 times less than the average human. Because mouse cells are about the same size as human cells, a section of coronary artery in the mouse contains about 3000 times fewer cells than an equivalent section of human coronary artery. Consequently, the endothelial layer in mice lies directly on the internal elastic lamina and the media consist of only a few layers of SMC. In contrast to humans, arteriosclerotic lesions in the mouse coronary artery often extend beyond the elastic lamina. Also, it is difficult in mice to make a distinction between plaque erosion, as defined by endothelial denudation, and complete rupture of the fibrous cap.58 Although classic eccentric atheromas with a single fibrous cap exist in lesion-prone murine models, multiple necrotic core areas with or without separate fibrous caps are the norm.31,59,60 Disruption of these lesions may not mimic plaque rupture in humans, placing a fundamental limit on the applicability of mouse models for the investigation of rupture mechanisms.58 In addition to the difficulties arising from the differences between mouse and human biology, problems also occur when two different genetic models of a particular illness are used to investigate the effect of a third genetic manipulation. Another major problem with the genetically manipulated mouse strains is the genetic heterogeneity between the strains used to generate transgenic and knockout mice. This may lead to a situation in which animals containing the same genetic manipulation exhibit profoundly different phenotypes when present on diverse genetic backgrounds.10 The ideal features of an animal model of plaque rupture are summarized in Table 40–2.

373

Table 40–2 Ideal features of an animal model of plaque rupture10 1. The atherosclerotic process in the animal model should be histologically identical to that in humans 2. The atherosclerotic plaque in the animal model should show the same vulnerability to rupture as its human counterpart 3. The events leading to vulnerability and rupture (i.e., cap/core ratio, cellular composition, collagen production, and breakdown) should as far as possible be identical to those in humans 4. Plaque rupture in the animal model should occur without the need for manipulations such as cuffing of the artery or insertion of balloon catheters 5. Plaque rupture should at least in some cases be accompanied by the formation of platelet-rich fibrin thrombi 6. Response to treatment in the animal model should have the potential for duplication in humans 7. The species should be available for research, easy and economical to maintain, and plaque rupture should occur under reproducible conditions within a reasonable time frame

CONCLUSIONS Arteriosclerosis in humans is a multifactorial condition that develops over many years and modeling it in animals is a notoriously tricky business.10 Reproducing the events leading up to occlusive thrombosis, the event that actually kills and disables humans, is even more challenging. Existing animal models are useful for studying certain features of arteriosclerosis in regard to the function of specific genes. However, because of the complexity of the processes involved in plaque rupture and thrombosis, it is not certain whether a good model of these complications will ever be developed.

RESTENOSIS BACKGROUND Restenosis in the form of accelerated arteriosclerosis and intimal hyperplasia constitutes the greatest threat to prolonged vessel patency following treatment of vascular occlusive diseases by percutaneous endovascular revascularization procedures. Although such procedures can achieve high initial success rates (>95%), a significant proportion of the treated vessels (20–50%) develops restenosis within 3–6 months.61–64 Animal models have been used successfully to study specific aspects of the early phase of the restenotic process (e.g., smooth muscle cell proliferation). Ideally, restenosis research should be performed on arteries with advanced arteriosclerotic disease. However, animal models with complex arteriosclerotic obstructive plaques are difficult to produce and require expensive, lengthy experiments. Complex lesions similar to those clinically significant in humans have been produced experimentally only in hyperlipemic pigs49 and in nonhuman primates.65 Although arteriosclerosis progresses over a lifetime in humans, experimental settings try to reproduce atherosclerotic lesions in weeks or months based on diet modifications, and then following vascular injury the restenosis process is studied over a period of weeks or months. Furthermore, there are significant interspecies differences relevant to vascular disease. While elastic arteries (aorta and its primary branches) from various species have been reported to demonstrate remarkable structural similarity, muscular arteries (coronary and femoral arteries) exhibit major differences between species with respect to their thickness, medial

374

SECTION V / MODELS OF BEHAVIOR

elastin content, and intimal cellularity.66 In addition, the type, duration, and number of successive injuries as well as the associated forces (e.g., shear stress) cause a proportional injury-related vascular response in various species, which is measured by an intima-to-media area ratio or luminal cross-sectional area narrowing by neointima. However, the relationships vary among species; the injury-neointimal thickness regression slope is steep in pigs, intermediate in baboons and rabbits, and almost flat in dogs.67–69 VASCULAR INJURY TECHNIQUES Localized arterial endothelial and mural injuries are primarily used for studying restenosis (morphology, mechanisms, and therapeutic strategies). Vascular injury accelerates lesion development and can be produced in animal models using a variety of methods including dietary manipulation. Some of the nondietary methods for inducing vascular injury include the Baumgartner balloon catheter technique,70 nylon and wire loop techniques,71,72 air desiccation,73 chemical irrigation,74 thermal drying,75 and irradiation.76 As the names imply, mechanical devices (balloon catheters or wire loops) or agents (chemical, electrical, thermal, or radiation) are used to create site-specific injury in the arterial tree, and lesions develop shortly thereafter. Restenosis can be studied on either normal or previously injured arteries. In the latter model, the animal may be placed on a high fat/high cholesterol (HFHC) diet after focal injury to enhance lesion formation. The most commonly employed vascular injury technique is focal balloon catheter injury. This technique involves inflating a Fogarty balloon at the desired site within the artery and then withdrawing the dilated balloon along a specific length of the vessel to create adequate shear stress on the wall to produce a significant injury. Nondietary methods such as this can be used either alone or in combination with an HFHC diet. The decision to use a particular method or a combined approach to induce vascular injury depends on the type of lesion that is required, which in turn depends on the purpose of the study. When combinations of techniques of disease induction are utilized, lesions develop at an accelerated pace and with greater similarity to the human prototype.77 Such manipulations also allow greater control over the type of lesion that can be created. The utilization of combined techniques has been more successful in creating a range of morphological lesions of desired severity.77–84 For instance, a combination of an atherogenic HFHC diet and balloonmediated endothelial injury in Yucatan microswine produces consistent high-grade arterial stenoses, and this model has been proposed for the study of cardiovascular interventional techniques.78 The creation of fatty streaks and plaques with minimal luminal narrowing may be sufficient for assessing the cellular and molecular roles in the histopathogenesis of restenosis,82 whereas a lesion of intermediate magnitude with moderate narrowing of the luminal diameter is needed for pharmacological therapy research.83 The investigation of endovascular recanalization devices requires consistent high-grade stenoses.84 Injury of the media is critical to the formation of such lesions and an integrated approach to their creation may be useful.85 ANIMAL MODELS Arterial injury models simulating vascular injury in humans resulting from revascularization procedures are created in animals with the aim of determining morphology, pathogenesis, and preventive as well as therapeutic strategies. However, there is no single ideal model. The rat, rabbit, swine, and nonhuman primate have all been used as models of

restenosis, with the last two affording the best approximation to humans. The rat carotid artery balloon injury model developed in the 1960s has been well characterized. However, the rat as well as the rabbit are small animals and do not serve all the demands of restenosis models. In this regard, the larger size of the Flemish Giant rabbit (6–7 kg) offers an advantage over the common New Zealand white rabbit (3–4 kg), and this strain of rabbits has been used to study the effects of percutaneous transluminal angioplasty (PTA) on the arterial wall.86 As for testing coronary stent designs, the rabbit iliac artery offers a reliable model.87,88 Studying restenosis following stent placement in peripheral vessels requires large animal models such as the sheep, dog, and pig.89–91 These models allow evaluation of postplacement healing, short- and mid-term stent patency, as well as mechanical integrity of the stent.92 They also allow determination of the mechanical factors dictated by the stent design that may play a significant role in the development of intimal hyperplasia. In large animal models, arterial size allows easy targeting of the selected vessel (e.g., coronary arteries), and this is one reason the porcine coronary artery model has become a common model for coronary restenosis studies.93,94 In addition, the neointima that forms in this model is identical to human restenotic neointima, and the amount of neointimal thickening is directly proportional to the degree of injury. Therefore, this model permits the creation of an injury-response regression relationship that can be used to quantitate the response to potential therapeutic strategies.95 Previously, research activities related to restenosis following balloon angioplasty focused on intimal hyperplasia and the smooth muscle cell as crucial players in its pathogenesis. Dissenting opinions, however, have emerged. The main reason for this has been that despite favorable results with numerous pharmacological strategies in many animal models, clinical trials in humans did not match the experimental success.96 This discrepancy led to a rethinking of the pathogenesis of restenosis, and arterial wall remodeling is now being considered as a major contributor to the underlying mechanisms of restenosis.97 Endovascular procedures in humans are carried out on atherosclerotic arteries with large plaques, whereas in the majority of animal experiments, procedures are used to create injury in the normal disease-free vascular wall. Therefore, factors contributing to the development of restenosis may be different in animal models, which would explain the lack of success of therapeutic strategies in humans. Angioplasty Until recently, PTA has been the most commonly used revascularization procedure in clinical practice. PTA alleviates luminal stenosis by affecting plaque fractures and remodeling with overstretching of the disease-free portion of the arterial wall.98,99 Acute restenosis is often the result of wall spasm, in situ thrombosis, and/or elastic recoil. Constrictive vascular remodeling, which is defined as a loss of vessel area within the external elastic lamina, is the major determinant of chronic restenosis in humans after balloon angioplasty. Excessive neointimal formation, including proliferation of smooth muscle cells and synthesis of the extracellular matrix, has traditionally been considered the major cause of restenosis. However, a third process, the formation of neoadventitia, has been suggested to play an active role in both vascular remodeling100 and neointimal formation.101,102 It was hypothesized that neoadventitia could act in a way similar to scar contraction leading to “strangulation” of the vessel.100 It was also shown that adventitial fibroblasts

CHAPTER 40 / ANIMAL MODELS FOR ATHEROSCLEROSIS, RESTENOSIS, AND ENDOVASCULAR ANEURYSM REPAIR

migrate toward the lumen and take on a myofibroblastic phenotype to form the neointima.101–103 A recent porcine model has provided more accurate insight into the pathophysiology of constrictive vascular remodeling.97 After 30% overdilation of the porcine coronary arteries, loss of lumen, constrictive remodeling, and formation of neointima occurred between days 7 and 28. These processes were preceded by the formation of neoadventitia and adventitial neovascularization occurring during the first 4 days after angioplasty. Neointimal formation began just after day 7; therefore, the constrictive remodeling coincided with the formation of neointima rather than the formation of neoadventitia. At late follow-up, loss of lumen was found to be correlated with remodeling, not neointimal formation. However, the neointimal area was not correlated with either late loss of lumen or vascular remodeling. These results indicate that qualitative rather than quantitative changes within the vessel wall neointima, media, and adventitia may play an important role in late luminal narrowing. In conclusion, the time course of events after PTA is as follows: (1) platelet adhesion/aggregation and in situ thrombosis occur minutes after a type II/III arterial injury,63,104 (2) formation of neoadventitia including adventitial neovascularization occurs within 4 days, and (3) late loss of lumen consisting of constrictive remodeling and formation of neointima occurs between 7 and 28 days.97 Late loss of lumen is more dependent on vascular remodeling than on the formation of neointima. The time course of remodeling, however, coincides with the growth of neointima rather than with changes in the adventitia, indicating that constrictive vascular remodeling might depend on the formation of neointima. Thus, the present knowledge of restenosis after balloon angioplasty indicates that in humans and pigs late loss of lumen after balloon angioplasty is caused more by constrictive remodeling than by neointimal formation. Stenting and Drug-Eluting Stents PTA has been replaced by arterial stenting as the most common revascularization procedure in certain instances. This is especially true for the coronary artery and the majority of the peripheral vascular territories where primary stenting has become the treatment of choice. Compared to percutaneous transluminal coronary angioplasty (PTCA), the introduction of intracoronary stents reduced the 3- to 6-month restenosis rate to 20–30%.105 To further reduce the “instent” restenosis rate, drug-eluting stents (DESs) have recently been introduced into clinical practice for coronary artery application. This change in clinical practice was made possible by extensive preclinical studies that required relevant changes in the animal models used to evaluate restenosis after stenting. The pathological responses to balloon dilatation (PTA) and stenting differ. Regional constrictive remodeling predominates after balloon angioplasty, whereas after stent implantation, restenosis is due solely to neointima thickening. Early studies in the porcine coronary injury model suggested that the deeper the arterial injury, the greater the neointimal thickening.106 This proportionality in the pig model resulted in the development of stent designs that reduce arterial injury. Early balloon expandable stents caused substantial injury when they were overexpanded, whereas the currently used self-expanding slotted tubular stent designs produce less injury. Overstretch injury to pig coronary arteries holds important lessons for neointimal response to injury. Simple overstretch without stent implantation causes medial fracture and laceration

375

with frequent dissections.107 A typical balloon/artery ratio is 1.2/1 or 1.3/1, which creates enough injury for acceptable neointimal thickening without the risk of large dissections. Larger balloon/ artery ratios yield the likelihood of severe dissection with resulting thrombosis and occlusion. When stents are implanted, dissections are usually controlled except at the stent margins. However, stent/artery ratios of >1.3/1 often result in chronic vessel injury dominated by a high degree of inflammation. This inflammation is undesirable because drug elution cannot overcome such severe injury, making assessment of stent/drug efficacy impossible. Therefore, for preclinical DES testing a stent/artery ratio of 1.1/1 is usually employed so as to produce only a minimal to mild degree of neointima formation, which in turn makes it possible to determine the efficacy of the drug used for coating the stent. The porcine coronary model suggests a temporal comparability for healing after stenting that can be predicted for human application. Stent healing occurs approximately six times faster in pigs compared to humans.107 This proven time relationship has established standardized times for porcine studies (1, 3, 6, and 12 months). Times points after 1 month are used principally to evaluate safety issues only because few changes occur in the pig model beyond this time with the exception of the slow healing process. Self-expanding stents induce a neointima composed of cells migrating from the media. Conversely, balloon overstretch injury resulting in complete rupture of the media and exposure of the adventitia to blood components can lead to SMC stimulation in addition to activation and proliferation of adventitial fibroblasts followed by their inward migration and participation in the formation of neointima. The different SMC markers characteristic of these two different pathomechanisms of neointimal formation were demonstrated in the established porcine coronary artery model.108 The lessons learned from animal restenosis models are summarized in Table 40–3. It is essential to rapidly and effectively screen candidate compounds for DES before large, expensive, and time-consuming preclinical trials take place. Moreover, it is necessary to evaluate

Table 40–3 Lessons learned from animal restenosis models107 1. Arterial injuries are major determinants of neointimal thickening; the optimization of stent design should limit arterial injury as much as possible 2. Neointimal formation on drug-eluting stents (DESs) develops the same as on bare-metal stents 3. Although DESs limit neointimal formation, they may also delay or cause incomplete healing to a greater degree than bare-metal stents 4. The porcine coronary and rabbit iliac artery models provide useful information regarding stent thrombosis risk, and thus can be used to measure safety 5. Significant lumen loss in animal models resulting from medial cell death, inflammation, and neointimal thickening should be taken into consideration when initiating clinical trials 6. Efficacy testing in currently used preclinical models (rabbit, porcine) has proven difficult to establish 7. Innovative preclinical model enhancements are required to achieve more rapid turnaround time of the results of experimental interventions

376

SECTION V / MODELS OF BEHAVIOR

safety-related pathological features of the existing antirestenotic compounds to properly define the therapeutic window of candidate compounds for DES.109 Therefore, it is important that the animal model used possesses the underlying atherosclerotic lesion. DES may not only affect the formation of in-stent restenosis, but could also affect the formation or progression of the underlying atherosclerotic stented lesions (stabilization vs. destabilization of the stented plaque). While the porcine coronary stent model appears predictive for stent thrombosis and adverse neointimal formation, this model is simply too expensive and time consuming to be used for screening drugs suitable for DES technology. Furthermore, the lack of arteriosclerosis in this native-vessel model is a major limitation to the proper evaluation of the efficacy and safety of DES strategies. Well-established genetically modified murine models appear to be helpful for the purpose of accurately screening candidate compounds for use on DES. Pires et al. recently described an inventive new model using an established transgenic mouse strain to screen possible drugs for DES.110 First, a nonconstrictive polyethylene perivascular cuff is surgically placed around the femoral artery of diet-driven hypercholesterolemic apoE*-Leiden transgenic mice, which results in the development of atheroscleroticlike lesions in 2 weeks. Then, the first cuff is replaced by a second drug-eluting cuff constructed from a poly(ε-caprolactone) polymer matrix that is loaded with the drug of interest. This novel mouse model enables cost-effective screening of candidate drugs for DES and the establishment of dose–response relationships with respect to both therapeutic and harmful effects on the vessel wall. The candidate drugs with reasonable potential for DES can subsequently be tested in large animal models. Evaluating DES in the presence of atherosclerosis results in new insights into restenosis and elucidates the effects of DES on atherosclerotic lesions. CONCLUSIONS Animal models have been used successfully to study specific aspects of the restenotic process. These arterial models are created to simulate the vascular injury seen in humans following revascularization techniques (i.e., balloon angioplasty and stenting) with the aim of determining morphology and pathogenesis as well as preventive and therapeutic strategies. However, none of the described methods for inducing restenosis produces a single ideal model.

ENDOVASCULAR ANEURYSM REPAIR BACKGROUND Among the animal models that have been created for studying aneurysm formation and repair, abdominal aortic aneurysm (AAA) models have dominated the field. The ideal animal model for AAA should possess all of the features of the human disease. While the manifestations of AAA may vary considerably depending on the stage of the disease, consistent features of the dilated segment are medial degeneration and adventitial inflammation. There are several small animal models (mouse, rat) in which biochemical or genetic perturbations have been used to develop AAAs. The interested reader is referred to detailed articles on the basic mechanical and clinical implications as well as small animal models.111–114 In the past decade, the use of stent grafts has become the therapeutic alternative of choice for the endovascular repair of AAAs, while minimally invasive transcatheter embolization techniques have become the treatment of choice for endovascular treatment of intracerebral aneurysms. Recent developments in these two fields of endovascular aneurysm repair have been based

on extensive experimental studies using a variety of animal models. An overview of these models follows. AORTIC ANEURYSM MODELS One of the most exciting developments in interventional imaging guided minimally invasive techniques in recent years is endovascular stent grafts for the endoluminal exclusion of aneurysms. Animal testing of endovascular grafts, and of the techniques employed in their deployment, is an essential step for the successful clinical application of these devices. Such experimental trials require the creation of an aneurysm in an animal model for effective testing. Since 1986, when Balko et al.115 reported the first endoluminal exclusion of an experimentally created AAA in sheep, several large animal aortic aneurysm models have been used for the study of endovascular grafts. These have included models of dissecting, saccular, fusiform, and acutely rupturing aneurysms. The choice of research animal and the choice of aneurysm model to be used for the study of endovascular grafts are two of the most important decisions to be made by an investigator. Animal models can serve as an adequate test for endovascular prostheses only to the extent that they simulate the human condition. Animals The choice of an experimental animal is especially important because of species differences relative to humans. Appreciable differences in the coagulation and fibrinolytic systems between humans and the experimental animal used to evaluate an endovascular prosthesis could significantly bias the interpretation of test results. Comparative studies have shown that the clotting and fibrinolytic systems of the calf and nonhuman primates are more similar to the human than to those of dogs or pigs.116 Interspecies differences also exist regarding neointimal formation. Apart from specific situations (subsequently described), all models reported to date have employed normotensive, nonatherosclerotic animals. In 1993, the Ad Hoc Committee of the Joint Councils of the Society for Vascular Surgery and the International Society for Cardiovascular Surgery, North American chapter, recommended that the canine be used for preclinical testing of arterial grafts in most instances.117 According to the committee’s report, the dog has two characteristics that make it a suitable animal model. One is the lack of significant spontaneous endothelialization of prosthetic surfaces, which is similar to humans. The other is a variable and relatively unpredictable tendency toward hypercoagulability, which presents a challenging test of device thrombogenicity. Other advantages of the dog include large peripheral arteries, ease of handling, and tolerance of prolonged anesthesia. However, the availability of dogs is being restricted by animalrights activists, and as a result, the cost of using dogs is rising sharply. Furthermore, the dog has an extremely potent fibrinolytic system.116 Pigs and sheep also have been used for the study of endovascular grafts, although not as frequently as dogs. The arterial morphology of the pig is most similar to humans; they exhibit a tendency toward hypercoagulability, and their fibrinolytic system is not as active as the canine. When compared with dogs, however, they are not as easy to handle, are less tolerant of anesthesia, and their arteries are smaller, making it difficult to use large introducer systems and prostheses. Sheep, however, are more similar in size to the human and their arteries are large enough to accommodate larger diameter prostheses (18 mm). In addition, their coagulation system is closer to the human than either dogs or pigs. The sheep is an easy animal to manage in the laboratory and to use for testing

CHAPTER 40 / ANIMAL MODELS FOR ATHEROSCLEROSIS, RESTENOSIS, AND ENDOVASCULAR ANEURYSM REPAIR

of vascular devices. However, sheep can harbor a rickettsial infection (Q fever) that is transmissible to humans. Experimental Models and Techniques Only a few published reports have dealt with the evaluation of endovascular grafts in animal models of dissecting and acutely rupturing aortic aneurysms. The standard technique for the creation of dissection aortic aneurysms continues to be the Blanton technique.118 The aorta is surgically exposed, cross-clamped, and a 120˚ transverse aortotomy incision is performed. An intramural pocket is formed by dissecting a free edge of the vessel caudally, and the outer wall of the free edge is sutured back to the proximal edge of the aortotomy site creating a dissecting aneurysm. Williams et al.119 reported the use of covered stents in a model of acutely rupturing thoracic aortic aneurysm. The model was created by puncturing the aortic wall with a Colapinto needle and then balloon dilating the tract. However, immediate treatment and homologous blood transfusions were needed to prevent rapid exsanguination. To date, endovascular grafts have been studied primarily for the purpose of intraluminal exclusion of saccular and fusiform aneurysms of the infrarenal abdominal aorta. Since the first published studies in the mid-1980s several experimental AAA models have been used to test the delivery, biocompatibility, and efficacy of endovascular grafts. Anterior Patch Model The anterior patch AAA model has been one of the most popular animal models for the study of endovascular grafts. Saccular AAAs have been created in dogs, pigs, and sheep using various patch materials including Dacron,115,120 rectus fascia without120,121 and with122 peritoneum, jejunum,123,124 iliac vein,125 and double layer peritoneum.126 The advantage of using autologous tissue as a patch material is that it can progressively enlarge and rupture similar to a human AAA. This has been shown to occur with fascial and jejunal patches. Although iliac vein patches enlarge, they do not rupture because scar tissue forms around the aneurysm. The double layer peritoneum model created in swine has been shown to have the closest behavior to human AAA in regard to gradual expansion and tendency for rupture. Its rupture rates (70% in long and 20% in short aneurysms within 2 weeks) are higher than the rate associated with rectus fascia with peritoneum (11% in 5 months)122 and with iliac vein (0% in 4 ± 2 months),125 much lower than that with untreated jejunum (100% within 42 h),123 and comparable to that with glutaraldehyde-treated jejunum (66% at 11 days).123 During the creation of the anterior patch aneurysm model, the aorta is isolated, the animal is heparinized, the aorta is crossclamped below the renal arteries and above the iliac bifurcation, and collaterals are temporarily controlled with vessel loops or hemoclips that are removed upon the completion of the aortic patch suture. A longitudinal aortotomy is created in the anterior aspect of the aorta and an elliptical-shaped patch is sutured into the incision. No mural thrombus has been reported within fascial or iliac vein patch aneurysms and only trace amounts of thrombus have been found in Dacron patch aneurysms prior to graft implantation. It is unclear whether this is a limitation of these models because the effect that thrombus deposition within an aneurysm has on the early healing of the graft is unknown. Aneurysms formed by a jejunal patch have been found to contain thrombus prior to grafting, but none has been completely thrombosed. Approximately 95% of the aortic collateral arteries associated with anterior patch AAAs have remained patent prior to graft replacement. AAA models without patent lumbar and inferior

377

mesenteric arteries are unrealistic. The absence of collateral branches prevents the study of their role in maintaining patency of the aneurysmal sac after endovascular grafting and the efficacy of any particular device in preventing aneurysm rupture. At 30 and 60 days after graft placement, approximately 80% of the lumbar arteries bridged by the prosthesis have reportedly thrombosed. Collateral flow has remained in some instances in which blood flow into and out of the excluded aneurysmal sac occurred via two separate vessels. Unfortunately, with the anterior patch model, lumbar arteries are usually flush with the endovascular graft and are more likely to thrombose than if they were more separated from the prosthesis, as may be the case in human aneurysms. An “endoleak” AAA model based on a modified anterior patch technique was created to yield a large aneurysmal sac after stentgraft placement for testing of techniques for its percutaneous occlusion.127 Infrarenal AAAs were created in dogs by anastomosis of an isolated segment of the inferior vena cava (IVC) to the right side of the abdominal aorta in combination with a large anterior patch from the external jugular vein. During surgery, the aorta and IVC were isolated from the renal arteries to the bifurcation, and both were cross-clamped at these levels. The aorta and IVC were opened along their apposing surfaces, anterior to the origin of the lumbar arteries and veins. The medial aspects of the aorta and IVC were joined over a distance of approximately 3 cm in a side-to-side anastomosis. At least one or two lumbar arteries and one or two lumbar veins were incorporated into the AAA sac formed. The anterior wall of the aorta was then partially resected and replaced with a large vein patch. The patch was sewn to the aorta and to the anterior aspect of the IVC incision to create the anterior aneurysmal pouch. The IVC was then ligated immediately below and above the level of the side-to-side anastomosis, thereby transferring the anastomosed segment of the IVC and its lumbar veins to the aorta. One hour later, the animals underwent percutaneous implantation of polytetrafluoroethylene-covered Z stent endografts with three 3-mm-diameter holes through the fabric. Immediately after endograft placement, all nine animals had artificial type III endoleaks with angiographic filling of lumbar arteries and veins. One aneurysm ruptured at 1 week. Of the remaining eight endografts, six remained patent for 1 week (n = 1), 1 month (n = 1), 2 months (n = 2), 3 months (n = 1), and 6 months (n = 1), and two were occluded at 3 and 6 months, respectively. The aneurysm sac had enlarged by approximately 50% in seven animals during the follow-up period. The model proved to be technically feasible, but was not found to be reliable for chronic evaluation because of rapid progressive thrombosis in most aneurysm sacs and occasional complete thrombosis of the AAA and endograft. The model was later modified by Hiraki et al. to eliminate the use of the jugular vein patch and thereby shorten the aortic clamping time to about 30 min.128 Mural-Stripping Model Economou et al.129 described a method for creating saccular aortic aneurysms in dogs that involved removal of the adventitia and the outer 60–70% of the media, while leaving the remaining media, internal elastic membrane, and subendothelial layer intact. After dissection of the adventitia, the media was cut through in layers with gentle strokes of the scalpel. By not occluding the aorta during dissection, the pressure within the vessel produced a bulge at the dissected area, which acted as a guide to the depth of cutting necessary. Once the desired depth of the cut was achieved, the media was peeled along

378

SECTION V / MODELS OF BEHAVIOR

a cleavage plane at the depth of the dissection. An ellipse of media was then cut away. Optimal aneurysm formation occurred when approximately 70% of the media had been excised. A lesser dissection failed to produce an aneurysm, whereas removal of more than 70% of the media resulted in acute rupture of the resultant aneurysm. All aneurysms persisted for up to 13 weeks and no evidence of spontaneous repair was noted microscopically. Moreover, production of hypertension before and after aneurysm creation did not result in larger aneurysms at the time of death. This method was used by Mirich et al.130 to create AAAs in dogs for the evaluation of an aortic stent graft. Although cross-clamping of the aorta and its side branches was not required, surgical stripping of the adventitia and the media was slow, uncertain, and difficult to perform. Furthermore, no definite aneurysm was identified in any dog 2 weeks after surgery and angioplasty was required to achieve mild aortic dilation. The mean aortic diameters after the procedures increased by only an average of 46% (range, 36–58%). Interposition Graft Model Parodi et al.131 described a fusiform AAA model for the study of endovascular grafts that involved replacement of a portion of infrarenal aorta with a large crimped artificial graft. To date, this model has been created in dogs, pigs, and sheep with interposition grafts to mimic an aneurysm made from crimped woven Dacron,131–135 biomedical grade elastomeric polyurethane,133 and segments of glutaraldehyde-treated bovine internal jugular vein.136 After isolation of the aorta, the animal is heparinized, the aorta is cross-clamped below the renal arteries and above the bifurcation, and collaterals are ligated and divided. A segment of infrarenal aorta 2–6 cm in length is excised. The artificial aneurysm is anastomosed end to end with the aorta as an interposition graft. All aneurysms created with an artificial conduit have remained patent prior to endovascular graft placement. A decrease in aneurysmal diameter has been reported, but it has been difficult to determine if this decrease was associated with the formation of mural thrombi or with fibrosis and shrinkage of the graft material. In some studies, nonsignificant mural thrombus formation was noted in the aneurysmal sac by the time of endovascular graft placement. A moderate to severe fibrous reaction was present around the artificial aneurysm in all animals. All aneurysms created with glutaraldehyde-treated bovine internal jugular vein were immediately pulsatile and remained so until endovascular exclusion.136 No significant change in maximum aneurysm diameter was reported, although 1 of 12 aneurysms ruptured. High-flow jets at the proximal and distal anastomoses with turbulent flow in the sac itself were noted prior to stent placement. This was due to fibrointimal hyperplasia at the anastomoses. No mural thrombus developed in the sac prior to stent placement, and in all cases the sac wall demonstrated a florid inflammatory reaction with foreign body giant cells. The interposition graft AAA model is useful for the evaluation of delivery systems and of graft deployment techniques; advantages of this model include an aneurysmal size and configuration that more closely resemble human AAAs. However, it is not appropriate for studying either the role of the biological aneurysmal wall in the healing process around the prosthetic bypass or the effects of the patent aortic side branches. Furthermore, scarring of the prosthetic aortic system occurs, which is an experimental variable not likely to be present in human AAA.

Elastase Model Martin et al.137 reported the creation of AAA in dogs by temporary exposure to the elastolytic action of pancreatic elastase. Segments of the abdominal aorta measuring 3 cm in length were exposed to 0.1% trypsin or 0.1% elastase for a period of 90 min while blood flow was arrested. All animals underwent thyroidectomy and were given an HFHC diet with a large dose of vitamin D. None of the dogs exposed to trypsin developed aneurysms, whereas 50% of the dogs exposed to elastase developed aneurysms of the exposed aortic segment 2 months after the procedure. The elastin content of the aorta in the exposed area averaged 51% of that in the unexposed area. The technique, however, proved to be inconsistent, and when successful resulted in the formation of small aneurysms. Boudghène et al.138 reported the use of elastase to create an AAA model in dogs for evaluation of endovascular grafting. All infrarenal lumbar arteries, except for one lumbar branch at the caudal end of the aortic segment, were ligated. After heparinization, a 4-cm length of infrarenal aorta was isolated between two clamps. This isolated segment was catheterized via the preserved lumbar artery, which was then tied to the catheter. The blood was removed from the isolated segment and the segment was flushed several times with saline. A solution of porcine type I elastase was then infused at a rate of 60 ml/h for 40 min. The dose of elastase was found to be critical. When low doses of elastase (3000 U) were used, the aneurysms ruptured within 24–72 h. All animals treated with 2800 U of elastase developed a 4-cm-long aneurysm of the infrarenal aorta. Aneurysmal diameter was approximately twice the size of the normal aorta and aortic wall thickness decreased by an average of 50%. Parietal thrombus was present in three of eight animals. Histological examination showed intense inflammation around the induced aneurysm. In addition, the elastic laminae were frayed and broken in all sections and had totally disappeared in some areas. These lesions were not uniform throughout the length or circumference of the perfused segment and are similar to acquired aneurysms in patients that result from the progressive degeneration of the arterial wall with diffuse loss of elastic tissue. Ligation of the lumbar arteries is a technical disadvantage in the assessment of their role in maintaining AAA patency after grafting and in the evaluation of the efficacy of the graft in preventing aneurysm rupture. Transluminally Created Model Hallisey139 reported the first transluminal creation of a fusiform AAA in dogs. After systemic heparinization, a Palmaz stent was deployed in the infrarenal aorta. The stent and aorta were then dilated to twice the diameter of the normal aorta with an appropriately sized angioplasty balloon. The animals were maintained on oral aspirin daily for the entire 30-day follow-up period. All lumbar arteries remained patent and the AAAs remained unchanged. No gross evidence of rupture or retroperitoneal hematoma was noted in any of the dogs. A less than 1-cm-long nonstented but dilated segment was seen at either end of the aneurysm. The normal mural architecture was present, although hidden by stent compression. Some degeneration of the smooth muscle and replacement by fibrous connective tissue were seen. The luminal surface of the AAA was smooth and there was no significant inflammation around the stent wires, even when they entered the tunica media of the aortic wall. This AAA model is easily created transluminally without the need for retroperitoneal dissection or major surgery. The aneu-

CHAPTER 40 / ANIMAL MODELS FOR ATHEROSCLEROSIS, RESTENOSIS, AND ENDOVASCULAR ANEURYSM REPAIR

rysms are short in length, with a normal neck of abdominal aorta above and below. Lumbar arteries and the lumen of the aneurysm remain patent. However, the model lacks true elastin breakdown, inflammatory cell reaction, and arterial wall degradation, all of which are found in human AAAs. The Palmaz stent also prevents any reduction in the size of the aneurysmal sac that might occur after endovascular graft placement. The model originally described by Hallisey was modified by Schoder et al. who created an infrarenal AAA sheep model.140 First, the aorta was overdilated with a transluminally placed Palmaz stent similar to the Hallisey model. Then an approximately 20% undersized endograft suspended between two stentgraft adapters was used to bridge the aneurysm in such a manner that two pairs of lumbar arteries remained patent within the residual aneurysm sac. The stent-graft adapters were large-vessel occluders consisting of a 20-mm-square stent with four barbs and a small intestinal submucosa (SIS) cover with a 6-mm-long central slit. The specific goal of this modification was to percutaneously create an AAA model that duplicates the anatomic and physiological events associated with endoleaks after endografting. CEREBRAL ANEURYSM MODELS The selection of an experimental in vivo cerebral aneurysm model (CAM) for evaluation of medical devices for repair requires two critical decisions: the first is the selection of the specific animal species and the second is the methodology of CAM creation. For example, device safety is tested in a swine model due to its hypercoagulative and hyperproliferative nature. However, the efficacy of the bioimplant is difficult to assess in pigs since aneurysms in these animals readily thrombose. In contrast, a device successful for aneurysm treatment in humans will more likely be effective following evaluation in canine models because of the dogs’ strong fibrolytic profile. High patency rates in canine sidewall CAMs have been demonstrated by angiography in excess of 6 months.141 Thus, the selection criteria of an experimental in vivo CAM are strongly dependent upon the study objective. Experimental Models and Techniques Numerous experimental in vivo CAMs have been reported in the literature, and the methods of creation may be divided into the categories of mechanical vessel wall trauma, chemical injury, enzymatic elastolysis, surgical anastomosis of a venous pouch, and the combination of induced renal hypertension, unilateral carotid ligation, and lathyrism. In the latter model, high hemodynamic stress and the administration of neurotoxic amino acids leading to angiolathyrism create an environment for the eventual formation of aneurysms. This model produced in nonhuman primates is the most representative model of the pathogenesis of human cerebral aneurysms; however, unpredictable aneurysm formation, high cost, and the length of time for aneurysmal dilations make this model unattractive.142 This model may be reliably produced in rats or mice, but the small size of the vessels makes evaluation of endovascular repair impractical.143–145 Only enzymatic digestion and surgical venous pouch construction have been shown to reliably produce practical CAMs. One of the oldest and most frequently reported ways to create a CAM involves an arteriotomy with a vein segment sutured to the margins of the incision.146 Many variations of this technique have been employed in multiple animal species including the rabbit, canine, swine, and rat. Furthermore, a myriad of configurations of both the venous pouch anastomosis as well as the parent artery have evolved to produce a variety of aneurysm types

379

including fusiform, saccular sidewall, bifurcation, and terminal bifurcation geometries. Stehbens was the first to describe a bifurcation venous pouch CAM made in the rabbit at aortic bifurcation.147 Strother et al. developed a method of constructing a bifurcation CAM by performing an end-to-side anastomosis of the left common carotid artery (CCA) to the right CCA and subsequently suturing a vein segment to the apex of this juncture.148 One key advantage of the lateral wall configuration of the venous pouch model is the ability to create similar CAMs bilaterally in the same animal allowing for the evaluation of the safety profile of a repair device compared with a control device in the contralateral CCA. However, the lateral wall model does not produce realistic intraaneurysmal flow characteristics, which may be simulated with the more complex bifurcation models. Although the surgical techniques used to create the bifurcation models allow excellent manipulation of the aneurysm-parent vessel geometry and the ability to create various aneurysm types in many different animal species allowing precise control of the local hemodynamics,148,149 the venous pouch CAMs are not histologically similar to the human aneurysms that they are intended to represent.150,151 One striking dissimilarity is the average aneurysmal wall thickness, which was found to be more than four times greater in the venous pouch CAM model compared with human aneurysms.150 Moreover, the attenuation or absence of the tunica media and the internal elastic lamina at the dome of the human aneurysm is not reproduced in the experimental CAM, which might explain the infrequent rupture of venous pouch CAMs (other than acutely at the suture line of the neck). Within 2 h of creation, the experimental CAM is extensively infiltrated by inflammatory leukocytes. Rapid, spontaneous, and progressive fibromuscular neointimal proliferation occurs shortly after the venous pouch is sutured to the arterial segment and has been shown to continue for as long as 2 years.152 It has been postulated that the disruption of the endothelium by suturing a venous patch to the margins of an arteriotomy causes the release of platelet-derived growth factor, which leads to fibrotic scarring.153,154 The presence of fibrotic scarring in the model, which is frequently absent in human aneurysms, could complicate treatment efficacy studies154 if they are not conducted over sufficient time, since mural thickening occurs over several months. More recently, a method of experimental CAM construction has been proposed wherein the wall of the aneurysm is arterial and not venous.153,154,155–158 The fundamental concept of the technique is to create an arterial “stump” in the CCA of the rabbit either by surgical ligation or by the use of an endovascular balloon, and then to incubate within the stump pancreatic elastase for a period between 10 and 45 min. This technique has been employed to create CAMs that resemble human cerebral aneurysms for testing of endovascular treatment modalities.141,156 The elastase-induced rabbit CAM has been extensively studied over the past decade, and the results indicate that this model offers certain advantages over the experimental vein pouch CAM. The technique is simple, the model has been shown to remain patent for more than 1 year, and depending on the artery used to construct the CAM, both lateral aneurysms located on the outside aspect of a curved parent vessel and bifurcation-type aneurysms may be induced that mimic the geometry typical of human intracranial aneurysms. Furthermore, the elastase CAM is histologically representative of human cerebral aneurysms, with a similar

380

SECTION V / MODELS OF BEHAVIOR

wall thickness, attenuation or absence of the internal elastic lamina beyond the neck, the absence of intramural inflammation, and only a moderate presence of cellular elements within the aneurysmal sac.150 The migration of fibroblasts and cellular elements into the sac of the venous pouch CAM due to the disruption of the basement membrane by the surgical wound is avoided by the elastase technique. Thus, it has been proposed that the elastaseinduced CAM shares more common morphological and histological characteristics with human cerebral aneurysms than the vein pouch model.150 However, control of the angioarchitecture of the elastase CAM is not reliable, as it has been discovered that hemodynamic factors determine the final dimensions of the CAM.159 Another point to keep in mind is that if this model is created in the CCA, the proximity of the model to the thorax creates vascular motion due to respiration. This motion can potentially affect the evaluation of treatment strategies since it is not present within the intracranial vessels. CONCLUSIONS In conclusion, judicious selection of an aortic or cerebral aneurysm animal model allows specific scientific hypotheses to be addressed. Therefore, the choice of the animal species and the technique employed to create the aneurysm must be based on the desired characteristics of the model that are compatible with the objectives of the experiment.

ACKNOWLEDGMENT This updated work was substantially based on the following prior review article: Narayanaswamy M, Wright KC, Kandarpa K. Animal Models for Atherosclerosis, Restenosis, and Endovascular Graft Research. JVIR 2000;11:5–17.

REFERENCES 1. Gimbrone MA Jr. Vascular endothelium, hemodynamic forces, and atherogenesis. Am J Pathol 1999;155:1–5. 2. Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med 1999;340:115–126. 3. Reardon CA, Getz GS. Mouse models of atherosclerosis. Curr Opin Lipidol 2001;12:167–173. 4. Rekhter M. Vulnerable atherosclerotic plaque: Emerging challenge for animal models. Curr Opin Cardiol 2002;17:626–632. 5. Libby P. Current concepts of the pathogenesis of the acute coronary syndromes. Circulation 2001;104:365–372. 6. Bennett M, Macdonald K, Chan SW, Luzio JP, Simari R, Weisberg P. Cell surface trafficking of Fas: A rapid mechanism of p53mediated apoptosis. Science 1998;282:290–293. 7. Boyle JJ, Bowyer DE, Weissberg PL, Bennett MR. Human bloodderived macrophages induce apoptosis in human plaque-derived vascular smooth muscle cells by Fas-ligand/Fas interactions. Arterioscler Thromb Vasc Biol 2001;21:1402–1407. 8. Libby P, Geng YJ, Aikawa M, Schoenbeck U, Mach F, Clinton SK, Sukhova GK, Lee RT. Macrophages and atherosclerotic plaque stability. Curr Opin Lipidol 1996;7:330–335. 9. Aikawa M, Rabkin E, Sugiyama S, Voglic SJ, Fukumoto Y, Furukawa Y, Shiomi M, Schoen FJ, Libby P. An HMG-CoA reductase inhibitor, cerivastatin, suppresses growth of macrophages expressing matrix metalloproteinases and tissue factor in vivo and in vitro. Circulation 2001;103:276–283. 10. Cullen P, Baetta R, Bellosta S, Bernini F, Chinetti G, Cignarella A, von Eckardstein A, Exley A, Goddard M, Hofker M, Hurt-Camejo E, Kanters E, Kovanen P, Lorkowski S, McPheat W, Pentikainen M, Rauterberg J, Ritchie A, Staels B, Weitkamp B, deWinther M; MAFAPS Consortium. Rupture of the atherosclerotic plaque: Does a good animal model exist? Arterioscler Thromb Vasc Biol 2003;23:535–542. 11. Fan J, Watanabe T. Cholesterol-fed and transgenic rabbits for the study of atherosclerosis. J Atheroscler Thromb 2000;7:26–32.

12. Herrera VL, Makrides SC, Xie HX, Adari H, Krauss RM, Ryan US, Ruiz-Opazo N. Spontaneous combined hyperlipidemia, coronary heart disease and decreased survival in Dahl salt-sensitive hypertensive rats transgenic for human cholesteryl ester transfer protein. Nat Med 1999;5:1383–1389. 13. Russel JC, Graham SE, Richardson M. Cardiovascular disease in the JCR:LA-cp rat. Mol Cell Biochem 1998;188:113–126. 14. Drobnik J, Dabrowski R, Szczepanowska A, Giernat L, Lorenc J. Response of aorta connective tissue matrix to injury caused by vasopressin-induced hypertension or hypercholesterolemia. J Physiol Pharmacol 2000;51:521–533. 15. Badimon L. Atherosclerosis and thrombosis: Lessons from animal models. Thromb Haemost 2001;86:356–365. 16. Watanabe Y. Serial inbreeding of rabbits with hereditary hyperlipidemia (WHHL-rabbit). Atherosclerosis 1980;36:261–268. 17. Feranandez ML. Guinea pigs as models for cholesterol and lipoprotein metabolism. J Nutr 2001;131:10–20. 18. Fuster V, Lie JT, Badimon L, Rosemark JA, Badimon JJ, Bowie EJ. Spontaneous and diet-induced coronary atherosclerosis in normal swine and swine with von Willebrand disease. Arteriosclerosis 1985;5:67–73. 19. Turk JR, Henderson KK, Vanvickle GD, Watkins J, Laughlin MH. Arterial endothelial function in a porcine model of early stage atherosclerotic vascular disease. Int J Exp Pathol 2005;86:335–345. 20. Hamilton AJ, Kim H, Nagaraj A, Mun JH, Yan LL, Roth SI, McPherson DD, Chandran KB. Regional material property alterations in porcine femoral arteries with atheroma development. J Biomech 2005;38:2354–2364. 21. Nagaraj A, Kim H, Hamilton AJ, Mun JH, Smulevitz B, Kane BJ, Yan LL, Roth SI, McPherson DD, Chandran KB. Porcine carotid arterial material property alterations with induced atheroma: An in vivo study. Med Eng Phys 2005;27:147–156. 22. Gerrity RG. The role of the monocyte in atherogenesis: I. Transition of blood-borne monocytes into foam cells in fatty lesions. Am J Pathol 1981;103:181–190. 23. Fagiotto A, Ross R, Harker L. Studies of hypercholesterolemia in the nonhuman primate. I. Changes that lead to fatty streak formation. Arteriosclerosis 1984;4:323–340. 24. Rosenfeld ME, Tsukada T, Gown AM, Ross R. Fatty streak initiation in Watanabe heritable hyperlipemic and comparably hypercholesterolemic fat-fed rabbits. Arteriosclerosis 1987;7:9–23. 25. Jawien J, Nastalek P, Korbut R. Mouse models of experimental atherosclerosis. J Physiol Pharmacol 2004;55:503–517. 26. Paigen B, Morrow A, Holmes PA, Mitchell D, Williams RA. Quantitative assessment of atherosclerotic lesions in mice. Atherosclerosis 1987;68:231–240. 27. Getz GS, Reardon CA. Diet and murine atherosclerosis. Arterioscler Thromb Vasc Biol 2006;26:242–249. 28. Zhang SH, Reddick RL, Piedrahita JA, Meada N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science 1992;258:468–471. 29. Piedrahita JA, Zhang SH, Hagaman JR, Oliver PM, Maeda N. Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells. Proc Natl Acad Sci USA 1992;89:4471–4475. 30. Plump AS, Smith JD, Hayek T, Aalto-Setala K, Walsh A, Verstuyft JG, Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell 1992;71:343–353. 31. Nakashima Y, Plump AS, Raines EW, Breslow JL, Ross R. ApoEdeficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb 1994;14: 133–140. 32. Kashyap VS, Santamaria-Fojo S, Brown DR, Parrott CL, Applebaum-Bowden D, Meyn S, Talley G, Paigeb B, Maeda N, Brewer HB Jr. Apolipoprotein E deficiency in mice: Gene replacement and prevention of atherosclerosis using adenovirus vectors. J Clin Invest 1995;96:1612–1620. 33. Ishibashi S, Brown MS, Goldstein JL, Gerard RD, Hammer RE, Herz J. Hypercholesterolemia in low density lipoprotein receptor

CHAPTER 40 / ANIMAL MODELS FOR ATHEROSCLEROSIS, RESTENOSIS, AND ENDOVASCULAR ANEURYSM REPAIR

34.

35.

36.

37.

38.

39.

40.

41.

42.

43. 44.

45.

46.

47. 48. 49.

50.

51.

knockout mice and its reversal by adenovirus-mediated gene delivery. J Clin Invest 1993;92:883–893. Ishibashi S, Herz J, Maeda N, Goldstein JL, Brown MS. The tworeceptor model of lipoprotein clearance: Tests of the hypothesis in “knockout” mice lacking the low density lipoprotein receptor, apolipoprotein E, or both proteins. Proc Natl Acad Sci USA 1994;91:4431–4435. Paszty C, Maeda N, Verstuyft J, Rubin EM. Apolipoprotein AI transgene corrects apolipoprotein E deficiency-induced atherosclerosis in mice. J Clin Invest 1994;94:899–903. Plump AS, Scott CJ, Breslow JL. Human apolipoprotein A-I gene expression increases high density lipoprotein and suppresses atherosclerosis in the apolipoprotein E-deficient mouse. Proc Natl Acad Sci USA 1994;91:9607–9611. Linton MF, Farese RV Jr, Chiesa G, Grass DS, Chin P, Hammer RE, Hobbs HH, Young SG. Transgenic mice expressing high plasma concentrations of human apolipoprotein B100 and lipoprotein(a). J Clin Invest 1993;92:3029–3037. Callow MJ, Stoltzfus LJ, Lawn RM, Rubin EM. Expression of human apolipoprotein B and assembly of lipoprotein(a) in transgenic mice. Proc Natl Acad Sci USA 1994;91:2130–2134. Bonthu S, Heistad DD, Chappel DA, Lamping KG, Faraci FM. Atherosclerosis, vascular remodeling, and impairment of endothelium-dependent relaxation in genetically altered hyperlipidemic mice. Arterioscler Thromb Vasc Biol 1997;17:2333–2340. Witting PK, Petterson K, Ostlund-Lindqvist AM, Westerlund C, Eriksson AW, Stocker R. Inhibition by a coantioxidant of aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E and low density lipoprotein receptor gene double knockout mice. FASEB J 1999;13:667–675. Veniant MM, Sullivan MA, Kim SK, Ambroziak P, Chu A, Wilson MD, Hellerstein MK, Rudel LL, Walzem RL, Young SG. Defining the atherogenicity of large and small lipoproteins containing apolipoprotein B100. J Clin Invest 2000;106:1501–1510. Rosenfeld ME, Polinsky P, Virmani R, Kauser K, Rubanyi G, Schwartz SM. Advanced atherosclerotic lesions in the innominate artery of the apoE knockout mouse. Arterioscler Thromb Vasc Biol 2000;20:2587–2592. Ishibashi S. Lipoprotein(a) and atherosclerosis. Arterioscler Thromb Vasc Biol 2001;21:1–2. Fan J, Shimoyamada H, Sun H, Marcovina S, Honda K, Watanabe T. Transgenic rabbits expressing human apolipoprotein(a) develop more extensive atherosclerotic lesions in response to a cholesterol-rich diet. Arterioscler Thromb Vasc Biol 2001;21:88– 94. Shimano H, Yamada N, Katsuki M, Yamamoto K, Gotoda T, Harada K, Shimada M, Yazaki Y. Plasma lipoprotein metabolism in transgenic mice overexpressing apolipoprotein E. Accelerated clearance of lipoproteins containing apolipoprotein B. J Clin Invest 1992;90:2084–2091. Fan J, Ji ZS, Huang Y, deSilva H, Sanan D, Mahley RW, Innerarity TL, Taylor JM. Increased expression of apolipoprotein E in transgenic rabbits results in reduced levels of very low density lipoproteins and an accumulation of low density lipoproteins in plasma. J Clin Invest 1998;101:2151–2164. Libby P. Changing concepts of atherogenesis. J Intern Med 2000;247:349–358. Rekhter MD. How to evaluate plaque vulnerability in animal models of atherosclerosis? Cardiovasc Res 2002;54:36–41. Prescott MF, McBride CH, Hasler-Rapacz J, Von Linden J, Rapacz J. Development of complex atherosclerotic lesions in pigs with inherited hyper-LDL cholesterolemia bearing mutant alleles for apolipoprotein B. Am J Pathol 1991;139:139–147. Johnson JL, Jackson CL. Atherosclerotic plaque rupture in the apolipoprotein E knockout mouse. Atherosclerosis 2001;154:399– 406. Williams H, Johnson JL, Carson KG, Jackson CL. Characteristics of intact and ruptured atherosclerotic plaques in brachiocephalic arteries of apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol 2002;22:788–792.

381

52. Reddick RL, Zhang SH, Maeda N. Aortic atherosclerotic plaque injury in apolipoprotein deficient mice. Atherosclerosis 1998;140: 297–305. 53. Gertz SD, Fallon JT, Gallo R, Taubman MB, Banai S, Barry WL, Gimple LW, Nemerson Y, Thiruvikraman S, Naidu SS, Chesebro JH, Fuster V, Sarembock IJ, Badimon JJ. Hirudin reduces tissue factor expression in neointima after balloon injury in rabbit femoral and porcine coronary arteries. Circulation 1998;98:580– 587. 54. Rekhter MD, Hicks GW, Brammer DW, Work CW, Kim JS, Gordon D, Keiser JA, Ryan MJ. Animal model that mimics atherosclerotic plaque rupture. Circ Res 1998;83:705–713. 55. von der Thusen JH, van Vlijmen BJ, Hoeben RC, Kockx MM, Havekes LM, van Beerkel TJ, Biessen EA. Induction of atherosclerotic plaque rupture in apolipoprotein E-/- mice after adenovirus-mediated transfer of p 53. Circulation 2002;105:2064– 2070. 56. Shiomi M, Ito T, Hasegawa M, Yoshida K, Gould KL. Correlation of vulnerable coronary plaques to sudden cardiac events. Lessons from a myocardial infarction-prone animal model (the WHHLMI rabbit). J Atheroscler Thromb 2004;11:184–189. 57. Virmani R, Kolodgie FD, Burke AP, Farb A, Schwartz SM. Lessons from sudden coronary death: A comprehensive morphological classification scheme for atherosclerotic lesions. Arterioscler Thromb Vasc Biol 2000;20:1262–1275. 58. Calara F, Silvestre M, Casanada F, Yuan N, Napoli C, Palinski W. Spontaneous plaque rupture and secondary thrombosis in apolipoprotein E-deficient and LDL receptor-deficient mice. J Pathol 2001;195:257–263. 59. Reddick RL, Zhang SH, Maeda N. Atherosclerosis in mice lacking apo E. Evaluation of lesional development and progression. Arterioscler Thromb 1994;14:141–147. 60. Palinski W, Ord VA, Plump AS, Breslow JL, Steinberg D. ApoEdeficient mice are a model of lipoprotein oxidation in atherogenesis. Demonstration of oxidation-specific epitopes in lesions and high titers of autoantibodies to malondialdehyde-lysine in serum. Arterioscler Thromb 1994;14:605–616. 61. Roubin GS, Douglas JS Jr, King SB III, Lin SF, Hutchison N, Thomas RG, Gruentzig AR. Influence of balloon size on initial success, acute complications and restenosis after percutaneous transluminal angioplasty: A prospective randomized study. Circulation 1988;78:557–565. 62. Mabin TA, Holmes DR Jr, Smith HC, Vlietstra RE, Reeder GS, Bresnahan JF, Bove AA, Hammes LN, Elveback LR, Orszulak TA. Follow-up clinical results in patients undergoing percutaneous transluminal angioplasty. Circulation 1985;71:754–760. 63. Phillips-Hughes J, Kandarpa K. Restenosis: Pathophysiology and preventive strategies. J Vasc Interv Radiol 1996;7:321–333. 64. Serruys PW, de Jasegere P, Kiemeneij F, Macaya C, Rutsch W, Heyndrickx G, Emanuelsson H, Marco J, Legrand V, Materne P, Belardi J, Sigwart U, Colombo A, Goy JJ, van den Heuvel P, Delcan J, Morel M-A. A comparison of balloon-expandable stent implantation with balloon angioplasty in patients with coronary artery disease. N Engl J Med 1994:331:489–495. 65. Vesselinovitch D. Animal models and the study of atherosclerosis. Arch Pathol Lab Med 1988;112:1011–1017. 66. Sims FH. A comparison of structural features of the walls of coronary arteries from 10 different species. Pathology 1989;21:115– 124. 67. Gertz SD, Gimple LW, Ragosta M, Roberts WC, Haber HL, Powers ER, Perez LS, Sarembock IJ. Response of femoral arteries of cholesterol-fed rabbits to balloon angioplasty with or without laser: Emphasis on the distribution of foam cells. Exp Mol Pathol 1993;59:225–243. 68. Schwartz RS, Edwards WD, Bailey KR, Camrud AR, Jorgenson MA, Holmes DR Jr. Differential neointimal response to coronary artery injury in pigs and dogs. Implications for restenosis models. Arterioscler Thromb 1994;14:395–400. 69. Schwartz RS. Neointima and arterial injury: Dogs, rats, pigs, and more. Lab Invest 1994;71:789–791.

382

SECTION V / MODELS OF BEHAVIOR

70. Baumgartner HR. The role of blood flow in platelet adhesion, fibrin deposition and formation of mural thrombi. Microvasc Res 1973;5:167–179. 71. Reidy MA, Schwartz SM. Endothelial regeneration. III. Time course of intimal changes after small defined injury to rat aortic endothelium. Lab Invest 1981;44:301–308. 72. Walker LN, Ramsay MM, Bower DE. Endothelial healing following defined injury to rabbit aorta: Depth of injury and mode of repair. Atherosclerosis 1983;47:123–130. 73. Fishman JA, Ryan GB, Karnovsky MJ. Endothelial regeneration in the rat carotid artery and the significance of endothelial denudation in the pathogenesis of myointimal thickening. Lab Invest 1975; 32:339–351. 74. Hardin NJ, Minick CR, Murphy GE. Experimental induction of atherosclerosis by the synergy of allergic injury to arteries and lipid rich diet. 3. The role of earlier acquired fibromuscular intimal thickening in the pathology of later developing atherosclerosis. Am J Pathol 1973;73:301–326. 75. Douek PC, Correa R, Neville R, Unger EF, Shou M, Banai S, Ferrans VJ, Epstein SE, Leon MB, Bonner RF. Dose-dependent smooth muscle cell proliferation induced the thermal injury with pulsed infrared lasers. Circulation 1992;86:1249–1256. 76. Fajardo LF, Berthrong M. Vascular lesions following radiation. Pathol Annu 1988;23:297–330. 77. Fritz KE, Daoud AS, Augustyn JM, Jarmolych J. Morphological and biochemical differences among grossly defined types of swine aortic atherosclerotic lesions induced by a combination of injury and atherogenic diet. Exp Mol Pathol 1980;32:61–72. 78. Gal D, Rongione AJ, Slovenkai GA, DeJesus ST, Lucas A, Fields CD, Isner JM. Atherosclerotic Yucatan microswine: An animal model with high-grade fibrocalcific, nonfatty lesions suitable for testing catheter-based interventions. Am Heart J 1990;119:291– 300. 79. Faxon DP, Weber VJ, Haudenschild C, Gottsman SB, McGovern WA, Ryan TJ. Acute effects of transluminal angioplasty in three experimental models of atherosclerosis. Arteriosclerosis1982;2:125– 133. 80. Lee WM, Lee KT. Advanced coronary atherosclerosis in swine produced by combination of balloon catheter injury and cholesterol feeding. Exp Mol Pathol 1975;23:491–499. 81. Lee KT, Lee WM, Han J, Jarmolych J, Bishop MB, Goel BG. Experimental model for study of “sudden death” from ventricular fibrillation or asystole. Am J Cardiol 1973;32:62–73. 82. Clowes AW, Reidy MA, Clowes MM. Mechanisms of stenosis after arterial injury. Lab Invest 1983;49:208–215. 83. Muller DW, Ellis SG, Topol EJ. Experimental models of coronary artery restenosis. J Am Coll Cardiol 1992;19:418–432. 84. Schatz RA, Palmaz J, Tio FO, Garcia F, Garcia O, Reuther SR. Balloon expandable intracoronary stents in the adult dog. Circulation 1987;76:450–457. 85. Fischell TA, Grant G, Johnson DE. Determinants of smooth muscle injury during balloon angioplasty. Circulation 1990;82:2170– 2184. 86. LeVeen RF, Wolf GL, Villanueva TG. New rabbit atherosclerosis model for the investigation of transluminal angioplasty. Invest Radiol 1982;17:470–475. 87. Garasic JM, Edelman ER, Squire JC, Seifert P, Williams MS, Rogers C. Swine and primates best approximate the requirements of a model of restenosis and demonstrate lesions that are markedly similar in physiology and morphology to their human counterpart. Circulation 2000;101:812–818. 88. LaDisa F Jr, Olson LE, Molthen RC, Hettrick DA, Pratt PF, Hardel MD, Kersten JR, Warltier DC, Pagel PS. Alterations in wall shear stress predict sites of neointimal hyperplasia after stent implantation in rabbit iliac arteries. Am J Physiol Heart Circ Physiol 2005;288: H2465–2475. 89. Cejna M, Virmani R, Jones R, Bergmeister H, Loewe C, Schoder M, Grgurin M, Lammer J. Biocompatibility and performance of the Wallstent and the Wallgraft, Jostent, and Hemobahn stent-grafts in a sheep model. J Vasc Interv Radiol 2002;13:823–830.

90. Barth KH, Virmani R, Froelich J, Takeda T, Lossef SV, Newsome J, Jones R, Lindisch D. Paired comparison of vascular wall reactions to Palmaz stents, Strecker tantalum stents, and Wallstents in canine iliac and femoral arteries. Circulation 1996;93:2161– 2169. 91. Fontaine AB, Spigos DG, Eaton G, Das Passos S, Christoforidis G, Khabiri H, Jung S. Stent-induced intimal hyperplasia: Are there fundamental differences between flexible and rigid stent designs? J Vasc Interv Radiol 1994;5:739–744. 92. Andrews RT, Venbrux AC, Magee CA, Bova DA. Placement of a flexible endovascular stent across the femoral joint: An in vivo study in the swine model. J Vasc Interv Radiol 1999;10:1219–1228. 93. Wanibuchi H, Dingemans KP, Becker AE, Ueda M, Naruko T, Tanizawa S, Nakamura K. Is the Watanabe heritable hyperlipidemic rabbit a suitable experimental model for percutaneous transluminal coronary angioplasty in humans? A light microscopic, immunohistochemical and ultrastructural study. J Am Coll Cardiol1993;21:1490– 1496. 94. Schwartz RS, Murphy JG, Edwards WD, Camrud AR, Vliestra RE, Holmes DR. Restenosis after balloon angioplasty: A practical proliferative model in porcine coronary arteries. Circulation 1990;82:2190–2200. 95. Schwartz RS, Topol EJ, Serrujs PW, Sangiorgi G, Holmes DR Jr. Artery size, neointima, and remodeling: Time for standards. J Am Coll Cardiol 1998;32:2087–2094. 96. O’Brien ERM, deBlois D, Schwartz SM. A critical examination of animal models of restenosis following angioplasty. In: Dobrin P, Ed. Intimal Hyperplasia. Austin, TX: R. G. Landes, 1994:229– 256. 97. Mæng M, Olesen PG, Emmertsen NC, Thorwest M, Nielsen TT, Kristensen BO, Falk E, Andersen HR. Time course of vascular remodeling, formation of neointima and formation of neoadventitia after angioplasty in a porcine model. Coron Artery Dis 2001;12: 285–293. 98. Kinney TB, Chin AK, Rurik GW, Finn JC, Shoor PM, Hayden WG, Fogarty TJ. Transluminal angioplasty: A mechanicalpathophysiological correlation of its physical mechanisms. Radiology 1984;153:85–89. 99. Castenada-Zuniga WR, Formanek A, Tadavarthy M, Vlodaver Z, Edwards JE, Zollikofer C, Amplatz K. The mechanism of balloon angioplasty. Radiology 1980;135:565–571. 100. Andersen HR, Mæng M, Thorwest M, Falk E. Remodeling rather than neointimal formation explains luminal narrowing after deep vessel wall injury. Insights from a porcine coronary restenosis model. Circulation 1996;93:1716–1724. 101. Shi Y, O’Brien JE, Fard A, Mannion JD, Wang D, Zalewski A. Adventitial myofibroblasts contribute to neointimal formation in injured porcine coronary arteries. Circulation 1996;94:1655–1664. 102. Scott NA, Cipolla GD, Ross CE, Dunn B, Martin FH, Simonet L, Wilcox JN. Identification of a potential role for the adventitia in vascular lesion formation after balloon overstretch injury of porcine coronary arteries. Circulation 1996;93:2178–2187. 103. Siow RC, Mallawaarachchi CM, Weisberg PL. Migration of adventitial myofibroblasts following vascular balloon injury: Insights from in vivo gene transfer to rat carotid arteries. Cardiovasc Res 2003;59:212–221. 104. Wilentz JR, Sanborn TA, Haudenschild CC, Valeri CR, Ryan TJ, Faxon DP. Platelet accumulation in experimental angioplasty: Time course and relation to vascular injury. Circulation 1987;75:636– 642. 105. Fischman DL, Leon MB, Baim DS, Schatz RA, Savage MP, Penn I, Detre K, Veltri L, Ricci D, Nobuyoshi M, Cleman M, Heuser R, Almond D, Teirstein PS, Fish RD, Colombo A, Brinker J, Moses J, Shaknovich A, Hirshfeld J, Bailey S, Ellis S, Rake R, Goldberg S for The Restenosis Study Investigators. A randomized comparison of coronary-stent placement and balloon angioplasty in the treatment of coronary artery disease. Stent Restenosis Study Investigators. N Engl J Med 1994;331:496–501. 106. Schwartz RS, Huber KC, Murphy JG, Edwards WD, Camrud AR, Vliestra RE, Holmes DR. Restenosis and the proportional neointi-

CHAPTER 40 / ANIMAL MODELS FOR ATHEROSCLEROSIS, RESTENOSIS, AND ENDOVASCULAR ANEURYSM REPAIR

107.

108.

109.

110.

111.

112.

113.

114. 115.

116. 117.

118. 119.

120.

121.

122.

123.

124.

125.

126.

mal response to coronary artery injury: Results in a porcine model. J Am Coll Cardiol 1992;19:267–274. Schwartz RS, Chronos NA, Virmani R. Preclinical restenosis models and drug-eluting stents. Still important, still much to learn. J Am Coll Cardiol 2004;44:1373–1385. Christen T, Verin V, Bochaton-Piallat ML, Popowski Y, Ramaekers F, Debruyne P, Camenzind E, van Eys G, Gabbiani G. Mechanisms of neointima formation and remodeling in the porcine coronary artery. Circulation 2001;103:882–888. Pires NMM, Jukema JW, Daemen JAP, Quax PHA. Drug-eluting stents studies in mice: Do we need atherosclerosis to study restenosis? Vascul Pharmacol 2006;44:257–264. Pires NMM, van der Hoeven BL, de Vries MR, Havekes LM, van Vlijmen BJ, Hennink WE, Quax PHA, Jukema JW. Local perivascular delivery of anti-restenotic agents from a drug-eluting poly(epsilon-caprolactone) stent cuff. Biomaterials 2005;26:5386– 5394. Thompson RW, Geraghty PJ, Lee JK. Abdominal aortic aneurysms: Basic mechanisms and clinical implications. Curr Probl Surg 2002;39:110–230. Carell TWG, Smith A, Burnand KG. Experimental techniques and models in the study of the development and treatment of abdominal aortic aneurysm. Br J Surg 1999;86:305–312. Manning MW, Cassis LA, Huang J, Szilvassy SJ, Daugherty A. Abdominal aortic aneurysms: Fresh insights from a novel animal model of the disease. Vasc Med 2002;7:45–54. Daugherty A, Cassis LA. Mouse models of abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol 2004;24:429–434. Balko A, Piasecki GJ, Shah DM, Carney WI, Hopkins RW, Jackson BT. Transfemoral placement of intraluminal polyurethane prosthesis for abdominal aortic aneurysm. J Surg Res 1986;40:305–309. Mason RG, Read MS. Some species differences in fibrinolysis and blood coagulation. J Biomed Mater Res 1971;5:121–128. Abbott WM, Callow A, Moore W, Rutherford R, Veith F, Weinberg S. Evaluation and performance standards for arterial prosthesis. J Vasc Surg 1993;17:746–756. Blanton FS Jr, Muller WH Jr, Warren WD. Experimental production of dissecting aneurysms of the aorta. Surgery 1959;45:81–90. Williams DM, Andrews JC, Chee SS, Marx MV, Abrams GD. Canine model of acute aortic rupture: Treatment with percutaneous delivery of a covered Z stent–work in progress. J Vasc Interv Radiol 1994;5:797–803. Verbin C, Donayre C, Kopchok G, Scoccianti M, White RA. Anterior patch aortic aneurysm model for the study of endoluminal grafts. J Invest Surg 1995;8:381–388. Ruiz CE, Zhang HP, Douglas JT, Zuppan CW, Kean CJ. A novel method for the treatment of abdominal aortic aneurysms using percutaneous implantation of a newly designed endovascular device. Circulation 1995;91:2470–2477. Palmaz JC, Tio FO, Laborde JC, Clem M, Rivera FJ, Murphy KD, Encarnacion CE. Use of stents covered with polytetrafluoroethylene in experimental abdominal aortic aneurysms. J Vasc Interv Radiol 1995;6:879–885. Criado E, Marston WA, Woosley JT, Ligush J, Chuter TA, Baird C, Suggs CA, Mauro MA, Keagy BA. An aortic aneurysm model for the evaluation of endovascular exclusion prostheses. J Vasc Surg 1995;22:306–315. Marston WA, Criado E, Baird CA, Keagy BA. Reduction of aneurysm pressure and wall stress after endovascular repair of abdominal aortic aneurysm in a canine model. Ann Vasc Surg 1996;10:166– 173. Eton D, Warner D, Owens C, McClenic B, Cava R, Ofek B, Borhani M, Baraniewski H, Schuler JJ. Results of endoluminal grafting in an experimental aortic aneurysm model. J Vasc Surg 1996;23:819– 831. Maynar M, Qian Z, Hernandez J, Sun F, DeMiguel C, Crisostomo V, Usón J, Pineda L-F, Espinoza CG, Castañeda WR. An animal model of abdominal aortic aneurysm created with peritoneal patch: Technique and initial results. Cardiovasc Intervent Radiol 2003;26:168–176.

383

127. Pavcnik D, Andrews RT, Yin Q, Uchida BT, Timmermans HA, Corless C, Toyota N, Nakata M, Kaufman J, MD, Keller FS, Rösch J. A canine model for studying endoleak after endovascular aneurysm repair. J Vasc Interv Radiol 2003;14:1303–1310. 128. Hiraki T, Pavcnik D, Uchida BT, Timmermans HA, Yin Q, Wu RH, Niyyati M, Keller FS, Rösch J. Prophylactic residual aneurysmal sac embolization with expandable hydrogel embolic devices for endoleak prevention: Preliminary study in dogs. Cardiovasc Intervent Radiol 2005;28:459–466. 129. Economou SG, Taylor CB, Beattie EJ Jr, Davis CB Jr. Persistent experimental aortic aneurysms in dogs. Surgery 1960;47:21–28. 130. Mirich D, Wright KC, Wallace S, Yoshioka T, Lawrence DD Jr, Charnsangavej C, Gianturco C. Percutaneously placed endovascular grafts for aortic aneurysms: Feasibility study. Radiology 1989;170: 1033–1037. 131. Parodi JC, Palmaz JC, Barone HD. Transfemoral intraluminal graft implantation for abdominal aortic aneurysms. Ann Vasc Surg 1991;5:491–499. 132. Laborde JC, Parodi JC, Clem MF, Tio FO, Barone HD, Rivera FJ, Encarnacion CE, Palmaz JC. Intraluminal bypass of abdominal aortic aneurysm: Feasibility study. Radiology 1992;184:185–190. 133. Hagen B, Harnoss BM, Trabhardt S, Ladeburg M, Fuhrmann H, Franck C. Self-expandable macroporous nitinol stents for transfemoral exclusion of aortic aneurysms in dogs: Preliminary results. Cardiovasc Intervent Radiol 1993;16:339–342. 134. Piquet P, Rolland PH, Bartoli JM, Tranier P, Moulin G, Mercier C. Tantalum-dacron coknit stent for endovascular treatment of aortic aneurysms: A preliminary experimental study. J Vasc Surg 1994; 19:698–706. 135. Gorin DR, Arbid EJ, D’Agostino R, Yucel EK, Solovay KS, La Morte WW, Quist WC, Mulligan N, Menzoian JO. A new generation endovascular graft for the treatment or repair of abdominal aortic aneurysms. Am J Surg 1997;173:159–164. 136. Whitbread T, Birch P, Rogers S, Majeed A, Rochester J, Beard JD, Gaines P. A new animal model for abdominal aortic aneurysms: Initial results using a multiple-wire stent. Eur J Vasc Endovasc Surg 1996;11:90–97. 137. Martin DE III, Nasbeth DC, Rowe MI. Production of experimental aneurysms with pancreatic elastase. Surg Forum 1962;8: 237–239. 138. Boudghene F, Anidjar S, Allaire E, Osborne-Pellegrin M, Bigot JM, Michel JB. Endovascular grafting in elastase-induced experimental aortic aneurysms in dogs: Feasibility and preliminary results. J Vasc Interv Radiol 1993;4:497–504. 139. Hallisey MJ. A transluminally created abdominal aortic aneurysm model. J Vasc Interv Radiol 1997;8:305–312. 140. Schoder M, Pavcnik D, Uchida BT, Corless C, Timmermans HA, Yin Q, Brountzos E, Nakata M, Hiraki T, Niyyati M, Kaufman JA, Keller FS, Rösch J. Small intestinal submucosa aneurysm sac embolization for endoleak prevention after abdominal aortic aneurysm endografting: A pilot study in sheep. J Vasc Interv Radiol 2004;15:69–83. 141. Kallmes DF, Altes TA, Vincent DA, Cloft HJ, Do HM, Jensen ME. Experimental side-wall aneurysms: A natural history study. Neuroradiology 1999;41:338–341. 142. Hashimoto N, Kim C, Kikuchi H, Kojima M, Kang Y, Hazama F. Experimental induction of cerebral aneurysms in monkeys. J Neurosurg 1987;67:903–905. 143. Hashimoto N, Handa H, Nagata I, Hazama F. Experimentally induced cerebral aneurysms in rats: Part V: Relation of hemodynamics in the circle of Willis to formation of aneurysms. Surg Neurol 1980;13:41–45. 144. Moriwaki T, Takagi Y, Sadamasa N, Aoki T, Nozaki K, Hashimoto N. Impaired progression of cerebral aneurysms in interleukin-1-Bdeficient mice. Stroke 2006;37:900–905. 145. Nagata I, Handa H, Hashimoto N, Hazama F. Experimentally induced cerebral aneurysms in rats: Part VI. Hypertension. Surg Neurol 1980;14:477–479. 146. German W, Black S. Experimental production of carotid aneurysms. N Engl J Med 1954;250:104–106.

384

SECTION V / MODELS OF BEHAVIOR

147. Stehbens WE. Experimental production of aneurysms by microvascular surgery in rabbits. Vasc Surg 1973;7:165–175. 148. Strother CM, Graves VB, Rappe A. Aneurysm hemodynamics: An experimental study. Am J Neuroradiol 1992;13:1089–1095. 149. Wakhloo AK, Schellhammer F, de Vries J, Haberstroh J, Schumacher M. Self-expanding and balloon-expandable stents in the treatment of carotid aneurysms: An experimental study in a canine model. Am J Neuroradiol 1994;5:493–502. 150. Abruzzo T, Shengelaia R, Dawson RC, Owens DS, Cawley CM, Gravanis MB. Histologic and morphologic comparison of experimental aneurysms with human intracranial aneurysms. Am J Neuroradiol 1998;19:1309–1314. 151. Miskolczi L, Guterman LR, Flaherty JD, Szikora I, Hopkins LN. Rapid saccular aneurysm induction by elastase application in vitro. Neurosurgery 1997;41:220–229. 152. Stehbens WE. Chronic changes in experimental saccular and fusiform aneurysms in rabbits. Arch Pathol Lab Med 1981;105: 603–607 153. Cloft HJ, Altes TA, Marx WF, Raible RJ, Hudson SB, Helm GA, Mandell JW, Jensen ME, Dion JE, Kallmes DF. Endovascular creation of an in vivo bifurcation aneurysm model in rabbits. Radiology 1999;213:223–238.

154. Crawley CM, Dawson RC, Shengelaia G, Bonner G, Barrow DL, Colohan AR. Arterial saccular aneurysm model in the rabbit. Am J Neuroradiol 1996;17:1761–1766. 155. Fujiwara NH, Cloft HJ, Marx WF, Short JG, Jensen ME, Kallmes DF. Serial angiography in an elastase-induced aneurysm model in rabbits: Evidence for progressive aneurysm enlargement after creation. Am J Neuroradiol 2001;22:698–703. 156. Marx WF, Cloft HJ, Helm GA, Short JG, Do HM, Jensen ME, Kallmes DF. Endovascular treatment of experimental aneurysms by use of biologically modified embolic devices: Coil-mediated intraaneurysmal delivery of fibroblast tissue allografts. Am J Neuroradiol 2001;22:323–333. 157. Miskolczi L, Guterman LR, Flaherty JD, Szikora I, Hopkins LN. Rapid saccular aneurysm induction by elastase application in vitro. Neurosurgery 1997;41:220–229. 158. Miskolczi L, Guterman LR, Flaherty JD, Hopkins LN. Saccular aneurysm induction by elastase digestion of the arterial wall: A new animal model. Neurosurgery 1998;43:595–600. 159. Onizuka M, Miskolczi L, Gounis MJ, Seong J, Lieber BB, Wakhloo AK. Elastase-induced aneurysms in rabbits—effect of postconstruction geometry on final size. Am J Neuroradiol 2006;27: 1129–1131.

41

Transgenic Mouse Models of HIV-1/AIDS and Cardiac Performance JAMES J. KOHLER AND WILLIAM LEWIS

ABSTRACT

focuses on mechanisms of CM in AIDS through the use of rodent and transgenic murine (TG) models.

Cardiomyopathy (CM) associated with HIV/AIDS is important clinically and increasingly may impact the epidemic of AIDS as survival with HIV/AIDS increases due to effective antiretroviral therapies. Animal models offer sophisticated in vivo biological systems to recapitulate many direct and indirect effects of HIV-1 infection or the effects of therapeutics on the host. Few small animal models are available to investigate cardiovascular parameters associated with HIV/AIDS or highly active antiretroviral therapy (HAART). Unlike nonhuman primates, murine models of AIDS offer a safe, cost-effective approach to evaluate cardiac structure and function in AIDS CM. Experimentally, the mouse serves as perhaps the foremost laboratory mammal for genetic and immunological research. Several models utilize the wellestablished approach of transgenesis for general expression or specific, cardiac-targeted expression of viral genes. Gene targeting to generate gain or loss of function mutations in the mouse has yielded remarkable advances in understanding the roles played by specific gene products. This chapter focuses on mechanisms of CM in AIDS through the use of transgenic murine (TG) models as an important biological tool to study the impact of HIV-1, its gene products, and antiretroviral therapy on the pathophysiology of CM. Key Words: Transgenic mouse, HIV/AIDS, Cardiomyopathy, Antiretrovirals, Mitochondria.

NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITOR–HIGHLY ACTIVE ANTIRETROVIRAL THERAPY TOXICITY AND SMALL RODENT MODEL Nucleoside analog reverse transcriptase inhibitors (NRTI) are a class of antiretrovirals commonly used as part of the combination known as highly active antiretroviral therapy (HAART). Compelling experimental and clinical evidence suggests that combined HAART is a formidable way to combat HIV-1 infection and to treat AIDS.3,4 Despite the benefits of HAART, adverse effects associated with antiretroviral therapies include clinical symptoms such as myopathy, CM, polyneuropathy, lactic acidosis, steatosis, pancreatitis, and lipodystrophy.5–9 The central pathogenetic mechanism in these complications is hypothesized to relate to mitochondrial dysfunction with morphologically damaged mitochondria, decreased mitochondrial DNA (mtDNA), and depletion of mitochondrial-encoded enzymes.6,10,11 Of note, these are all findings in clinical mitochondrial genetic diseases.12,13 A supply of nucleotides and balanced nucleotide pools is required for mtDNA replication.14–27 Even in postmitotic cells, mtDNA replication is continuous.28 Phosphorylated nucleotides within mitochondria comprise a distinct pool.28,29 The clinical impact of NRTI therapy on this pool is largely unknown, despite extensive therapeutic use of NRTIs. Some NRTIs exhibit cardiac toxicity, and may be etiologically linked to AIDS CM, the latter of which is a focus of our laboratory studies. Our group has identified deleterious cardiac effects from NRTIs (including zidovudine, fialuridine, stavudine, and others; see Table 41–1), which cause cardiac mitochondrial dysfunction and related effects in other tissues.10,11,20,30–35 Because we focus sharply on NRTI toxicity in the heart, an overview of mechanisms of NRTI toxicity is presented here. A number of mechanisms may underlie NRTI mitochondrial toxicity. mtDNA polymerase-gamma (pol-γ), for example, is the eukaryotic mtDNA replication enzyme and is thought to play a key role in NRTI MT.36 “The DNA pol-γ hypothesis” was proposed over a decade ago.5 It postulates that inhibition of pol-γ leads to the depletion of mtDNA and thereby causes mitochondrial dysfunction. Specifically, possible mechanisms of mitochondrial toxicity include direct inhibition of mtDNA pol-γ without

INTRODUCTION Few small animal models are available to investigate cardiovascular parameters associated with HIV/AIDS. Animal models ideally completely recapitulate HIV-1 infection, its systemic or cardiovascular effects, or the effects of therapeutics. The use of nonhuman primates, for example, offers a model that closely reflects many virological and immunological effects of human HIV-1 but with limited availability, high cost, and intrinsic complexity that make cardiac studies in this model more difficult.1,2 Murine models of AIDS, on the other hand, offer a safe, costeffective approach to the evaluation of cardiac structure and function in AIDS cardiomyopathy (CM), particularly if they capture features of the disease and/or its therapy. Therefore, this chapter

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

385

386

SECTION V / MODELS OF BEHAVIOR

Table 41–1 Nucleoside Reverse Transcriptase Inhibitors with mitochondrial toxicity Zidovudine (AZT)14,15 Tenofovir (TDF)16,17 Zalcitabine (ddC)18 Fialuridine (FIAU)19–21 Stavudine (d4T)22,23 Lodenosine (FddA)24 Adefovir (Adf )26,27

NRTI incorporation, chain termination by incorporation of NRTI triphosphate into mtDNA by DNA pol-γ, persistence of incorporated analogs in mtDNA because of inefficient excision by DNA pol-γ and resultant defective template mtDNA, or a combination of these mechanisms.30 Additionally, the hypothesis addresses the impact of NRTIs on pools of native nucleotides in mitochondria. With respect to intramitochondrial nucleotide pools, four potential pathways exist from which phosphorylated nucleotides arise within mitochondria. First, phosphorylated nucleotides could be synthesized in the cytosol and taken up by specific transport systems.37 Second, cytosolic phosphorylated nucleotides could be dephosphorylated and then the corresponding deoxyribonucleoside diphosphates (dNDPs) taken up by the deoxynucleotide transporter (DNC)38 and converted to triphosphorylated nucleotides intramitochondrially. Third, ribonucleoside diphosphates could be imported by DNC, then reduced within the mitochondrion by mitochondrial ribonucleotide reductase,39 but evidence to support the latter moiety is sketchy. Last, mitochondria could directly import deoxyribonucleosides using the equilibrative nucleoside transporter (ENT1),40–43 although evidence is emerging for speciesrelated differences.44

USE OF SPECIFIC MARKERS IN GENETICALLY MANIPULATED MOUSE Experimentally, the mouse serves as perhaps the foremost laboratory mammal for genetic and immunological research.45,46 Biochemical screening of genetically manipulated mice offers the distinct advantage of a scientifically panoramic, integrative view of the general health of the living, genetically manipulated mouse while focusing on a parametric evaluation of cardiovascular dysfunction. The approach offers the advantages of broad screening for a variety of analytes, both murine general health and disease (cardiac)-related screening. Analogous screenings are routinely performed for human patients for the presence of health and disease, and serve as important tools for the presence of cardiovascular diseases, their prognosis, and efficacy of therapy. It follows that use of mouse models to elucidate mechanisms of AIDS CM in humans includes such screening methods and data analysis of markers in mice. These screening tests offer the investigator an opportunity to explore pathophysiological events in the cardiovascular system while maintaining its integrity in vivo in a mammalian system. Rapid nonlethal analyses are performed while simultaneous examination of the general well being of the genetically manipulated mouse is monitored. It should be understood that such analyses would likely not fall under routine moni-

toring procedures and animal care, and specialized breeding, husbandry, and care of the mice are required. With respect to heart failure studies, one clinically relevant approach is to evaluate the presence of heart failure through the screening of plasma or serum analyses and immunoassay. This approach offers the advantage of defining illness early in the genetically manipulated mouse where sequential blood samples may be obtained. Cytokines, in particular, can be measured. Circulating or locally acting cytokines including endothelin (ET-1) and tumor necrosis factor-α (TNF-α) play key roles in cardiac dysfunction, cardiomyopathy, myocarditis, and other cardiovascular diseases.47–50 TNF-α, interleukin (IL)-1β, and interferon (IFN)-γ can lead to increased levels of inducible nitric oxide syntheses (iNOS), increased nitric oxide (NO) synthesis, and myocyte death.51 Cytokines do not cause myocyte dysfunction or necrosis directly. However, cytokines may alter myocyte function, through the β1-adrenoceptor-G-protein-adenylyl cyclase axis.52–54 From in vitro studies, long-term treatment of cardiomyocytes with immune cell supernatants containing IL-1 and TNF-α reduced contractility and cyclic adenosine monophosphate (cAMP) accumulation by inhibition of β-adrenergic responsiveness.55 Furthermore, TNF-α depressant effects on cardiomyocytes have been demonstrated in culture and lead to left ventricle (LV) dysfunction in vivo.56,57 Plasma ET-1 concentrations are increased in patients with cardiac heart failure (CHF)58–60 and suggest poor prognosis in patients with New York Heart Association (NYHA) class III or IV CHF. ET receptors are expressed on several cell types in the myocardium, including myocytes, fibroblasts, and endothelial cells. Stimulation with the ET-1 ligand, endothelin, stimulates myocyte hypertrophy61,62 and impacts the synthesis and degradation of the extracellular matrix.63 The progressive nature of cardiac dysfunction encompasses a complex of molecular and cellular events termed “remodeling” that leads to changes in the structure, function, and phenotype of myocardium.64 These changes include hypertrophy and death of myocytes, regression to a molecular phenotype characterized by the expression of fetal genes and proteins, and alterations in the quantity and composition of the extracellular matrix. NF-κB substantially increases the progression of cardiac remodeling.65 LV expression of atrial natriuretic factor (ANF), a fetal gene product, is a marker of LV hypertrophy (LVH) and cardiac dysfunction.66 Thus, ANF serum and/or plasma levels offer affective markers for consideration. ANF is a 28-amino-acid peptide that is normally synthesized in the atria and to a lesser extent in the ventricles and is released into the circulation during atrial distention.67 In patients with CHF, plasma ANF concentrations rise as atrial pressure increase. Increased secretion of atrial natriuretic peptide in early CHF may attenuate or delay systemic and renal arterial vasoconstriction, venoconstriction with increased cardiac preload, and renal sodium retention. In animals with CHF, administration of antibodies to ANF peptide or antagonists to ANF receptors decreases renal blood flow; increases right atrial pressure, plasma rennin activity, and sodium retention; and worsens diastolic dysfunction by impairing myocardial relaxation.68 Plasma ANF and brain natriuretic peptide (BNP) levels increase in patients with heart failure with progression of clinical symptoms.69,70 Therefore, monitoring plasma BNP may be useful as a noninvasive biomarker for identifying possible cardiac dysfunction. Echocardiography (ECHO) is a noninvasive, highly accurate

CHAPTER 41 / TRANSGENIC MOUSE MODELS OF HIV-1/AIDS AND CARDIAC PERFORMANCE

and quick assessment of the overall health of the heart through generation of a two-dimensional ultrasound image of the heart, providing a quick assessment of the heart valves and degree of heart muscle contraction. Together with echocardiography, the routine assay of plasma BNP offers important markers to monitor cardiac function in mouse models. Increased local and circulating concentrations of norepinephrine (NE) may also contribute to myocyte hypertrophy, but are difficult to measure in the mouse. Their action can occur directly through stimulation of α1- and β-adrenergic receptors or secondarily by activating renin–angiotensin–aldosterone.71,72 NE’s toxicity to myocardial cells is mediated through calcium overload and/or the induction of apoptosis.48 Blocking β-adrenergic receptors can prevent NE-induced myocyte death. Patients with plasma NE concentrations >800 pg/ml (4.7 nmol/liter) have a 1 year survival rate of less than 40%. Thus, a simple method of screening of plasma NE levels may be effective for identification of CHF in mice, but has not been widely used. As mentioned, renin–angiotensin–aldosterone system activity is also increased in CHF. Together with plasma NE and ANF, measurement of plasma renin activity provides a prognostic index in patients.73 If such measurements were possible in microvolume samples, this could prove a useful screen of cardiovascular disease in mice.

TRANSGENIC MURINE STRATEGIES GENERAL EXPRESSION TGs are well-established, efficient models of living systems that are useful to evaluate viral gene function and pathogenesis in AIDS and other viral illnesses.74–76 Transgenesis, particularly in the models with HIV, is predicated on the cellular incorporation of viral sequences into genetic material. This technique eliminates possible problems associated with viral administration since the viral sequences are contained within every target cell. The extensive use of TG in cardiovascular research and HIV/AIDS is established.46,77 The power of transgenesis provides a unique experimental system to dissect complex aspects of AIDS CM. For the purposes of studying HIV-1 effects in the heart, the challenge has been to develop a TG harboring a defective provirus that results in expression in the heart. An early model developed by Iwakura et al.,78 while expressed primarily in the eye, had no viral mRNA detected in the heart, kidney, liver, or bone marrow. Alternatively, a hemizygous NL4-3∆ gag/pol TG mouse, which contains an internal deletion that eliminates most of the gag/pol coding sequence, was developed,79 which has become an important generalized model of AIDS that can be used to evaluate CM in AIDS. No founder mice carrying the transgene developed disease during their life span. Three founders (Tg22, Tg25, and Tg26) produced progeny that developed renal disease, and one founder (Tg26) developed myopathy/myositis.80 Variable tissue distribution and level of viral transgene expression occurred among the three lines. Three mRNA species were detected (fulllength, single and double spliced messages, respectively) with widespread expression but varied tissue distribution (high levels in skin and muscle, followed by moderate levels in thymus, gastrointestinal tract, kidney, eye, brain and spleen, but low levels in heart). More importantly, NL4-3∆ gag/pol TG demonstrate key features of HIV/AIDS including AIDS nephropathy, wasting, and skin diseases that phenotypically resemble clinical counterparts in AIDS. Specific viral protein expression patterns, as determined

387

by immunoblotting, were tissue specific. Altered Rev functions in different tissues may explain the apparent disparate protein expression patterns. GENE-SPECIFIC EXPRESSION TGs expressing HIV Tat have also been generated.81,82 In this model, a single gene from HIV (Tat) is driven by the viral long terminal repeat (LTR) and by the β-actin promoter. Tat TG mice develop hepatocellular carcinomas, endothelial proliferation, and skin lesions that resemble Kaposi’s sarcoma.83–85 Some Tat TG lines appear to potentiate zidovudine (AZT)-induced toxicity and oxidative damage.86 Other Tat-TG murine models have demonstrated a molecular mechanism of reduced glutathione levels and induction of B-lymphomas.87,88 Another replication-defective HIV-1 TG created by Gilbert Jay is based on a construct with a gag/pol/env deletion. Viral mRNA expression occurred in all tissues with a perivascular inflammation as a prominent phenotypic feature.77 These TGs had minimal cardiac physiological changes, compared to NL4-3∆ gag/pol TG, which have striking defects in contractility and relaxation.33 Lastly, a series of HIV-1 TG lines was generated by Paul Jolicoeur’s group that expresses coding sequences of HIV-1 regulated by the CD4 gene.89–92 Thus, expression of HIV-1 genes occurs in the same subset of immune cells that is infected by HIV-1 (CD4+CD8+ immature thymic T cells, circulating CD4+CD8T cells, and CD4+ monocytes/macrophages). Interestingly, these TG mice spontaneously develop severe cardiac inflammatory lesions. Thus, this TG line may constitute an animal model of inflammatory cardiomyopathy, a subset of the clinically relevant heart diseases found in human AIDS. CARDIAC-SPECIFIC TARGETING OF INDIVIDUAL GENES Targeting transgenic expression exclusively to a specific tissue target is a more focused, organ-relevant approach. Although plausible, a complete infectious HIV-1 genome is technically possible, but problematic. Such a system employing an intact human provirus in rodents would warrant serious concerns for laboratory safety. Mice expressing intact HIV-1 may constitute animal reservoirs for virus and potentially be infectious to humans.93 The fundamental approach taken to develop TG CM models is based on the fact that a specific protein relates to cardiac function and dysfunction.94 Modification of the cardiac protein repertoire in a defined manner through stable transgenesis facilitates expression of engineered proteins in the heart. The most widely used and successful promoter candidate for these purposes is the α-myosin heavy chain promoter (α-MyHC), although it is not the only approach available. This promoter meets the criteria of the key features desired, including a high degree of cardiac specificity, high levels of expression in adult atria and ventricles, the presence of sequences necessary for copy-number dependent and position-independent expression, and homogeneous expression across the cardiac myocyte population. Then, cardiac contractility and structure can be examined exclusively.94 We have utilized Robbins’ approach95 to achieve cardiac targeted transgenesis. In principle, key features included selection of an ideal promoter for cardiac expression that (1) would elicit a high level of cardiacspecific gene expression at appropriate times of development with a minimum of position-sensitive effects of chromosomal context and (2) would be copy number dependent and (3) position independent.

388

SECTION V / MODELS OF BEHAVIOR

Table 41–2 HIV-1 viral proteins used in transgenics (in the Lewis laboratory) Protein

Function

Tat Nef Vpr Vpu

Transactivation98–101 Pleiotropic, can increase or decrease virus102,103 Helps in virus replication104,105 Helps in virus release; disrupts gp160–CD4 complexes; expression regulated by Vpr106

Applying this TG approach, we targeted selected HIV-1 transgenes to cardiac ventricular myocytes in mice. Targeting singlegene TG with cardiac myocyte-specific expression of individual HIV-1 genes has proven to be a unique and powerful model system to define structural and functional effects of HIV-1 gene products on cardiac performance and myocyte structure. Granted the disadvantages of the single-gene-targeted transgenesis may be that the interaction of multiple viral proteins may be required to induce the observed clinical pathological or physiological (phenotypic) changes. Moreover, isolation of a gene from its genomic context may alter or eliminate critical cis-acting control elements required for effective, native gene expression. Despite some potential limitations of single-gene-targeted transgenesis, we successfully generated several TG lines expressing HIV-1 gene products using this cardiac-targeted approach (see Table 41–2). TG AIDS lines that express HIV-1 Tat in ventricular cardiac myocytes with different levels of Tat expression were created. Progeny of founders demonstrate exclusive expression of HIV-1 Tat mRNA in cardiac myocytes. Hemizygotes demonstrate left ventricle hypertrophy pathologically, and ultrastructural defects in mitochondrial cristae with enlarged, abnormal mitochondria.96 Six TG lines targeting HIV-1 viral protein R (Vpr) to cardiomyocytes have been developed; each develops congestive heart failure (∼8 weeks of age), abnormal cardiomyocyte nuclei, and mitoses.97 In addition, Vpr TGs have induced four-chamber dilation, defective contraction, and atrial masses. We developed TG lines each expressing HIV-1 Nef exclusively targeted in ventricular cardiac myocytes. Phenotypic analysis of mice up to 12 months of age reveals no gross pathological changes in hemizygous Nef TG.98–106 ORGANELLE-SPECIFIC TARGETING As mentioned above, because our focus in AIDS has been to examine the effects of antiretroviral agents on mtDNA replication, other models of mitochondrial biogenesis may be important tools. A number of other transgenic and knockout mouse models have been examined to delineate the role of specific, related signaling pathways on cardiac performance and CM.107 Sah et al.108 generated transgenic mice in which constitutively activated RhoA or wild-type (WT) RhoA were expressed in a cardiac-specific manner through the α-MHC promoter to determine the role of Rho family GTPases on cardiac performance. Results demonstrated evidence of heart failure seen in RhoA transgenics, with severe edema, ventricular chamber dilation, increased cardiac fibrosis, atrial enlargement, and decreased fractional shortening. Unlike Rho, Rac1 proved to be more challenging by use of conventional gene targeting approaches as deletion results in embryonic lethality.109 Instead, genetic manipulation of Rac1 to study the consequences of

increased myocardial Rac1 activity was accomplished by cardiacspecific transgenesis with an unusual dichotomy: (1) rapid-onset, high-level postnatal expression that led to lethal dilated CM or (2) slow-onset postnatal expression leading to transient hypertrophy in juvenile mice that resolved with age.110 Transgenic mice have also been created to investigate the role of cardiac regulatory proteins, protein kinase C (PKC) and troponin I (cTNI).111 Pioneering work in the laboratory of Chris Vlahos112 has shown that PKC is upregulated in the human heart in end-stage failure. To demonstrate causality of PKC in heart failure, transgenic mice have been created in which the PKC isoform is overexpressed in the heart, often in a constitutively active form.113–116 Collectively, studies with these TG show that chronic PKC overexpression can lead to features of cardiac failure including hypertrophy, reexpression of fetal genes, wall thinning, and left ventricular dysfunction. Mouse transgenic models also may alter expression of genes involved in cardiac uptake and metabolism of either lipid or carbohydrate.117 Disruption of the insulin-sensitive GLUT4, for example, by various transgenic techniques results in cardiac hypertrophy.118,119 Studies by Yang et al. utilized TG murine models to investigate the role of myosin-binding protein C and molecular regulation of lipotoxicity in the heart.120,121 Results from these and many other TG models demonstrate that alterations in cardiac uptake and metabolism may precede both the functional and morphological alterations that accompany diabetic cardiomyopathy and heart failure. Understanding the critical role of NO signaling in myocardial function has also advanced with targeted overexpression of endothelial NOS (NOS3).122 In the hearts of three lines of mice carrying the αMHC-NOS3 TG, pressure development of the LV was inversely correlated with NOS3 transgene expression.123,124 On the other hand, the contractile response to norepinephrine and acetylcholine was not affected by TG expression. Janssens et al.125 and Champion et al.126 confirmed overexpressed NOS3 localized to caveolae by confocal microscopy and immunoprecipitation studies. In short, the availability of mice in which NOS3 is overexpressed has enabled investigators to characterize the function of this enzyme in mouse models of cardiovascular disease. CONDITIONAL EXPRESSION Gene targeting to generate gain or loss of function mutations in the mouse has yielded remarkable advances in understanding the roles played by specific gene products.127 Not all biological processes, however, can be accessed and studied by gene-inactivation or transgeneexpression strategies. Insights have been gained into both embryogenesis and later gene function; however obstacles exist that include indirect systemic defects that can complicate transgenic constructs in the germ line to elicit the role of genes in a specific pathophysiological context. For example, many genes have several roles during embryogenesis and adulthood for which their ablation results in an embryonic-lethal phenotype, precluding an analysis of its function in later development. To circumvent these limitations, mouse geneticists developed strategies that allow genes to be activated or silenced where and when the investigator chooses. The ideal genetic “switch” results in low or zero basal gene expression when “off” and high levels of gene expression when “on.” Furthermore, the switch ideally is reversible and specific for the target gene, and should not interfere with other cellular components or with general metabolism. Early attempts included use

CHAPTER 41 / TRANSGENIC MOUSE MODELS OF HIV-1/AIDS AND CARDIAC PERFORMANCE

of promoters that could be induced by heavy metals, heat shock, interferon, or steroids. Unfortunately, these strategies fell short due to high basal activity in the absence of induction.128 More recently, success was reached with binary transgenic systems. Gene expression is controlled by the interaction of two components: an “effector” transgene and a “target” transgene.45 The most widely used binary transcription transactivation systems are the tetracycline-dependent regulatory systems developed by Mandfred Gossen and Hermann Bujard.129 The effector is a fusion of sequences that encodes the VP16 transactivation domain and the Escherichia coli tetracycline repressor (TetR) protein, which specifically binds both tetracycline and the 19-bp operator sequences (tetO) of the tet operon in the target transgene, resulting in transcription. Two versions exist: (1) the tetracycline controlled transactivator (tTA) protein cannot bind DNA when the inducer is present (“tet-off”), and (2) the “reverse tTA” (rtTA) binds DNA only when the inducer is present (“tet-on”). Conditional expression with this tetO promoter system was used in a recent study to induce cardiac fibrosis and heart failure using an antisense mRNA of mineralocorticoid receptor targeted in cardiomyocytes.130 The first nonviral binary system to be used in mice was the Gal4/upstream activator sequence (UAS) system. Gal4, an isolated transcriptional activator of Saccharomyces cerevisiae, directs the transcription of Gal4-responsive genes by binding to (UASs). Two members of the integrase family of site-specific recombinases, Cre from bacteriophage P1131 and Flp from S. cerevisiae132 are currently used to conditionally control gene expression by site-specific DNA recombination. Somatic, tissue-specific recombination has gained appeal more recently. One strategy for this exploits the cre-loxP system derived from bacteriophage P1.133,134 The 38-kDa Cre protein functions as a site-specific recombinase, splicing DNA between specific 34-bp sequences known as loxP sites. Mating mice that contain a loxPflanked gene with mice expressing Cre under the control of a cell specific-promoter result in Cre-mediated excision selectively in targeted cells. The cre–loxP system is entirely contingent on the exogenous recombines, so that recombination can be regulated via the timing and tissue specificity of Cre transgene expression.135,136 A strategy has been generated for cell type-specific plus temporal control by tissue-restricted expression of a conditionally functional chimeric Cre protein, comprising a fusion between Cre and the mutated ligand binding domain (LBD) of a steroid hormone receptor.135–140 These chimeric proteins become selectively active on binding a synthetic ligand, in preference to endogenous progesterone and estrogen, respectively.

PHARMACOLOGICAL MANIPULATION OF AIDS TRANSGENIC MURINE MODELS We extensively used the NL4-3∆ gag/pol TG and the WT littermates (FVB/n mice) in both AZT and d4T monotherapy and combination therapy studies. TG and WT received water ad libitum with and without AZT, for example, for 21 or 35 days. After 21 days, molecular indicators of cardiac dysfunction were identified. Abundances of mRNA for cardiac sarcoplasmic reticulum calcium ATPase (SERCA2), sodium calcium exchanger (NCX1), and ANF were determined individually. Depressed SERCA2 and increased ANF mRNA abundance were found in LV from AZT-treated TG. NCX1 abundance remained unchanged.

389

Eccentric LV hypertrophy was determined echocardiographically. After 35 days, cardiac dysfunction was worse in TG, with or without AZT treatment. Decreases in the first derivative of the maximal change in LV systolic pressure with respect to time (+dP/dt) occurred at baseline in TG. The increased half-time of relaxation and ventricular relaxation (−dP/dt) occurred in TG as well, independent of treatment. However, increased time to peak pressure was found only in AZT-treated TG. Histopathological and ultrastructural (transmission electron microscopy) changes were also identified. Ultrastructurally, mitochondrial destruction was most pronounced in AZT-treated TG, but was also found in AZT treated WT. In short, TG mice that express HIV-1 demonstrate cardiac dysfunction. AZT-treatment of WT causes mitochondrial ultrastructural alterations and −dP/dt changes. Therefore, data collectively suggest that HIV-1 and AZT each contributes to cardiac dysfunction in this transgenic model of AIDS CM.33 Additional studies have been performed using a combination HAART [AZT, lamivudine (3TC), and indinavir] or vehicle control treatment for 10 or 35 days in NL4-3∆ gag/pol TG and FVB/n WT mice. This “2 × 2” protocol provides a systematic determination of the effect(s) of TG and/or treatment. At the termination of the experiments (day 35), mice underwent echocardiography, quantitation of ANF, SERCA2, and determination of plasma lactate.141 Myocardial histological features were also analyzed semiquantitatively and results were confirmed by transmission electron microscopy. Echocardiographic analysis revealed a 160% increase in LV mass in the TG + HAART cohort. Molecularly, ANF mRNA increased 250% and SERCA2 mRNA decreased 57%. Biochemically, plasma lactate was elevated (8.5 ± 2.0 mM). Pathologically, granular cytoplasmic changes were found in cardiac myocytes indicating enlarged, damaged mitochondria. Findings were confirmed ultrastructurally. Surprisingly, no changes occurred in the other cohorts. Cumulative HAART causes mitochondrial CM in AIDS TG mice.

GENERAL HEALTH OF THE GENETICALLY MANIPULATED MOUSE Mouse genetic research has provided an important biological tool resulting in remarkable advances in the understanding of disease, pathogenesis, and treatment strategies. Important concerns remain, however, regarding the specificity of genetic manipulations of the cardiovascular system and other physiological systems. Concerns that the resultant changes yield an authentic model of human disease and that the degree of accurate phenotypic characteristics of the disease the genetic manipulation displays have been raised by clinicians and researchers alike. More importantly is the concern that nontargeted organ systems remain intact and functional. This is the rationale for a complete evaluation for general health parameters in genetically manipulated mice. Establishing a mouse “strain-specific baseline” for routine parameters is the key to identifying and ensuring that phenotypic characteristics of the particular strain remain, overall, intact when evaluating the phenotypic changes that occur as a result of a particular genetic manipulation. Phenotypic parameters of size, weight-to-age ratios, for example, can offer basic, observational, noninvasive assessments when comparing a native (WT) strain to a genetically manipulated mouse line. Overall appetite, feeding and activity patterns can also serve as observational comparisons.

390

SECTION V / MODELS OF BEHAVIOR

Table 41–3 Relevant murine cardiac analytesa 9.

Atrial natriuretic peptide (ANF)142 Brain natriuretic peptide (BNP)143 Troponin I144 Troponin T145 Lactic acid146 Lactate dehydrogenase (LDH)147 Creatinine kinase148 a All of the above assays are based on enzymatic colorimetric or immunoturbidometric assays and have been tested for feasibility.

Commercially available assays exist to address the general health of domestic animals. A slight modification of these assays could prove specifically useful in the mouse model, for various strains. Blood sampling, for example, is ideal for repetitive sampling as a function of age and disease development or in response to treatment of disease while preserving the living organism. Previous experience with obtaining blood from FVB/n and our own genetically manipulated mice indicates that retroorbital bleeding was rapid, safe, and effective. Collection of 200 µl of blood into heparinized capillary tubes yields approximately 100– 120 µl of plasma. From this total volume, ∼90 µl is usable for the analyte panel and ∼10–30 µl plasma for other analyses. The panel of analytes shown in Table 41–3 may serve as a marker for cardiovascular disease to evaluate the genetically manipulated mouse and the controls.142–148

CONCLUSIONS Cardiomyopathy associated with HIV-1/AIDS is important clinically, and increasingly may impact the epidemic of AIDS as survival with HIV-1 increases due to effective antiretroviral therapies. The mechanisms of AIDS CM are not yet completely understood. TG models, however, particularly murine models, provide useful biological tools to study the impact of HIV-1, its gene products, as well as side effects of NRTI-HAART therapy on the pathophysiology and pathogenesis of CM.

REFERENCES 1. Shannon RP. SIV cardiomyopathy in non-human primates. Trends Cardiovasc Med 2001;11(6):242–246. 2. Yearley JH, Pearson C, Carville A, Shannon RP, Mansfield KG. SIV-associated myocarditis: Viral and cellular correlates of inflammation severity. AIDS Res Hum Retroviruses 2006;22(6):529– 540. 3. Hammer SM, Saag MS, Schechter M, et al. Treatment for adult HIV infection: 2006 recommendations of the International AIDS SocietyUSA panel. JAMA 2006;296(7):827–843. 4. Resino S, Resino R, Maria Bellon J, et al. Clinical outcomes improve with highly active antiretroviral therapy in vertically HIV type-1-infected children. Clin Infect Dis 2006;43(2):243–252. 5. Lewis W, Dalakas MC. Mitochondrial toxicity of antiviral drugs. Nat Med 1995;1(5):417–422. 6. Brinkman K, ter Hofstede HJ, Burger DM, Smeitink JA, Koopmans PP. Adverse effects of reverse transcriptase inhibitors: Mitochondrial toxicity as common pathway. AIDS 1998;12(14):1735–1744. 7. Moyle G. Clinical manifestations and management of antiretroviral nucleoside analog-related mitochondrial toxicity. Clin Ther 2000; 22(8):911–936; discussion 898. 8. Villarroya F, Domingo P, Giralt M. Lipodystrophy associated with highly active anti-retroviral therapy for HIV infection: The adipo-

10.

11.

12.

13.

14.

15.

16.

17. 18.

19.

20.

21.

22.

23.

24. 25.

26. 27.

28.

29.

cyte as a target of anti-retroviral-induced mitochondrial toxicity. Trends Pharmacol Sci 2005;26(2):88–93. Sharma PL, Nurpeisov V, Hernandez-Santiago B, Beltran T, Schinazi RF. Nucleoside inhibitors of human immunodeficiency virus type 1 reverse transcriptase. Curr Top Med Chem 2004;4(9): 895–919. Lewis W, Day BJ, Copeland WC. Mitochondrial toxicity of NRTI antiviral drugs: An integrated cellular perspective. Nat Rev Drug Discov 2003;2(10):812–822. Lewis W, Copeland WC, Day BJ. Mitochondrial DNA depletion, oxidative stress, and mutation: Mechanisms of dysfunction from nucleoside reverse transcriptase inhibitors. Lab Invest 2001;81(6): 777–790. Sukernik RI, Derbeneva OA, Starikovskaia EB, et al. [The mitochondrial genome and human mitochondrial diseases]. Genetika 2002;38(2):161–170. Wallace DC. The mitochondrial genome in human adaptive radiation and disease: On the road to therapeutics and performance enhancement. Gene 2005;354:169–180. Arnaudo E, Dalakas M, Shanske S, Moraes CT, DiMauro S, Schon EA. Depletion of muscle mitochondrial DNA in AIDS patients with zidovudine-induced myopathy. Lancet 1991;337(8740):508–510. Dalakas MC, Illa I, Pezeshkpour GH, Laukaitis JP, Cohen B, Griffin JL. Mitochondrial myopathy caused by long-term zidovudine therapy. N Engl J Med 1990;322(16):1098–1105. Murphy MD, O’Hearn M, Chou S. Fatal lactic acidosis and acute renal failure after addition of tenofovir to an antiretroviral regimen containing didanosine. Clin Infect Dis 2003;36(8):1082–1085. De Clercq E. New approaches toward anti-HIV chemotherapy. J Med Chem 2005;48(5):1297–1313. Dubinsky RM, Yarchoan R, Dalakas M, Broder S. Reversible axonal neuropathy from the treatment of AIDS and related disorders with 2′,3′-dideoxycytidine (ddC). Muscle Nerve 1989;12(10): 856–860. Colacino JM. Mechanisms for the anti-hepatitis B virus activity and mitochondrial toxicity of fialuridine (FIAU). Antiviral Res 1996;29(2–3):125–139. Lewis W, Griniuviene B, Tankersley KO, et al. Depletion of mitochondrial DNA, destruction of mitochondria, and accumulation of lipid droplets result from fialuridine treatment in woodchucks (Marmota monax). Lab Invest 1997;76(1):77–87. Lewis W, Meyer RR, Simpson JF, Colacino JM, Perrino FW. Mammalian DNA polymerases alpha, beta, gamma, delta, and epsilon incorporate fialuridine (FIAU) monophosphate into DNA and are inhibited competitively by FIAU triphosphate. Biochemistry 1994;33(48):14620–14624. Dalakas MC, Semino-Mora C, Leon-Monzon M. Mitochondrial alterations with mitochondrial DNA depletion in the nerves of AIDS patients with peripheral neuropathy induced by 2′3′-dideoxycytidine (ddC). Lab Invest 2001;81(11):1537–1544. Lim SE, Ponamarev MV, Longley MJ, Copeland WC. Structural determinants in human DNA polymerase gamma account for mitochondrial toxicity from nucleoside analogs. J Mol Biol 2003; 329(1):45–57. Highleyman L. Lodenosine trials stopped due to safety concerns. BETA 1999;12(4):4. Moreno A, Quereda C, Moreno L, et al. High rate of didanosinerelated mitochondrial toxicity in HIV/HCV-coinfected patients receiving ribavirin. Antiviral Ther 2004;9(1):133–138. Baker R. Kidney dysfunction: A safety update on adefovir (preveon). BETA 1998;Jul:9–10. Ho ES, Lin DC, Mendel DB, Cihlar T. Cytotoxicity of antiviral nucleotides adefovir and cidofovir is induced by the expression of human renal organic anion transporter 1. J Am Soc Nephrol 2000;11(3):383–393. Song S, Wheeler LJ, Mathews CK. Deoxyribonucleotide pool imbalance stimulates deletions in HeLa cell mitochondrial DNA. J Biol Chem 2003;278(45):43893–43896. Pontarin G, Gallinaro L, Ferraro P, Reichard P, Bianchi V. Origins of mitochondrial thymidine triphosphate: Dynamic relations to

CHAPTER 41 / TRANSGENIC MOUSE MODELS OF HIV-1/AIDS AND CARDIAC PERFORMANCE

30.

31.

32.

33.

34.

35.

36. 37.

38.

39.

40.

41.

42.

43.

44.

45. 46.

47. 48. 49.

50.

51.

cytosolic pools. Proc Natl Acad Sci USA 2003;100(21): 12159–12164. Lewis W. Mitochondrial dysfunction and nucleoside reverse transcriptase inhibitor therapy: Experimental clarifications and persistent clinical questions. Antiviral Res 2003;58(3):189–197. Lewis W. Mitochondrial DNA replication, nucleoside reversetranscriptase inhibitors, and AIDS cardiomyopathy. Prog Cardiovasc Dis 2003;45(4):305–318. Lewis W, Gonzalez B, Chomyn A, Papoian T. Zidovudine induces molecular, biochemical, and ultrastructural changes in rat skeletal muscle mitochondria. J Clin Invest 1992;89(4):1354–1360. Lewis W, Grupp IL, Grupp G, et al. Cardiac dysfunction occurs in the HIV-1 transgenic mouse treated with zidovudine. Lab Invest 2000;80(2):187–197. Lewis W, Papoian T, Gonzalez B, et al. Mitochondrial ultrastructural and molecular changes induced by zidovudine in rat hearts. Lab Invest 1991;65(2):228–236. Lewis W, Simpson JF, Meyer RR. Cardiac mitochondrial DNA polymerase-gamma is inhibited competitively and noncompetitively by phosphorylated zidovudine. Circ Res 1994;74(2):344–348. Kaguni LS. DNA polymerase gamma, the mitochondrial replicase. Annu Rev Biochem 2004;73:293–320. Bridges EG, Jiang Z, Cheng YC. Characterization of a dCTP transport activity reconstituted from human mitochondria. J Biol Chem 1999;274(8):4620–4625. Dolce V, Fiermonte G, Runswick MJ, Palmieri F, Walker JE. The human mitochondrial deoxynucleotide carrier and its role in the toxicity of nucleoside antivirals. Proc Natl Acad Sci USA 2001;98(5):2284–2288. Young P, Leeds JM, Slabaugh MB, Mathews CK. Ribonucleotide reductase: Evidence for specific association with HeLa cell mitochondria. Biochem Biophys Res Commun 1994;203(1):46–52. Chang C, Swaan PW, Ngo LY, Lum PY, Patil SD, Unadkat JD. Molecular requirements of the human nucleoside transporters hCNT1, hCNT2, and hENT1. Mol Pharmacol 2004;65(3): 558–570. Lai Y, Bakken AH, Unadkat JD. Simultaneous expression of hCNT1-CFP and hENT1-YFP in Madin-Darby canine kidney cells. Localization and vectorial transport studies. J Biol Chem 2002; 277(40):37711–37717. Lai Y, Tse CM, Unadkat JD. Mitochondrial expression of the human equilibrative nucleoside transporter 1 (hENT1) results in enhanced mitochondrial toxicity of antiviral drugs. J Biol Chem 2004;279(6):4490–4497. SenGupta DJ, Lum PY, Lai Y, et al. A single glycine mutation in the equilibrative nucleoside transporter gene, hENT1, alters nucleoside transport activity and sensitivity to nitrobenzylthioinosine. Biochemistry 2002;41(5):1512–1519. Lee EW, Lai Y, Zhang H, Unadkat JD. Identification of the mitochondrial targeting signal of the human equilibrative nucleoside transporter 1 (hENT1): Implications for interspecies differences in mitochondrial toxicity of fialuridine. J Biol Chem 2006;281(24): 16700–16706. Lewandoski M. Conditional control of gene expression in the mouse. Nat Rev Genet 2001;2(10):743–755. Klotman PE, Notkins AL. Transgenic models of human immunodeficiency virus type-1. Curr Top Microbiol Immunol 1996;206:197– 222. Bristow MR. Tumor necrosis factor-alpha and cardiomyopathy. Circulation 1998;97(14):1340–1341. Mann DL. Stress-activated cytokines and the heart: From adaptation to maladaptation. Annu Rev Physiol 2003;65:81–101. Welch S, Plank D, Witt S, et al. Cardiac-specific IGF-1 expression attenuates dilated cardiomyopathy in tropomodulin-overexpressing transgenic mice. Circ Res 2002;90(6):641–648. Dorn GW 2nd. Physiologic growth and pathologic genes in cardiac development and cardiomyopathy. Trends Cardiovasc Med 2005;15(5):185–189. Pinsky DJ, Cai B, Yang X, Rodriguez C, Sciacca RR, Cannon PJ. The lethal effects of cytokine-induced nitric oxide on cardiac

52.

53. 54. 55.

56.

57.

58. 59.

60.

61.

62. 63.

64. 65.

66. 67.

68.

69.

70.

71.

72.

391

myocytes are blocked by nitric oxide synthase antagonism or transforming growth factor beta. J Clin Invest 1995;95(2):677– 685. Levine B, Kalman J, Mayer L, Fillit HM, Packer M. Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med 1990;323(4):236–241. Barry WH. Mechanisms of immune-mediated myocyte injury. Circulation 1994;89(5):2421–2432. Lange LG, Schreiner GF. Immune mechanisms of cardiac disease. N Engl J Med 1994;330(16):1129–1135. Gulick T, Chung MK, Pieper SJ, Lange LG, Schreiner GF. Interleukin 1 and tumor necrosis factor inhibit cardiac myocyte betaadrenergic responsiveness. Proc Natl Acad Sci USA 1989;86(17): 6753–6757. Pagani FD, Baker LS, Hsi C, Knox M, Fink MP, Visner MS. Left ventricular systolic and diastolic dysfunction after infusion of tumor necrosis factor-alpha in conscious dogs. J Clin Invest 1992;90(2): 389–398. Pagani FD, Baker LS, Knox MA, Cheng H, Fink MP, Visner MS. Load-insensitive assessment of myocardial performance after tumor necrosis factor-alpha in dogs. Surgery 1992;111(6):683–693. Pacher R, Stanek B. Prostaglandin E1 in heart failure: The Vienna experience. Wien Klin Wochenschr 1996;108(16):491–495. Pacher R, Stanek B, Hulsmann M, et al. Prognostic impact of big endothelin-1 plasma concentrations compared with invasive hemodynamic evaluation in severe heart failure. J Am Coll Cardiol 1996;27(3):633–641. Cody RJ, Haas GJ, Binkley PF, Capers Q, Kelley R. Plasma endothelin correlates with the extent of pulmonary hypertension in patients with chronic congestive heart failure. Circulation 1992;85(2):504–509. Shubeita HE, McDonough PM, Harris AN, et al. Endothelin induction of inositol phospholipid hydrolysis, sarcomere assembly, and cardiac gene expression in ventricular myocytes. A paracrine mechanism for myocardial cell hypertrophy. J Biol Chem 1990;265(33): 20555–20562. Wei CM, Lerman A, Rodeheffer RJ, et al. Endothelin in human congestive heart failure. Circulation 1994;89(4):1580–1586. Weber KT. Extracellular matrix remodeling in heart failure: A role for de novo angiotensin II generation. Circulation 1997;96(11): 4065–4082. Colucci WS. Molecular and cellular mechanisms of myocardial failure. Am J Cardiol 1997;80(11A):15L–25L. Onai Y, Suzuki J, Maejima Y, et al. Inhibition of NF-{kappa}B improves left ventricular remodeling and cardiac dysfunction after myocardial infarction. Am J Physiol Heart Circ Physiol 2006;292(1): H530–538. Rosenzweig A, Seidman CE. Atrial natriuretic factor and related peptide hormones. Annu Rev Biochem 1991;60:229–255. Feringa HH, Schouten O, Dunkelgrun M, et al. Plasma N-terminal pro-B-type natriuretic peptide as long-term prognostic marker after major vascular surgery. Heart 2006;93(2):226–231. Drexler H, Hirth C, Stasch HP, Lu W, Neuser D, Just H. Vasodilatory action of endogenous atrial natriuretic factor in a rat model of chronic heart failure as determined by monoclonal ANF antibody. Circ Res 1990;66(5):1371–1380. Clerico A, Iervasi G, Del Chicca MG, et al. Circulating levels of cardiac natriuretic peptides (ANP and BNP) measured by highly sensitive and specific immunoradiometric assays in normal subjects and in patients with different degrees of heart failure. J Endocrinol Invest 1998;21(3):170–179. Date T, Shinozaki T, Yamakawa M, et al. Elevated plasma brain natriuretic peptide level in cardiac sarcoidosis patients with preserved ejection fraction. Cardiology 2006;107(4):277–280. Bristow MR, Abraham WT, Yoshikawa T, et al. Second- and thirdgeneration beta-blocking drugs in chronic heart failure. Cardiovasc Drugs Ther 1997;11(Suppl. 1):291–296. Abraham WT, Raynolds MV, Gottschall B, et al. Importance of angiotensin-converting enzyme in pulmonary hypertension. Cardiology 1995;86(Suppl. 1):9–15.

392

SECTION V / MODELS OF BEHAVIOR

73. Francis GS, Benedict C, Johnstone DE, et al. Comparison of neuroendocrine activation in patients with left ventricular dysfunction with and without congestive heart failure. A substudy of the Studies of Left Ventricular Dysfunction (SOLVD). Circulation 1990;82(5): 1724–1729. 74. Small JA, Scangos GA, Cork L, Jay G, Khoury G. The early region of human papovavirus JC induces dysmyelination in transgenic mice. Cell 1986;46(1):13–18. 75. Hinrichs SH, Nerenberg M, Reynolds RK, Khoury G, Jay G. A transgenic mouse model for human neurofibromatosis. Science 1987;237(4820):1340–1343. 76. Kim CM, Koike K, Saito I, Miyamura T, Jay G. HBx gene of hepatitis B virus induces liver cancer in transgenic mice. Nature 1991;351(6324):317–320. 77. Tinkle BT, Ngo L, Luciw PA, Maciag T, Jay G. Human immunodeficiency virus-associated vasculopathy in transgenic mice. J Virol 1997;71(6):4809–4814. 78. Iwakura Y, Shioda T, Tosu M, et al. The induction of cataracts by HIV-1 in transgenic mice. AIDS 1992;6(10):1069–1075. 79. Dickie P, Felser J, Eckhaus M, et al. HIV-associated nephropathy in transgenic mice expressing HIV-1 genes. Virology 1991;185(1): 109–119. 80. Kopp JB, Klotman ME, Adler SH, et al. Progressive glomerulosclerosis and enhanced renal accumulation of basement membrane components in mice transgenic for human immunodeficiency virus type 1 genes. Proc Natl Acad Sci USA 1992;89(5):1577–1581. 81. Cai SR, Xu G, Becker-Hapak M, Ma M, Dowdy SF, McLeod HL. The kinetics and tissue distribution of protein transduction in mice. Eur J Pharm Sci 2006;27(4):311–319. 82. Gibellini D, Vitone F, Schiavone P, Re MC. HIV-1 tat protein and cell proliferation and survival: A brief review. New Microbiol 2005;28(2):95–109. 83. Vogel J, Hinrichs SH, Napolitano LA, Ngo L, Jay G. Liver cancer in transgenic mice carrying the human immunodeficiency virus tat gene. Cancer Res 1991;51(24):6686–6690. 84. Corallini A, Altavilla G, Pozzi L, et al. Systemic expression of HIV-1 tat gene in transgenic mice induces endothelial proliferation and tumors of different histotypes. Cancer Res 1993;53(22): 5569–5575. 85. Barbanti-Brodano G, Sampaolesi R, Campioni D, et al. HIV-1 tat acts as a growth factor and induces angiogenic activity in BK virus/ tat transgenic mice. Antibiot Chemother 1994;46:88–101. 86. Prakash O, Teng S, Ali M, et al. The human immunodeficiency virus type 1 Tat protein potentiates zidovudine-induced cellular toxicity in transgenic mice. Arch Biochem Biophys 1997;343(2):173– 180. 87. Choi J, Liu RM, Kundu RK, et al. Molecular mechanism of decreased glutathione content in human immunodeficiency virus type 1 Tat-transgenic mice. J Biol Chem 2000;275(5):3693–3698. 88. Kundu RK, Sangiorgi F, Wu LY, et al. Expression of the human immunodeficiency virus-Tat gene in lymphoid tissues of transgenic mice is associated with B-cell lymphoma. Blood 1999;94(1):275– 282. 89. Hanna Z, Kay DG, Rebai N, Guimond A, Jothy S, Jolicoeur P. Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice. Cell 1998;95(2): 163–175. 90. Hanna Z, Priceputu E, Hu C, Vincent P, Jolicoeur P. HIV-1 Nef mutations abrogating downregulation of CD4 affect other Nef functions and show reduced pathogenicity in transgenic mice. Virology 2006;346(1):40–52. 91. Jolicoeur P, Kay DG, Cool M, Jothy S, Rebai N, Hanna Z. A novel mouse model of HIV-1 disease. Leukemia 1999;13(Suppl. 1): S78–80. 92. Zhong J, Zuo Y, Ma J, et al. Expression of HIV-1 genes in podocytes alone can lead to the full spectrum of HIV-1-associated nephropathy. Kidney Int 2005;68(3):1048–1060. 93. Locardi C, Puddu P, Ferrantini M, et al. Persistent infection of normal mice with human immunodeficiency virus. J Virol 1992;66(3):1649–1654.

94. Robbins J. Remodeling the cardiac sarcomere using transgenesis. Annu Rev Physiol 2000;62:261–287. 95. Subramaniam A, Jones WK, Gulick J, Wert S, Neumann J, Robbins J. Tissue-specific regulation of the alpha-myosin heavy chain gene promoter in transgenic mice. J Biol Chem 1991;266(36):24613– 24620. 96. Raidel SM, Haase C, Jansen NR, et al. Targeted myocardial transgenic expression of HIV Tat causes cardiomyopathy and mitochondrial damage. Am J Physiol Heart Circ Physiol 2002;282(5):H1672– 1678. 97. Lewis W, Miller YK, Haase CP, et al. HIV viral protein R causes atrial cardiomyocyte mitosis, mesenchymal tumor, dysrhythmia, and heart failure. Lab Invest 2005;85(2):182–192. 98. Karn J, Dingwall C, Finch JT, Heaphy S, Gait MJ. RNA binding by the tat and rev proteins of HIV-1. Biochimie 1991;73(1): 9–16. 99. Kim YS, Panganiban AT. The full-length Tat protein is required for TAR-independent, posttranscriptional trans activation of human immunodeficiency virus type 1 env gene expression. J Virol 1993;67(7):3739–3747. 100. Marciniak RA, Calnan BJ, Frankel AD, Sharp PA. HIV-1 Tat protein trans-activates transcription in vitro. Cell 1990;63(4): 791–802. 101. Vives E, Charneau P, van Rietschoten J, Rochat H, Bahraoui E. Effects of the Tat basic domain on human immunodeficiency virus type 1 transactivation, using chemically synthesized Tat protein and Tat peptides. J Virol 1994;68(5):3343–3353. 102. Aiken C, Trono D. Nef stimulates human immunodeficiency virus type 1 proviral DNA synthesis. J Virol 1995;69(8):5048–5056. 103. Romero IA, Teixeira A, Strosberg AD, Cazaubon S, Couraud PO. The HIV-1 nef protein inhibits extracellular signal-regulated kinasedependent DNA synthesis in a human astrocytic cell line. J Neurochem 1998;70(2):778–785. 104. Elder RT, Benko Z, Zhao Y. HIV-1 VPR modulates cell cycle G2/M transition through an alternative cellular mechanism other than the classic mitotic checkpoints. Front Biosci 2002;7:d349–357. 105. Subbramanian RA, Kessous-Elbaz A, Lodge R, et al. Human immunodeficiency virus type 1 Vpr is a positive regulator of viral transcription and infectivity in primary human macrophages. J Exp Med 1998;187(7):1103–1111. 106. Willey RL, Maldarelli F, Martin MA, Strebel K. Human immunodeficiency virus type 1 Vpu protein regulates the formation of intracellular gp160-CD4 complexes. J Virol 1992;66(1):226–234. 107. Brown JH, Del Re DP, Sussman MA. The Rac and Rho hall of fame: A decade of hypertrophic signaling hits. Circ Res 2006;98(6): 730–742. 108. Sah VP, Minamisawa S, Tam SP, et al. Cardiac-specific overexpression of RhoA results in sinus and atrioventricular nodal dysfunction and contractile failure. J Clin Invest 1999;103(12):1627–1634. 109. Sugihara K, Nakatsuji N, Nakamura K, et al. Rac1 is required for the formation of three germ layers during gastrulation. Oncogene 1998;17(26):3427–3433. 110. Sussman MA, Welch S, Walker A, et al. Altered focal adhesion regulation correlates with cardiomyopathy in mice expressing constitutively active rac1. J Clin Invest 2000;105(7):875–886. 111. Walker JW. Protein kinase C, troponin I and heart failure: Overexpressed, hyperphosphorylated and underappreciated? J Mol Cell Cardiol 2006;40(4):446–450. 112. Vlahos CJ, McDowell SA, Clerk A. Kinases as therapeutic targets for heart failure. Nat Rev Drug Discov 2003;2(2):99–113. 113. Goldspink PH, Montgomery DE, Walker LA, et al. Protein kinase Cepsilon overexpression alters myofilament properties and composition during the progression of heart failure. Circ Res 2004;95(4):424–432. 114. Huang L, Wolska BM, Montgomery DE, Burkart EM, Buttrick PM, Solaro RJ. Increased contractility and altered Ca(2+) transients of mouse heart myocytes conditionally expressing PKCbeta. Am J Physiol Cell Physiol 2001;280(5):C1114–1120. 115. Takeishi Y, Chu G, Kirkpatrick DM, et al. In vivo phosphorylation of cardiac troponin I by protein kinase Cbeta2 decreases cardiomyo-

CHAPTER 41 / TRANSGENIC MOUSE MODELS OF HIV-1/AIDS AND CARDIAC PERFORMANCE

116.

117.

118.

119.

120.

121.

122.

123.

124.

125.

126.

127. 128. 129.

130.

131.

132.

cyte calcium responsiveness and contractility in transgenic mouse hearts. J Clin Invest 1998;102(1):72–78. Wakasaki H, Koya D, Schoen FJ, et al. Targeted overexpression of protein kinase C beta2 isoform in myocardium causes cardiomyopathy. Proc Natl Acad Sci USA 1997;94(17):9320–9325. Hartil K, Charron MJ. Genetic modification of the heart: Transgenic modification of cardiac lipid and carbohydrate utilization. J Mol Cell Cardiol 2005;39(4):581–593. Abel ED, Kaulbach HC, Tian R, et al. Cardiac hypertrophy with preserved contractile function after selective deletion of GLUT4 from the heart. J Clin Invest 1999;104(12):1703–1714. Katz EB, Stenbit AE, Hatton K, DePinho R, Charron MJ. Cardiac and adipose tissue abnormalities but not diabetes in mice deficient in GLUT4. Nature 1995;377(6545):151–155. Yang Q, Sanbe A, Osinska H, Hewett TE, Klevitsky R, Robbins J. A mouse model of myosin binding protein C human familial hypertrophic cardiomyopathy. J Clin Invest 1998;102(7):1292–1300. Yang Q, Hewett TE, Klevitsky R, Sanbe A, Wang X, Robbins J. PKA-dependent phosphorylation of cardiac myosin binding protein C in transgenic mice. Cardiovasc Res 2001;51(1):80–88. Bloch KD, Janssens S. Cardiomyocyte-specific overexpression of nitric oxide synthase 3: Impact on left ventricular function and myocardial infarction. Trends Cardiovasc Med 2005;15(7):249– 253. Brunner F, Andrew P, Wolkart G, Zechner R, Mayer B. Myocardial contractile function and heart rate in mice with myocyte-specific overexpression of endothelial nitric oxide synthase. Circulation 2001;104(25):3097–3102. Brunner F, Maier R, Andrew P, Wolkart G, Zechner R, Mayer B. Attenuation of myocardial ischemia/reperfusion injury in mice with myocyte-specific overexpression of endothelial nitric oxide synthase. Cardiovasc Res 2003;57(1):55–62. Janssens S, Pokreisz P, Schoonjans L, et al. Cardiomyocyte-specific overexpression of nitric oxide synthase 3 improves left ventricular performance and reduces compensatory hypertrophy after myocardial infarction. Circ Res 2004;94(9):1256–1262. Champion HC, Georgakopoulos D, Takimoto E, Isoda T, Wang Y, Kass DA. Modulation of in vivo cardiac function by myocytespecific nitric oxide synthase-3. Circ Res 2004;94(5):657–663. Rajewsky K, Gu H, Kuhn R, et al. Conditional gene targeting. J Clin Invest 1996;98(3):600–603. Yarranton GT. Inducible vectors for expression in mammalian cells. Curr Opin Biotechnol 1992;3(5):506–511. Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 1992;89(12):5547–5551. Beggah AT, Escoubet B, Puttini S, et al. Reversible cardiac fibrosis and heart failure induced by conditional expression of an antisense mRNA of the mineralocorticoid receptor in cardiomyocytes. Proc Natl Acad Sci USA 2002;99(10):7160–7165. Sauer B, Henderson N. Cre-stimulated recombination at loxPcontaining DNA sequences placed into the mammalian genome. Nucleic Acids Res 1989;17(1):147–161. O’Gorman S, Fox DT, Wahl GM. Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science 1991;251(4999):1351–1355.

393

133. Lakso M, Sauer B, Mosinger B Jr, et al. Targeted oncogene activation by site-specific recombination in transgenic mice. Proc Natl Acad Sci USA 1992;89(14):6232–6236. 134. Sauer B, Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci USA 1988;85(14):5166–5170. 135. Kellendonk C, Tronche F, Monaghan AP, Angrand PO, Stewart F, Schutz G. Regulation of Cre recombinase activity by the synthetic steroid RU 486. Nucleic Acids Res 1996;24(8):1404– 1411. 136. Kellendonk C, Tronche F, Casanova E, Anlag K, Opherk C, Schutz G. Inducible site-specific recombination in the brain. J Mol Biol 1999;285(1):175–182. 137. Reichardt HM, Kellendonk C, Tronche F, Schutz G. The Cre/loxP system—a versatile tool to study glucocorticoid signalling in mice. Biochem Soc Trans 1999;27(2):78–83. 138. Tsujita M, Mori H, Watanabe M, Suzuki M, Miyazaki J, Mishina M. Cerebellar granule cell-specific and inducible expression of Cre recombinase in the mouse. J Neurosci 1999;19(23):10318– 10323. 139. Indra AK, Warot X, Brocard J, et al. Temporally-controlled sitespecific mutagenesis in the basal layer of the epidermis: Comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res 1999;27(22): 4324–4327. 140. Brocard J, Warot X, Wendling O, et al. Spatio-temporally controlled site-specific somatic mutagenesis in the mouse. Proc Natl Acad Sci USA 1997;94(26):14559–14563. 141. Lewis W, Haase CP, Raidel SM, et al. Combined antiretroviral therapy causes cardiomyopathy and elevates plasma lactate in transgenic AIDS mice. Lab Invest 2001;81(11):1527–1536. 142. Milne R, Gutkowska J, Thibault G, et al. A murine monoclonal antibody against rat atrial natriuretic factor (ANF) which crossreacts with mouse ANF. Mol Immunol 1987;24(2):127–132. 143. Nakagawa M, Tanaka I, Suga S, et al. Preparation of a monoclonal antibody against mouse brain natriuretic peptide (BNP) and tissue distribution of BNP in mice. Clin Exp Pharmacol Physiol Suppl 1995;22(1):S186–187. 144. O’Brien PJ, Dameron GW, Beck ML, Brandt M. Differential reactivity of cardiac and skeletal muscle from various species in two generations of cardiac troponin-T immunoassays. Res Vet Sci 1998;65(2):135–137. 145. Lim SS, Tu ZH, Lemanski LF. Anti-troponin-T monoclonal antibody crossreacts with all muscle types. J Muscle Res Cell Motil 1984;5(5):515–526. 146. Kromenaker SJ, Srienc F. Effect of lactic acid on the kinetics of growth and antibody production in a murine hybridoma: Secretion patterns during the cell cycle. J Biotechnol 1994;34(1): 13–34. 147. Sigal LJ, Berens S, Wylie D. A lactate dehydrogenase (LDH)-based immunoassay for detection of cell surface antigens and its application to the study of MHC class I-binding peptides. J Immunol Methods 1994;177(1–2):261–268. 148. Laurino JP, Fischberg-Bender E, Galligan S, Chang J. An immunochemical mass assay for the direct measurement of creatine kinase MB2. Ann Clin Lab Sci 1995;25(3):252–263.

42

Primate Models for the Assisted Reproductive Technologies and Embryonic Stem Cell Biology DON P. WOLF, SHOUKHRAT MITALIPOV,

ABSTRACT

AND JAMES

A. BYRNE

and cognitive skills that are difficult to discern in lower mammals. This feature is particularly important in assessing whether treatments designed to ameliorate subtle cognitive defects are safe and effective. Other unique characteristics of NHPs include their susceptibility to human infectious agents and their responses to experimentally induced diseases.1 They also serve as relevant models for stress or nutrition-related research. In the more specific context of assisted reproductive technologies (ARTs) and stem cell biology, NHPs models are relevant to research interests as diverse as the etiology and treatment of infertility and contraceptive development to evaluations of cell or tissue-based treatments of disease using embryonic stem cell (ESC)-derived phenotypes. The ARTs are used in the production of desired genotypes for HIV vaccine development and could be used to produce genetically identical animals for nature versus nurture comparisons. When also considering somatic cell nuclear transfer (SCNT) or reproductive cloning, the rescue of the unique genetics of aging or even deceased animals is possible as is the creation of genetically identical animals, testing the efficacy of therapeutic cloning and in combination with gene targeting, producing monkey models of genetically based diseases. ADVANTAGES OF THE MODEL Rhesus monkeys are recognized as an important preclinical model. For instance, the methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkey is the best animal model of Parkinson’s disease (see Chapter 35, this volume, on transgenic models of neurodegenerative diseases). With regard to the reproductive and embryonic stem cell technologies, monkeys show menstrual cycles that are similar to humans in length and characteristics. This analogy is also true for spermatogenesis, early preimplantation development/ embryology, and placentation. ESCs derived from NHP embryos are similar to those from humans and markedly different from murine ESCs. Relevant areas of research include intracytoplasmic sperm injection (ICSI) or spermatid injection,2 in vitro oocyte maturation,3–7 optimizing embryo culture conditions, therapeutic and reproductive cloning, and models of contraception and implantation. NHP ESCs could also be employed in preclinical studies involving cell or tissue-based treatments of human degenerative diseases, in the prospective systematic evaluation of health consequences of the ARTs, and even in the development of female fertility preservation strategies.8 The availability of NHP ESCs and the ability to use federal monies to generate addi-

Nonhuman primates (NHPs) represent clinically relevant animal models used in studies on the etiology and treatment of human diseases. In the context of reproduction, NHP models are relevant to research interests as diverse as the etiology and treatment of infertility and contraceptive development to an evaluation of cell or tissue-based therapies of disease employing embryonic stem cell-derived phenotypes. The assisted reproductive technologies (ARTs) are used in the production of animals carrying desired MHC alleles for HIV vaccine development and could be used in the production of genetically identical animals by somatic cell nuclear transfer (SCNT). SCNT in turn is relevant to rescuing the unique genetics of aging or even deceased animals, to testing the efficacy of therapeutic cloning, and in combination with gene targeting, to creating monkey models of genetically based, neurodegenerative diseases. Since most NHP experience in applying the ARTs and in deriving embryonic stem cells (ESCs) involves Old World monkeys, principally rhesus and cynomolgus macaques and baboons, studies with these species will be featured in this chapter. Key Words: Nonhuman primates, Assisted reproductive technology, Embryonic stem cells, Cloning.

GENERAL CHARACTERISTICS Nonhuman primates (NHPs) are among the least available and most costly of animal models employed in biomedical research and their use is carefully regulated and should be exhaustively justified. While there are approximately 200 species in the order Primates, representatives from seven genera are most commonly used in research including both Old World and New World primates (see Chapter 28, this volume, on nonhuman primates). Among the great apes, the chimpanzee (Pan troglodytes) has played a unique role in biomedical research predicated on its susceptibility to human infectious diseases including HIV.1 Of obvious relevance to their role in biomedical research, NHPs resemble human beings much more closely in anatomy and physiology than do other commonly employed animals such as rodents. A case in point, because NHP brains more closely resemble those of humans in size and complexity, they exhibit complex behavior From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

397

398

SECTION V / MODELS OF BEHAVIOR

tional lines represent advantages over current efforts with human ESCs. Applications of the ARTs in conjunction with SCNT should eventually allow the creation of NHP models, for instance, of genetically based diseases. DISADVANTAGES ASSOCIATED WITH NONHUMAN PRIMATE RESEARCH Features of NHP research include high costs in animal lease and per diem fees and limited access or availability. Obviously, unique facilities are required in terms of animal husbandry, caging, and surgical facilities. There is also substantial public concern over the use of such highly sentient animals in biomedical research. As is true for all animal models employed in research there are and should be restrictions on use, that is, when rodent models are not available or when the unique features of the NHP necessitate it. Other restrictions that impact the use of the model include limits on the number and types of surgeries to which animals can be exposed and the large number of animals required in animal production using the ARTs—egg donors and embryo transfer recipients—or in studies involving infrequent events, for instance, investigations into intracytoplasmic sperm injection-related abnormalities in newborns.

HUSBANDRY AND AVAILABLE STOCKS As significant increases in our knowledge of the biology and care of NHPs have been realized, the number of NHPs imported has declined (the 2002 estimation of the number of NHPs imported annually is 50–60,000) because of bans on exportation from countries of origin (case in point, Indian-origin rhesus macaques), the impact of conservation movements, animal activist group activities, and more efficient animal use.1 Domestic breeding programs initiated in the 1970s have become the major supply source of rhesus monkeys, baboons, chimpanzees and squirrel monkeys (consult Chapter 28, this volume, on NHPs and [email protected]. washington.edu for an Annual Resource Guide concerning animal suppliers). The National Primate Research Center system is a major supplier of animals for National Institutes of Health (NIH)sponsored research with infrastructural support from National Center for Research Resources (NCRR). There are currently eight Centers in this system with a major emphasis on the propagation of rhesus and cynomolgus macaques and baboons. Specific pathogen-free production colonies are currently under development with NCRR sponsorship that might, parenthetically, benefit from the use of the ARTs, and should eventually become the standard resource. For ARTs and ESC biology, only a few NHP models have been developed sufficiently to allow itemization of reproducible protocols. In several contexts, the human serves as the model for the NHP as clinical applications of the ARTs are now responsible for the production of over 1% of the births in Western nations accounting, cumulatively, for millions of children. Furthermore, federally sponsored research on human ESCs dwarfs funded efforts employing NHP ESCs. The great apes can be eliminated from consideration simply based on economic, practical, and ethical arguments. Indeed, in this case, it is more likely that the ARTs may ultimately be used to propagate endangered animals rather than serve as useful models in translational research. As for the more commonly available New World monkeys, such as owl or squirrel monkeys, extensive efforts to establish routine protocols for the ARTs have met with limited success, allocating this group into species currently requiring protocol development.9 In a few applications, specific advantages may be compelling in the selection of a

species for study, for instance in polyovulatory marmosets where dizygotic twinning is the rule1 or baboons where external signs, such as sex skin changes, can be used to conveniently monitor the menstrual cycle or where seasonal anovulation may not be limiting as it is in rhesus macaques. Based on the extensive experience and success achieved in the past two decades, Old World macaques, rhesus and cynomolgus monkeys, will be featured in this chapter. The baboon is still limited by suboptimal in vitro embryo culture conditions and while proof of principle experimentation has been reported recently in Macaca nemenstrina, the experience is limited.10 As an example of the robust experience now available with the rhesus monkey, a highly unique and valuable resource exists in the ART Core at Oregon National Primate Research Center (ONPRC) (http://www.onprc.ohsu.edu/) established in 1988 under the codirection of Drs. Don Wolf and Richard Stouffer. The focus of the core has been and continues to be on utilizing the rhesus macaque as a NHP model for research on gametogenesis, fertilization, embryogenesis, and implantation. This facility is responsible for 11 of the first 15 test tube rhesus monkeys produced in the world. Parenthetically, Dr. Barry Bavister’s group at the University of Wisconsin is responsible for the other four including Petri, the first success.11 The ART Core was, in part, responsible for the first monkey produced following the transfer of an embryo that had been frozen, cryobanked, and thawed after successful in vitro fertilization,12 the first twin pregnancies,12 the first infants produced by nuclear transfer of embryonic cells,13 the first infant born following the transfer of an ICSI-produced blastocyst employing a nonsurgical procedure,14 the first live birth resulting from the transfer of a demiembryo created by blastomere separation at the two-cell stage,15 the first animal produced from transplanted ovarian tissue,8 and the first pregnancy to result from embryo transfer (ET) during a controlled ovarian stimulation (COS) cycle (unpublished result). The program has contributed to improvements in COS16 and in its more recent configuration has added an ESC component to its activities. Currently the ART/ESC Core program provides oocytes, sperm, embryos, follicular fluid, cumulus, and granulosa cells and distributes rhesus monkey ESC lines along with mouse embryonic fibroblasts, media, protocols, and expertise to the global scientific community. It is worth noting that a major limitation in developing the ARTs or ESC technology in NHPs is the focus by NIH scientific reviewers on basic or applied research as opposed to protocol development. One approach to overcoming this limitation has been to couple protocol development with the production of a highly desirable but rare product such as monkeys with specific major histocompatibility alleles or with efforts to produce specific pathogen-free colonies, in both cases reflecting the unmet needs of the HIV vaccine development community. However, despite these limitations remarkable progress has been made in the rhesus monkey with implantation rates from ART-produced embryos comparable to those obtained in humans at approximately one in six embryos transferred. Of relevance to investigators interested in gene expression is the recently released gene chip with probe sets interrogating over 47,000 rhesus monkey transcripts (www. affymetrix.com/products/arrays/specific/rhesus_macaque.affx).

METHODS ASSISTED REPRODUCTIVE TECHNOLOGIES A general pitfall in the application of ARTs in NHPs is the realization that

CHAPTER 42 / PRIMATE MODELS FOR THE ASSISTED REPRODUCTIVE TECHNOLOGIES AND EMBRYONIC STEM CELL BIOLOGY 399

it is not necessarily possible to extrapolate technologies from one species to another. Typically there are advantages and disadvantages associated with each species, for instance, in their availability, health, size, relevance to a specific project, and availability of established disease models. However, the trump card at present in selecting an NHP animal model has to be the existence of established protocols and relevant experience in that species. Fortunately, as noted above, much of the methodology involved in the routine application of the ARTs in rhesus monkeys has been worked out and published in detail.17–19 A comprehensive itemization of references available in NHPs on gamete collection, fertilization, embryo culture, and transfer is included in Table 42–1.20–72 A few specific recommendations or potential concerns are discussed below. Generally sperm collection involves penile or rectal electroejaculation or needle biopsy of the caudal epididymas or vas deferens. Rhesus monkey sperm collected by penile electroejaculation is notable for its uniform nature and high motility, for example, at ONPRC, only animals with sperm concentrations greater that 60 million/ml and with more that 70% motile cells with normal morphology are acceptable. Inducing sperm capacitation in the rhesus monkey is more that simply washing sperm as is the case in humans. Typically, washed sperm must be exposed to an intracellular cAMP enhancer such as caffeine or the cell-permeable dibutyryl derivative.37 This exposure is associated with a transition in sperm motility and a tendency of sperm to agglutinate, which can create real problems when in vitro insemination is involved. While there are several reports on the cryostorage of NHP sperm, a notably loss of motility in the rhesus macaque makes the prospect of using frozen/thawed sperm for artificial insemination by donor (AID) or in vitro fertilization (IVF) from a wide spectrum of donors questionable. In situations in which IVF is involved, ICSI can be employed to overcome sperm motility limitations (see below). Egg collection for ARTs in rhesus macaques now typically involves subjecting females to COS using recombinant human follicle-stimulating hormone, luteinizing hormone, and chorionic gonadotropin. COS does not always result in the achievement of

preestablished thresholds in animal response and the percentage of “nonresponders” varies by season showing an increase during the summer months, reaching over 35% in June and July. During summer, despite housing in controlled environments, many females also become anovulatory and it is impractical to attempt COS. Females can be recycled for COSs, however, the response to recombinant human gonadotropins is gradually decreased with increasing numbers of stimulations, apparently due to an immune reaction.16,73 Practically, up to three stimulations on average can be performed per female with the recovery of a reasonable number of high-quality oocytes. The availability of monkey recombinant gonadotropins may allow the more efficient and extended use of females. This immune response, although greatly reduced compared to when animal or human urinary preparations were employed, still limits animal responses and the number of repeat cycles that are possible. The egg yield is highly variable in rhesus monkeys from a few to over 100 using such fixed stimulation protocols and, not surprisingly, oocyte quality is also variable.14 The recovery of high oocyte numbers, in our hands, is associated with poor performance as measured by fertilization and/or in vitro developmental potential. Fertilization by IVF or ICSI is feasible with the latter conveniently used with frozen-thawed sperm or whenever poor motility compromises IVF. We have previously reported that fertilization rates by ICSI with cryopreserved sperm injected shortly after thaw are significantly lower than with fresh sperm.19 However, preincubation times of more than 3 h after thaw were associated with ICSI fertilizing capacity similar to that seen with fresh sperm. Piercing the plasma membrane during the ICSI procedure is critical and often difficult to assess since the needle can grossly invaginate the membrane without breaking through. For that reason, the injection pipette should be inserted through the zona pellucida and “into” the oocyte across approximately one-third of the egg’s diameter. Cytoplasm is slowly aspirated into the ICSI pipette (as far back as the needle junction with the zona pellucida) until the plasma membrane breaks. A “pop” or sudden movement of cytoplasm into the pipette indicates the release of membrane tension. Once the membrane is penetrated, the sperm can be expelled into the cytoplasm.

Table 42–1 Gamete collection, fertilization, embryo culture, and transfer in nonhuman primatesa ART procedures Species

Semen recovery and cryostorage

COS and egg collection

Fertilization

Embryo culture

Embryo transfer; pregnancy outcome

21, 22, 28 41, 48, 49 10, 51 53, 54 60

20, 22, 29–38 48 10 56 60

13, 20–22, 30, 39, 40 41, 50 10 57 60

63

62, 64

Old World macaques Rhesus Cynomolgus Pig tailed Baboon Vervet

11, 20–25 41–46 10, 51, 52 53 58–60

20, 26, 27 41, 47 10, 51 54, 55 60, 61

New World macaques Marmoset Squirrel

62, 63 65, 66

63 67, 68

62, 63 65, 66

Great apes Chimpanzee Gorilla

69 70–72

72

70–72

a

ART, assisted reproductive technology; COS, controlled ovarian stimulation.

70

400

SECTION V / MODELS OF BEHAVIOR

Embryo culture has progressed from BRL cell coculture to reliance on chemically defined medium such as HECM-9 or -10 fortified with a protein source, usually fetal bovine serum (FBS), at later stages in preimplantation development. Development of the primate embryo as in other mammals includes morphologically distinct developmental stages from the formation of the zygote after fertilization to cleavage, morula formation, compaction, and finally cavitation of the blastocyst. In HECM-9aa + FBS, blastocysts containing 100 or so cells can be seen by day 6 or 7 in vitro at a control rate of approximately 50% in the rhesus monkey. Embryo transfer in the context of producing animals can be accomplished by laparoscopy or by minilaparotomy with direct deposition into the oviduct. A nonsurgical transcervical route, although evaluated, has proven troublesome in the rhesus monkey secondary to difficulties in passing a cannula through the tortuous cervix in this species.14,19 For information on optimal timing of embryo transfer see Wolf et al.19 Pregnancy outcomes in NHPs following ET of ART-produced embryos have been evaluated; however, a significant experience is available only in the rhesus macaque where approximately 100 infants have been born following IVF/ICSI.17–19 PRODUCTION OF IDENTICAL ANIMALS The ARTs can, in theory, be employed for the production of genetically identical animals by twinning, embryonic cell nuclear transfer (ECNT), or SCNT. This would be a highly significant accomplishment as there is a need among the research community for monkeys with specific genotypes that cannot be satisfied by the importation of animals from the wild or by the identification and propagation of founder animals using conventional breeding. Moreover, spontaneous or naturally occurring NHP models of human genetic diseases are exceedingly rare and not currently available in useful numbers. If a technology for preserving, propagating, and indeed creating NHP models was available, it could be a key to understanding factors involved in disease progression, and it would also provide a relevant platform for testing therapies. Embryo twinning alternatives, as perfected in domestic animal species, have been evaluated in efforts to create genetically identical monkeys. The limitation of this approach is that sets of only two or possibly four identical animals could be produced by embryo separation at early developmental stages or by blastocyst splitting. Monkeys have been produced following the transfer of demiembryos or split embryos15,18,74 into a single recipient; however, the outcomes have been disappointing with only a few twin pregnancies resulting and none progressing to term. Nuclear transfer using embryonic blastomeres is an alternative option for the production of identical animals, but it is also restricted theoretically by a small clone size.13,17–19,75 Protocols for ECNT in the rhesus monkey, including oocyte and embryo production, micromanipulation, and embryo transfer, are described in detail in the Methods in Molecular Biology Series.76 With success in producing identical animals by SCNT in the past decade, attention has focused on the use of this technology in nonhuman primates. Several advantages accrue to SCNT including the potential to introduce precise genetic modifications into cultured cells, and then use these cells as nuclear donors for the production of transgenic or loss-of-function monkey models of human diseases. SCNT embryos could also provide a resource for the derivation of autologous ESCs and the subsequent thera-

peutic use of these cells in preclinical trials of therapeutic cloning. Initial efforts at SCNT by conventional protocols in the rhesus monkey were disappointing because SCNT embryos seldom progressed beyond the eight-cell stage in vitro when fetal fibroblasts were used as the source of donor nuclei.75 While this failure to develop was likely related to incomplete or erroneous reprogramming, the depletion of essential microtubule and centrosomal proteins during oocyte enucleation has also been implicated,75 to the extent that reproductive cloning in nonhuman primates was thought to be impossible.77 However, these obstacles were subsequently overcome, to some degree, by technique alteration78 with normal spindles present after the introduction of somatic nuclei into enucleated cytoplasts.79,80 An encouraging outcome was achieved in a prospective study comparing SCNT outcome in the monkey with fibroblasts as the nuclear donor source using a conventional protocol versus a one-step method. SCNT-derived embryos developed to the blastocyst stage at a rate in excess of 20% using the one-step method.80 In the latest rendition of this technique, we incorporated several modifications.81 First, to prevent premature degradation of cytoplasmic reprogramming factors, the manipulation medium was modified to exclude calcium and magnesium. As before, the electrofusion step was replaced by direct donor cell injection. The latter was performed with a Piezo drill similar to procedures described for mouse SCNT. Cultured donor cells were dispersed and a blunt transfer pipette (5–7 µm outer diameter) was used to disrupt plasmalemma integrity by aspiration before a lysed cell with intact nucleus was injected into a cytoplast. A second modification was implemented to avoid possible deleterious effects of Hoechst staining and ultraviolet (UV) exposure on cytoplast integrity. Visualization of the metaphase spindle during enucleation was accomplished using Oosight Imaging System (CRI, Inc., Woburn, MA) that allowed noninvasive, polarized light imaging and detection of the spindle based on birefringence. Using this innovative approach, we were able to locate and quickly enucleate the spindle, real-time with 100% efficiency. Several fetal and adult somatic cell types supported significant blastocyst development and these achievements represent breakthroughs that allow, for the first time, the in vivo and in vitro characterization of SCNT blastocysts in this species. EMBRYONIC STEM CELLS ESCs can proliferate indefinitely, maintain an undifferentiated pluripotent state, and differentiate into any cell type. These pliable embryonic cells are derived from the inner cell mass (ICM) of mammalian blastocyststage embryos. Embryonic stem cells—or at least ES-like cells— have been derived from the embryos of multiple species, most notably mice,82 rhesus monkeys,83 marmoset monkeys,84 cynomolgus monkeys,85 and humans.86 The ability of ESCs to differentiate into any cell type means we may be able to utilize these cells to cure or alleviate the symptoms of many degenerative diseases. However, unresolved issues regarding cellular survival, maintenance of functionality, and tumor formation mean a prudent approach should be taken toward advancing ESCs into human clinical trials. The short-term and long-term efficacy and safety of ESCs must be thoroughly examined. Due to practical and ethical considerations these studies cannot be performed in vivo in humans and clinically relevant nonhuman models are therefore essential. ESCs derived from NHP or human blastocysts demonstrate extensive similarities not observed in murine ESCs.87,88

CHAPTER 42 / PRIMATE MODELS FOR THE ASSISTED REPRODUCTIVE TECHNOLOGIES AND EMBRYONIC STEM CELL BIOLOGY 401

There are currently 42 NHP ESC lines available: 26 in the rhesus monkey, of which eight were derived from in vivo produced embryos,83,89 and 18 from in vitro efforts,90 five cynomolgus monkey ESC lines, of which four were derived from in vitro fertilized embryos85 and one from a parthenogenetically generated embryo,91 and 11 common marmoset ESC lines that were all derived from in vivo produced embryos.84,92 A summary of the ESC lines currently available in the nonhuman primate is presented in Table 42–2. To produce these lines, blastocysts were either obtained following in vivo fertilization and nonsurgical uterine flushing83 or though in vitro production90 including conventional in vitro fertilization,85 ICSI,90 and parthenogenesis.91,93 Once the blastocyst is obtained, the same basic technique is generally used to derive an ESC line. Zonae pellucidae are removed by a brief exposure to pronase. The blastocysts are then exposed to an animal serum containing anti-rhesus antibodies that can bind to the trophectoderm cells that surround the embryo’s ICM. Next, the embryos are moved into serum complement. The complement serum proteins respond to the trophectoderm-bound antibodies and induce lysis. The lysed trophectodermal cells are gently removed with a small bore pipette and the isolated ICM is plated onto dishes containing a monolayer of mitotically inactivated mouse embryonic fibroblast (MEF) feeder cells in ESC culture media. The ICMs attach to the feeder layer and produce small outgrowths after a few days that are manually dissociated into small cell clumps and replated onto fresh dishes of MEFs. Colonies with an ESC-like morphology (flat monolayer colony with distinctive cobbled stem cell morphology, prominent nucleoli, and a high nucleocytoplasmic ratio) are manually selected for further propagation, characterization, and low temperature storage. Medium is changed daily and ESC colonies are split every few days onto dishes with fresh MEF feeder layers. The nondividing MEF feeder cells provide various growth factors that help maintain the NHP ESCs in an undifferentiated state and encourage proliferation. It should be noted that NHP ESC culture is technically difficult. The majority of novice groups supplied with NHP ESCs have had severe difficulty in maintaining their colonies in an undifferentiated state. It is therefore highly recommended that investigators interested in NHP ESCs seek out and attend a relevant training workshop such as an NIH-sponsored workshop on human ESCs or an NHP ESC workshop provided by an active laboratory in this field. Manual passaging of ESCs is, of course, labor intensive and is incompatible with the scaled up production numbers required for in vivo transplantation. Theoretically, combining enzymatic passage to produce multiple subclones with continuous passageby-passage karyotypic analysis could be used to discard aneuploid

subclones and select for an enzymatically passaged karyotypically stable NHP ESC line that would be ideal for scaled up production. This type of NHP ESC line would be an important addition to any NHP ESC bank. NHP ESC lines have, for the most part, been maintained on mouse feeder layers that may contaminate the primate cells with murine pathogens and/or xenoepitopes, either of which could induce a severe immune response following transplantation. Possible solutions to this problem would be to grow NHP ESCs either on NHP feeders94 or in feeder-free conditions.95,96 Our research has suggested that NHP ESCs are best cultured on MEFs in DMEM/F12 medium supplemented with 15% FBS, 0.1 mM 2-mercaptoethanol, 1% nonessential amino acids, 2 mM l-glutamine, 4 ng/ml fibroblast growth factor 2 (FGF2) (pH adjusted to 7.2 with NaOH) at 37°C in 3% CO2, 5% O2, and 92% N2.90 NHP ESC lines derived from in vivo-produced embryos have been characterized by a normal karyotype; they express the same stem cell markers as human ESCs, such as OCT-4, SSEA-3, SSEA-4, TRA-1–60, and TRA-1–81, and possess the ability to differentiate into multiple cell types in vitro and into teratomas in vivo.83,84,92 NHP ESC lines derived from in vitro-produced embryos also express the aforementioned stem cell markers and can differentiate into multiple cell types both in vitro and in vivo.85,90–92,97 However, abnormal karyotypes were discovered in three of the six in vitro-derived rhesus monkey ESC lines that were enzymatically passaged during their initial derivation. Primate ESCs appear to be particularly prone to karyotypic aneuploidy during enzymatic passaging as none of the 12 in vitro-derived rhesus monkey ESC lines that were manually passaged demonstrated any aneuploidy.90 Most of the other biological characteristics of NHP ESCs have been elucidated using rhesus monkey ESCs. The cell cycle of rhesus monkey ESC is characterized by an extremely rapid transition though G1, which is not arrested by serum starvation, pharmacological inhibition, or gamma irradiation, reflecting an absence of a G1 checkpoint similar to murine ESCs.98 NHP ESCs can be either differentiated in vitro or in vivo. For in vitro differentiation, embryoid bodies (EBs) are formed and then placed into various culture conditions until the desired cell types are obtained. For in vivo differentiation, the ESCs are injected into a mouse and allowed to spontaneously form a tumor. To obtain embryoid bodies, entire ESC colonies are detached from the feeder cells and manually transferred into feeder-free ultralow adhesion dishes and cultured in suspension in ESC medium without FGF for several days. The NHP ESCs initially clump up into a ball and then cavitate to form a blastocyst-like embryoid body. To induce spontaneous differentiation, the embryoid bodies are attached to gelatin-coated culture dishes and cultured for 1 month in FGF-free ESC culture media. This allows

Table 42–2 A summary of the NHP ESC lines currently availablea Primate species

Blastocyst origin

Rhesus monkey (Macaca mulata) Rhesus monkey (Macaca mulata) Cynomolgus monkey (Macaca fascicularis) Cynomolgus monkey (Macaca fascicularis) Common marmoset (Callithrix jacchus)

In vivo In vitro In vitro In vitro In vivo

a

NHP, nonhuman primate; ESC, embryonic stem cell.

ESC lines (number)

Reference

8 18 4 1 (parthenote derived) 11

83, 89 90 85 91, 93 84, 92

402

SECTION V / MODELS OF BEHAVIOR

the ESCs to differentiate down all possible pathways and produces a wide spectrum of cell types. To form teratomas, large numbers of NHP ESCs (usually 106–107) are injected into the leg muscle of a severe combined immune deficient (SCID) mouse. These mice have no functional immune systems, so the NHP ESCs are tolerated and survive. Over 2 months the NHP ESC differentiate into a tumor composed of many different cell types and containing representatives of all three germ layers. This type of tumor is referred to as a teratoma. Finally a few of the limitations associated with NHP ESCs will be noted. ESC cloning strategies have proved successful using human ESCs,99 but are not yet available in the NHP. However, the initial difficulties in genetically modifying NHP ESCs have been largely solved using lentiviral vectors for transfection, resulting in high-level sustained transgene expression in both NHP and human ESCs.100,101 This lentiviral-based ability to produce gene knockout and transgenic NHP ESCs has significant implications for the use of NHP ESCs in developmental biology, disease pathology, and gene therapy. In the mouse, genetically modified ESCs are inserted into tetraploid host blastocysts and the implanted chimeras produce transgenic and gene knockout animals.102 If these chimera techniques eventually proved transferable to NHP ESCs then it would permit the generation of gene knockout and transgenic primates. Chimera research in the NHP is still in the preliminary stages. We have demonstrated that membrane marked NHP ESCs—when transferred to a host embryo at the four- to eight-cell stage—have the capacity to proliferate and contribute to the ICM and trophectoderm of the resulting NHP chimeras90 and when green fluorescent protein (GFP)-marked NHP ESCs have been transferred into NHPs at the fetal stages they have successfully integrated into the host tissues.103 Recent evidence has demonstrated that multiple NHP ESC lines demonstrate a loss of imprinting.104 Aberrant imprinting has also been observed in mouse ESCs105 and in high passage human ESCs,106 although it should be noted that low-passage human ESCs appear to demonstrate normal imprinting.106 How this loss of imprinting will affect the therapeutic potential of ESCs is still unknown, but as the loss of imprinting has been associated with germ cell tumors107 this question becomes yet another reason for extensive research into the safety of ESC-based therapy in the NHP before human clinical trials are initiated.

REFERENCES 1. Bernacky BJ, Gibson SV, Keeling ME, Abee CR. Nonhuman primates. In: Fox JG, Anderson LC, Loew FM, Quimby FW, Eds. Laboratory Animal Medicine, 2nd ed. London: Academic Press, 2002:676–791. 2. Hewitson L. Primate models for assisted reproductive technologies. Reproduction 2004;128:293–299. 3. Schramm RD, Bavister BD. A macaque model for studying mechanisms controlling oocyte development and maturation in human and non-human primates. Hum Reprod 1999;14:2544–2555. 4. Schramm RD, Paprocki AM. Birth of rhesus monkey infant after transfer of embryos derived from in-vitro matured oocytes: Short communication. Hum Reprod 2000;15:2411–2414. 5. Zheng P, Si W, Bavister BD, Yang J, Ding CH, Ji W. 17β-Estradiol and progesterone improve in-vitro cytoplasmic maturation of oocytes from unstimulated prepubertal and adult rhesus monkeys. Hum Reprod 2003;18:2137–2144. 6. Yin H, Duffy DM, Gosden RG. Comparative maturation of cynomolgus monkey oocytes in vivo and in vitro. Reprod Biol Endocrinol 2006;4:14.

7. Chen C, Liow S-L, Abdullah RB, Embong WKW, Yip W-Y, Tan L-G, Tong G-Q, Ng SC. Developmental competence of transported in vitro matured macaque oocytes. Reprod Biol Endocrinol 2006;23:50–59. 8. Lee DM, Yeoman RR, Battaglia DE, Stouffer RL, Zelinski-Wooten MB, Fanton JW, Wolf DP. Live birth after ovarian tissue transplant. Nature 2004;428:137–138. 9. Dukelow WR, Pierce DL, Roudebush WE, Jarosz SJ, Sengoku K. In vitro fertilization in nonhuman primates. J Med Primatol 1990;19:627–639. 10. Kubisch HM, Gagliardi C, Williams VM, Ribka EP, Ratterree MS. In vitro fertilization in the pigtailed macaque (Macaca nemestrina). Theriogenology 2006;66(4):749–754. 11. Bavister BD, Boatman DE, Collins K, Dierschke DJ, Eisele SG. Birth of rhesus monkey infant after in vitro fertilization and nonsurgical embryo transfer. Proc Natl Acad Sci USA 1984;81:2218–2222. 12. Wolf DP, Thomson JA, Zelinski-Wooten MB, Stouffer RL. In vitro fertilization-embryo transfer in nonhuman primates: The technique and its applications. Mol Reprod Dev 1990;27:261–280. 13. Meng L, Ely JJ, Stouffer RL, Wolf DP. Rhesus monkeys produced by nuclear transfer. Biol Reprod 1997;57:454–459. 14. Nusser KD, Mitalipov S, Widmann A, Gerami-Naini B, Yeoman RR, Wolf DP. Developmental competence of oocytes after ICSI in the rhesus monkey. Hum Reprod 2001;16:130–137. 15. Mitalipov SM, Yeoman RR, Kuo HC, Wolf DP. Monozygotic twinning in rhesus monkeys by manipulation of in vitro-derived embryos. Biol Reprod 2002;66:1449–1455. 16. Stouffer RL, Zelinski-Wooten M, Chandrasekher YA, Wolf DP. Stimulation of follicle and oocyte development in macaques for IVF procedures. In: Wolf DP, Stouffer RL, Brenner RM, Eds. In Vitro Fertilization and Embryo Transfer in Primates. New York: SpringerVerlag, 1992:124–141. 17. Ouhibi N, Zelinski-Wooten M, Thomson JA, Wolf DP. Assisted fertilization and nuclear transfer in non human primates. In: Wolf DP, Zelinski-Wooten M, Eds. Assisted Fertilization and Nuclear Transfer in Mammals. Totowa, NJ: Humana Press, 2001:253–284. 18. Wolf DP. Assisted reproductive technologies in rhesus macaques. Reprod Biol Endocrinol 2004;2:37. 19. Wolf DP, Thormahlen S, Ramsey C, Fanton J, Mitalipov S. Use of assisted reproductive technologies in the propagation of rhesus macaque offspring. Biol Reprod 2004;71:486–493. 20. Wolf DP, Vandevoort CA, Meyer-Haas GR, Zelinski-Wooten MB, Hess DL, Baughman WL, Stouffer RL. In vitro fertilization and embryo transfer in the rhesus monkey. Biol Reprod 1989;41:335– 346. 21. Lanzendorf SE, Zelinski-Wooten MB, Stouffer RL, Wolf DP. Maturity at collection and the developmental potential of rhesus monkey oocytes. Biol Reprod 1990;42:703–711. 22. Boatman DE. In vitro growth of nonhuman primate pre- and periimplantation embryos. In: Bavister BD, Ed. The Mammalian Preimplantation Embryo: Regulation of Growth and Differentiation In Vitro. New York: Plenum, 1987:273–308. 23. Roussel JD, Austin CR. Preservation of primate spermatozoa by freezing. J Reprod Fertil 1967;13:333–335. 24. Leverage WE, Valerio DA, Schultz AP, Kingsbury E, Dorey C. Comparative study on the freeze preservation of spermatozoa. Primate, bovine, and human. Lab Anim Sci 1972;22:882–889. 25. Yeoman RR, Mitalipov S, Gerami-Naini B, Nusser KD, Wolf DP. Low temperature storage of rhesus monkey spermatozoa and fertility evaluation by intracytoplasmic injection. Theriogenology 2005;63:2356–2371. 26. VandeVoort CA, Baughman WL, Stouffer RL. Comparison of different regimens of human gonadotropins for superovulation of rhesus monkeys: Ovulatory response and subsequent luteal function. J In Vitro Fert Embryo Transfer 1989;6:85–91. 27. Abassi R, Kenigsberg D, Danforth D, Falk RJ, Hodgen HG. Cumulative ovulation rate in human menopausal/human chorionic gonadotropin-treated monkeys: “Step-up” versus “step-down” dose regimens. Fertil Steril 1987;47:1019–1024.

CHAPTER 42 / PRIMATE MODELS FOR THE ASSISTED REPRODUCTIVE TECHNOLOGIES AND EMBRYONIC STEM CELL BIOLOGY 403

28. Boatman DE, Morgan PM, Bavister BD. Variables affecting the yield and developmental potential of embryos following superstimulation and in vitro fertilization in rhesus monkeys. Gamete Res 1986;13:327–338. 29. Zhang L, Weston AM, Denniston RS, Goodeaux LL, Godke RA, Wolf DP. Developmental potential of rhesus monkey embryos produced by in vitro fertilization. Biol Reprod 1994;51:433– 440. 30. Weston AM, Zelinski-Wooten MB, Hutchison JS, Stouffer RL, Wolf DP. Developmental potential of embryos produced by in-vitro fertilization from gonadotrophin-releasing hormone antagonisttreated macaques stimulated with recombinant human follicle stimulating hormone alone or in combination with luteinizing hormone. Hum Reprod 1996;11:608–613. 31. Seshagiri PB, Hearn JP. In-vitro development of in-vivo produced rhesus monkey morulae and blastocysts to hatched, attached, and post-attached blastocyst stages: Morphology and early secretion of chorionic gonadotrophin. Hum Reprod 1993;8:279–287. 32. Weston AM, Wolf DP. Differential preimplantation development of rhesus monkey embryos in serum-supplemented media. Mol Reprod Dev 1996;44:88–92. 33. Schramm RD, Bavister BD. Development of in-vitro-fertilized primate embryos into blastocysts in a chemically defined, proteinfree culture medium. Hum Reprod 1996;11:1690–1697. 34. Alak BM, Wolf DP. Rhesus monkey oocyte maturation and fertilization in vitro: Roles of the menstrual cycle phase and of exogenous gonadotropins. Biol Reprod 1994;51:879–887. 35. Goodeaux L, Thibodeaux JK, Voelkel S, Anzalone CA, Roussel JD, Cohen JC, Menezo Y. Collection, co-culture and transfer of rhesus preimplantation embryos. ARTA 1990;1:370–379. 36. Seshagiri PB, Dierschke DJ, Eisele SG, Scheffler J, Hearn JP. Recovery of rhesus monkey preimplantation embryos by non-surgical uterine flushing. Biol Reprod 1991;44(Suppl. 1):157. 37. Boatman DE, Bavister BD. Stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators. J Reprod Fertil 1984;71:357–366. 38. Morgan PM, Boatman DE, Bavister BD. Relationships between follicular fluid steroid hormone concentrations, oocyte maturity, in vitro fertilization and embryonic development in the rhesus monkey. Mol Reprod Dev 1990;27:145–151. 39. Goodeaux LL, Anzalone CA, Thibodeaux JK, Menezo Y, Roussel JD, Voelkel SA. Successful non-surgical collection of Macaca mulatta embryos. Theriogenology 1990;34:1159–1167. 40. Hewitson L, Dominko T, Takahashi D, Martinovich C, RamalhoSantos J, Sutovsky P, Fanton J, Jacob D, Monteith D, Neuringer M, Battaglia D, Simerly C, Schatten G. Unique checkpoints during the first cell cycle of fertilization after intracytoplasmic sperm injection in rhesus monkeys. Nat Med 1999;5:431–433. 41. Balmaceda JP, Pool TB, Arana JB, Heitman TS, Asch RH. Successful in vitro fertilization and embryo transfer in cynomolgus monkeys. Fertil Steril 1984;42:791–795. 42. Kreitman O, Lynch A, Nixon WE, G.D. H. Ovum collection, induced luteal dysfunction, in vitro fertilization, embryo development and low tubal ovum transfer in primates. In: Hafez ESE, Semm K, Eds. In Vitro Fertilization and Embryo Transfer. Lancaster, UK: MTP Press Ltd., 1982:303–324. 43. Cho F, Honjo S, Makita T. Fertility of frozen-preserved spermatozoa of cynomolgus monkeys. In: Kondo S, Kawai M, Ehara A, Eds. Contemporary Primatology. Basel: S. Karger, 1975:125–133. 44. Cho F, Honjo S. A simplified method for collecting and preserving cynomolgus macaque semen. Jpn J Med Sci Biol 1973;26: 261–268. 45. Mahone JP, Dukelow WR. Semen preservation in Macaca fascicularis. Lab Anim Sci 1978;28:556–561. 46. Tollner TL, VandeVoort CA, Overstreet JW, Drobnis EZ. Cryopreservation of spermatozoa from cynomolgus monkeys (Macaca fascicularis). J Reprod Fertil 1990;90:347–352. 47. Schenken RS, Williams RF, Hodgen GD. Ovulation induction using “pure” follicle-stimulating hormone in monkeys. Fertil Steril 1984;41:629–634.

48. Fujisaki M, Suzuki M, Kohno M, Cho F, Honjo S. Early embryonal culture of the cynomolgus monkey (Macaca fascicularis). Am J Primatol 1989;18:303–313. 49. Balmaceda JP, Gastaldi C, Ord T, Borrero C, Asch RH. Tubal embryo transfer in cynomolgus monkeys: Effects of hyperstimulation and synchrony. Hum Reprod 1988;3:441–443. 50. Balmaceda JP, Heitman TO, Garcia MR, Pauerstein CJ, Pool TB. Embryo cryopreservation in cynomolgus monkeys. Fertil Steril 1986;45:403–406. 51. Cranfield MR, Schaffer N, Bavister BD, Berger NG, Boatman DE, Kempske SE, et al. Assessment of oocytes retrieved from stimulated and unstimulated ovaries of pig-tailed macaques (Macaca nemestrina) as a model to enhance the genetic diversity of captive liontailed macaques (Macaca silenus). Zool Biol 1989;Suppl. 1:33–46. 52. Cranfield MR, Bavister BD, Boatman DE, Berger NG, Schaffer N, Kempske SE, et al. Assisted reproduction in the propagation management of the endangered lion-tailed macaque (Macaca silenus). In: Wolf DP, Stouffer RL, Brenner RM, Eds. In vitro Fertilization and Embryo Transfer in Primates. New York: Springer-Verlag, 1993:331–348. 53. Clayton O, Kuehl TJ. The first successful in vitro fertilization and embryo transfer in a nonhuman primate. Theriogenology 1984; 21:228. 54. Fourie FR, Snyman E, van der Merwe JV, Grace A. Primate in vitro fertilization research: Preliminary results on the folliculogenic effects of three different ovulatory induction agents on the chacma baboon, Papio ursinus. Comp Biochem Physiol 1987;87:889–893. 55. Kraemer DC, Vera Cruz NC. Collection, gross characteristics and freezing of baboon semen. J Reprod Fertil 1969;20:345–348. 56. Pope V, Pope E, Beck L. In vitro development of the primate embryo. In: Brans YW, Kuehl TJ, Eds. Nonhuman Primates in Perinatal Research. New York: John Wiley & Sons; 1988: 161–174. 57. Pope C, Pope V, Beck LR. Live birth following cryopreservation and transfer of a baboon embryo. Fertil Steril 1984;42:143–145. 58. Hess DL, Hendrickx AG, Stabenfeldt GH. Reproductive and hormonal patterns in the African green monkey (Cercopithecus aethiops). J Med Primatol 1979;8:273–281. 59. Van Der Horst G, Seier JV, Spinks AC, Hendricks S. The maturation of sperm motility in the epididymis and vas deferens of the vervet monkey, Cercopithecus aethiops. Int J Androl 1999;22:197–207. 60. Sparman ML, Ramsey C, Thomas CM, Mitalipov S, Fanton J, Maginnis GM, Stouffer RL, Wolf DP. Evaluation of the vervet (Cercopithecus aethiops) as a model for the assisted reproductive technologies. Am J Primatol 2007;69:1–13. 61. Sankai T, Cho F, Yoshikawa Y. In vitro fertilization and preimplantation embryo development of African green monkeys (Cercopithecus aethiops). Am J Primatol 1997;43:43–50. 62. Lopata A, Summers PM, Hearn JP. Births following the transfer of cultured embryos obtained by in vitro and in vivo fertilization in the marmoset monkey (Callithrix jacchus). Fertil Steril 1988;50: 503–509. 63. Wilton LJ, Marshall VS, Piercy EC, Moore HD. In vitro fertilization and embryo development in the marmoset monkey (Callithrix jacchus). J Reprod Fertil 1993;97:481–486. 64. Marshall VS, Kalishman J, Thomson JA. Nonsurgical embryo transfer in the common marmoset monkey. J Med Primatol 1997; 26:241–247. 65. Gould KG, Cline EM, Williams WL. Observations on the induction of ovulation and fertilization in vitro in the squirrel monkey (Saimiri sciureus). Fertil Steril 1973;24:260–268. 66. Kuehl TJ, Dukelow WR. Time relations of squirrel monkey (Saimiri sciureus) sperm capacitation and ovum maturation in an in vitro fertilization system. J Reprod Fertil 1982;64:135–137. 67. Dukelow WR. Human chorionic gonadotropin: Induction of ovulation in the squirrel monkey. Science 1979;206:234–235. 68. Denis LT, Poindexter AN, Ritter MB, Seager SWJ, Deter RL. Freeze preservation of squirrel monkey sperm for use in timed fertilization studies. Fertil Steril 1976;27:723–729.

404

SECTION V / MODELS OF BEHAVIOR

69. Gould KG. Ovum recovery and in vitro fertilization in the chimpanzee. Fertil Steril 1983;40:378–383. 70. Pope CE, Dresser BL, Chin NW, Liu JH, Loskutoff NM, Behnke EJ, Brown C, McRae MA, Sinoway CE, Campbell MK, Cameron KN, Owens OM, Johnson CA, Evans RR, Cedars MI. Birth of a western lowland gorilla (Gorilla gorilla gorilla) following in vitro fertilization and embryo transfer. Am J Primatol 1997;41: 247–260. 71. Lanzendorf SE, Holmgren WJ, Schaffer N, Hatasaka H, Wentz AC. In vitro fertilization and gamete micromanipulation in lowland gorilla. J Assist Reprod Genet 1992;9:358–364. 72. Huntress SL, Loskutoff NM, Raphael BL, Yee B, Bowsher TR, Putman JM, Kraemer DC. Pronucleus formation following in vitro fertilization of oocytes recovered from a gorilla (Gorilla gorilla gorilla) with unilateral endometroid adenocarcinoma of the ovary. Am J Primatol 1989;18:259–266. 73. Iliff SA, Molskness TA, Stouffer RL. Anti-human gonadotropin antibodies generated during in vitro fertilization (IVF)-related cycles: Effect on fertility of rhesus macaques. J Med Primatol 1995;24:7–11. 74. Chan AW, Dominko T, Luetjens CM, Neuber E, Martinovich C, Hewitson L, Simerly CR, Schatten GP. Clonal propagation of primate offspring by embryo splitting. Science 2000;287:317–319. 75. Mitalipov SM, Yeoman RR, Nusser KD, Wolf DP. Rhesus monkey embryos produced by nuclear transfer from embryonic blastomeres or somatic cells. Biol Reprod 2002;66:1367–1373. 76. Mitalipov S, Wolf DP. Nuclear transfer in non-human primates. In: Verna P, Trounson A, Eds. Nuclear Transfer Protocols: Cell Reprogramming and Transgenesis. Totowa, NJ: Humana Press, 2006. 77. Simerly C, Dominko T, Navara C, Payne C, Capuano S, Gosman G, Chong KY, Takahashi D, Chace C, Compton D, Hewitson L, Schatten G. Molecular correlates of primate nuclear transfer failures. Science 2003;300:297. 78. Simerly C, Navara C, Hyun SH, Lee BC, Kang SK, Capuano S, Gosman G, Dominko T, Chong KY, Compton D, Hwang WS, Schatten G. Embryogenesis and blastocyst development after somatic cell nuclear transfer in nonhuman primates: Overcoming defects caused by meiotic spindle extraction. Dev Biol 2004;276:237–252. 79. Ng SC, Chen N, Yip WY, Liow SL, Tong GQ, Martelli B, Tan LG, Martelli P. The first cell cycle after transfer of somatic cell nuclei in a non-human primate. Development 2004;131:2475–2484. 80. Zhou Q, Yang SH, Ding CH, He XC, Xie YH, Hildebrandt TB, Mitalipov SM, Tang XH, Wolf DP, Ji WZ. A comparative approach to somatic cell nuclear transfer in the rhesus monkey. Hum Reprod 2006;22:22. 81. Mitalipov SM, Zhou Q, Byrne J, Ji WZ, Wolf DP. Reprogramming events and developmental competence of rhesus monkey embryos produced by somatic cell nuclear transfer. Annual Conference of the International Embryo Transfer Society, Orlando, FL. 7–11 January 2006, 139. 82. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154–156. 83. Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Becker RA, Hearn JP. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci USA 1995;92:7844–7848. 84. Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Hearn JP. Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts. Biol Reprod 1996;55:254–259. 85. Suemori H, Tada T, Torii R, Hosoi Y, Kobayashi K, Imahie H, Kondo Y, Iritani A, Nakatsuji N. Establishment of embryonic stem cell lines from cynomolgus monkey blastocysts produced by IVF or ICSI. Dev Dyn 2001;222:273–279. 86. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147. 87. Donovan PJ, Gearhart J. The end of the beginning for pluripotent stem cells. Nature 2001;414:92–97. 88. Ginis I, Luo Y, Miura T, Thies S, Brandenberger R, Gerecht-Nir S, Amit M, Hoke A, Carpenter MK, Itskovitz-Eldor J, Rao MS. Dif-

89. 90.

91.

92.

93.

94.

95.

96.

97. 98.

99.

100.

101.

102.

103.

104.

105.

106.

107.

ferences between human and mouse embryonic stem cells. Dev Biol 2004;269:360–380. Marshall VS, Waknitz MA, Thomson JA. Isolation and maintenance of primate embryonic stem cells. Methods Mol Biol 2001;158:11–18. Mitalipov S, Kuo HC, Byrne J, Clepper L, Meisner L, Johnson J, Zeier R, Wolf D. Isolation and characterization of novel rhesus monkey embryonic stem cell lines. Stem Cells 2006;1:1. Cibelli JB, Grant KA, Chapman KB, Cunniff K, Worst T, Green HL, Walker SJ, Gutin PH, Vilner L, Tabar V, Dominko T, Kane J, Wettstein PJ, Lanza RP, Studer L, Vrana KE, West MD. Parthenogenetic stem cells in nonhuman primates. Science 2002;295:819. Sasaki E, Hanazawa K, Kurita R, Akatsuka A, Yoshizaki T, Ishii H, Tanioka Y, Ohnishi Y, Suemizu H, Sugawara A, Tamaoki N, Izawa K, Nakazaki Y, Hamada H, Suemori H, Asano S, Nakatsuji N, Okano H, Tani K. Establishment of novel embryonic stem cell lines derived from the common marmoset (Callithrix jacchus). Stem Cells 2005;23:1304–1313. Vrana KE, Hipp JD, Goss AM, McCool BA, Riddle DR, Walker SJ, Wettstein PJ, Studer LP, Tabar V, Cunniff K, Chapman K, Vilner L, West MD, Grant KA, Cibelli JB. Nonhuman primate parthenogenetic stem cells. Proc Natl Acad Sci USA 2003;100: 11911–11916. Li T, Wang S, Xie Y, Lu Y, Zhang X, Wang L, Yang S, Wolf D, Zhou Q, Ji W. Homologous feeder cells support undifferentiated growth and pluripotency in monkey embryonic stem cells. Stem Cells 2005;23:1192–1199. Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971–974. Levenstein ME, Ludwig TE, Xu RH, Llanas RA, VanDenHeuvelKramer K, Manning D, Thomson JA. Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells 2006;24:568–574. Byrne J, Mitalipov S, Wolf D. Current progress with primate embryonic stem cells. Curr Stem Cell Res Ther 2006;1:127–138. Fluckiger AC, Marcy G, Marchand M, Negre D, Cosset FL, Mitalipov S, Wolf D, Savatier P, Dehay C. Cell cycle features of primate embryonic stem cells. Stem Cells 2006;24:547–556. Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP, Waknitz MA, Itskovitz-Eldor J, Thomson JA. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 2000;227: 271–278. Takada T, Suzuki Y, Kondo Y, Kadota N, Kobayashi K, Nito S, Kimura H, Torii R. Monkey embryonic stem cell lines expressing green fluorescent protein. Cell Transplant 2002;11:631–635. Ma J, Ramezani A, Lewis R, Hawley RG, Thomson JA. High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors. Stem Cells 2003;21:111–117. Wang Z, Jaenisch R. At most three ES cells contribute to the somatic lineages of chimeric mice and of mice produced by EStetraploid complementation. Dev Biol 2004;275:192–201. Asano T, Ageyama N, Takeuchi K, Momoeda M, Kitano Y, Sasaki K, Ueda Y, Suzuki Y, Kondo Y, Torii R, Hasegawa M, Ookawara S, Harii K, Terao K, Ozawa K, Hanazono Y. Engraftment and tumor formation after allogeneic in utero transplantation of primate embryonic stem cells. Transplantation 2003;76:1061–1067. Fujimoto A, Mitalipov SM, Kuo HC, Wolf DP. Aberrant genomic imprinting in rhesus monkey embryonic stem cells. Stem Cells 2006;24:595–603. Dean W, Bowden L, Aitchison A, Klose J, Moore T, Meneses JJ, Reik W, Feil R. Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: Association with aberrant phenotypes. Development 1998;125:2273–2282. Rugg-Gunn PJ, Ferguson-Smith AC, Pedersen RA. Epigenetic status of human embryonic stem cells. Nat Genet 2005;37: 585–587. Nonomura N, Nishimura K, Miki T, Kanno N, Kojima Y, Yokoyama M, Okuyama A. Loss of imprinting of the insulin-like growth factor II gene in renal cell carcinoma. Cancer Res 1997;57:2575–2577.

43

Rat Models of Polycystic Ovary Syndrome KRISHNA B. SINGH

ABSTRACT

tion. The focus of the review will be on morphological and hormonal data on the validated rat PCO models. In the final section, the key issues pertaining to animal models in PCO research are discussed.

Polycystic ovary (PCO) syndrome is a common endocrine disorder of unknown etiology. This condition is a major cause of menstrual irregularity and infertility in women with chronic anovulation. This chapter reviews the literature on methods of producing experimental PCO models in the laboratory rat, and the findings that support their resemblance to the human condition. The principal rat PCO models that have been validated include constant light exposure, hypothalamic lesions, sex steroid-induced models, and the mifepristone (RU486) model. The biological systems involved in developing chronic anovulation and PCO in these models include the hypothalamus, the pituitary gland, the adrenals, and the ovary with its paracrine, autocrine, and intracrine regulators. Animal models are central to making the transition from scientific concepts to understanding the reality of a human disease. Validated animal models can be used for therapeutic screens, in preclinical trials, and for basic research in reproductive biology. However, animal models of chronic anovulation and PCO may not fully reproduce the reproductive events seen in the human syndrome. Key Words: Animal models, Persistent estrus, Polycystic ovary, Polycystic ovary syndrome, Chronic anovulation.

ESTROUS CYCLE The short estrous cycle (4–5 days) in rats and mice makes them ideal for exploring many physiological changes that occur during a reproductive cycle.12 The ovarian cycle once initiated at puberty is characterized by repeating patterns of cellular growth, differentiation, and transformation of ovarian tissues. The dynamic events include follicular development and ovulation with subsequent formation and regression of the corpora lutea. The sequence of these events is orchestrated by several interacting ovarian factors and the cyclic production of hormones from the anterior pituitary and hypothalamus. Estrogen exerts a critical regulatory influence upon the biosynthetic and secretory activity of the gonadotropin-releasing hormone (GnRH) neurons. It seems likely that estrogen regulates the behavior of the GnRH neuron through multiple transsynaptic, neuronal–glial, and direct membrane modes of action. Preovulatory GnRH and luteinizing hormone (LH) surges depend on activation of estrogen (E2)-inducible progesterone (P) receptors in the preoptic area. Surges do not occur in males or in perinatally androgenized females (see below).

INTRODUCTION Polycystic ovary (PCO) syndrome is a common but complex endocrine disorder among women of reproductive age. Women with this disorder present with menstrual irregularity, hirsutism, chronic anovulation, and infertility. Despite many clinical and basic animal studies reported in the literature, the etiology and pathogenesis of chronic anovulation and PCO syndrome remain enigmatic.1,2 The first systematic studies on a rat PCO model were reported in the 1960s.3–5 Since then newer rat PCO models have been developed and validated. These studies provide unique concepts in understanding the nature of chronic anovulation and polycystic ovary.6–11 This chapter first provides background information on the estrous cycle in rats and explains the terminology frequently used in basic research. It then reviews the literature on various methods of producing experimental PCO models in the laboratory rat, and the findings that support their resemblance to the human condi-

TERMINOLOGY The term persistent estrus means failure of two or more consecutive estrous cycles in adult rats. Persistent estrus is documented by precornified and cornified vaginal smears lasting for more than 10 days. The term PCO disease means the presence of persistent estrus, chronic anovulation, and polycystic or polyfollicular ovaries.4,5,7,11 The term polycystic ovary syndrome refers to the clinical disorder or syndrome in women of reproductive age. The following section describes studies in female rats according to the methods for induction of persistent estrus, chronic anovulation, and polycystic ovaries.

CONSTANT LIGHT The constant-light rat model is a simple, reversible, inexpensive, and reproducible model to study the pathogenesis of chronic anovulation and its reproductive outcomes. The exposure of young female Sprague–Dawley rats to continuous light gradually

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

405

406

SECTION V / MODELS OF BEHAVIOR

causes failure of the estrous cycle and infertility because of chronic anovulation associated with persistent estrus. During the early stages of constant light exposure the rats show regular estrous cycles, but the cycles become irregular and persistent estrus develops with increased duration of constant light. On changing the light schedule from constant light to light and dark, persistent estrus was interrupted in 32 of 34 (94.1%) rats after an average of 7.5 days. The ovaries of rats exposed to constant light for 56 days, then to light and dark for 28 days, were normal in appearance.3–5 When placed in darkness for 10 h, 80% of the animal ovulated 46 h later.13 The onset of persistent estrus in female rats depends on the intensity, duration, and spectral characteristics of the environmental light. The absence of 24-h time cues contributes to the induction of persistent estrus when the animals are housed under constant light.14 Constant light exposure of female rats for as little as 3 days causes a shift in phase of the LH surge related to decreased sensitivity to E2/P involved in GnRH release at the hypothalamus.15 The exposure of constant light disrupts the estrous cycle and decreases fertility in young female rats by inducing chronic anovulation associated with persistent estrus. However, light–dark cyclicity is not critical for postpartum ovulation in constant-light rats.16 Histological examinations of the ovary in constant-light rats show occasional primordial or secondary follicles, several cystic follicles, and the absence of corpora lutea.4–6 Electron microscopic studies have further revealed abnormal findings in the tunica albuginea, granulosa cells, and theca cells compared with agematched controls.17 The hormonal findings reported in constant-light rats have been consistent among published studies by various authors. Compared with control values, serum LH levels are normal, follicle-stimulating hormone (FSH) levels are low, E2 levels are high and P levels are low.17–20 With high E2/P ratios the constant-light rat model represents an estrogen-dominant condition. In our laboratory, the constant-light PCO rat models have been studied for several medical and surgical methods of ovulation induction, and for mating and pregnancy outcomes. Results show the rats exposed to constant light resume estrous cycles when exposed to the light and dark laboratory environment. The constant-light rats ovulate following mating with healthy male partners, and when treated with injections of P and human chorionic gonadotropin (hCG) or unilateral ovariectomy. Pregnancy outcomes are normal, and the pups raised in the constant-light environment do not develop polycystic ovaries until they reach puberty.3–5,21,22 The mechanism of chronic anovulation in constant-light rats is not fully understood. The GnRH sensitivity of the pituitary gland for releasing LH is decreased in constant-light rats with persistent estrus.3 Giving injections of naloxone elevates serum FSH and LH during ovulation and significantly increases the hypothalamic levels of β-endorphin compared with saline-injected controls. Blockade of opiate receptors increases β-endorphin production and uptake and/or decreases its release from the hypothalamus.23 There is evidence that anovulation in constant-light rats may result from activation of the sympathetic nervous system that results in adrenal gland hypertrophy during light exposure.4,5 In animals exposed to chronic lighting, the absolute and relative volumes of zona fasciculata are significantly increased. The serum

concentration of corticosterone is also significantly increased in these rats in comparison with controls. These findings suggest that exposure of female rats to constant light increases growth and secretory activity of zona fasciculata cells, which may be related to chronic stress.24 Chronic anovulation during continuous light exposure in rats may also be caused by decreased pineal gland activity and suppression of melatonin production. Treatment with melatonin induces ovulation in about 70% of constant-light rats.25 Feeding precooked maize devoid of tryptophan to light-exposed rats suppresses persistent estrus and significantly reduces estrus–proestrus frequency.26

LIMBIC SYSTEM The limbic system of the brain consists of the hippocampus, hypothalamus, thalamus, amygdala, and the pineal gland. The neural elements obligatory for GnRH release in female rats are found within and or immediately close to the medial preoptic nucleus of the hypothalamus. Preovulatory GnRH and LH surges depend on activation of estrogen-inducible P receptors in the preoptic area of the hypothalamus. Estrogen signaling to GnRH neurons is critical for coordinating the preovulatory surge release of LH that leads to full follicular maturation and ovulation. The preovulatory surges of GnRH and LH are activated by increased E2 concentrations in blood.27 The mechanisms for the blockade of the GnRH surge in persistent estrus rats are poorly understood. Several experimental approaches to manipulate the limbic system have been taken to induce persistent estrus in female rats. Electrolytic lesions using 5 and 10 µA of direct current into the anterior medial preoptic nucleus or the suprachiasmatic nucleus of female rats induced persistent estrus or irregular estrous cycles about 5 weeks after lesions. The preovulatory surges of LH and prolactin (PRL) were eliminated in both groups of persistent estrus rats.28 Amygdala kindling in adult female rats causes persistent estrus and development of PCOs associated with high serum E2 and increased pituitary weight. The ovary shows many cystic follicles, as well as follicles in various stages of growth and atresia. Treatment with P restored cyclicity in the persistent estrus kindled rats. The amygdaloid-lesioned rats provide powerful tools to investigate the association of PCO and epilepsy.29,30 Pinealectomy in female rats induces persistent estrus with morphological features of PCO as seen in the constant light rat model.31

ANDROGENS Several studies have shown that giving an androgen such as testosterone propionate (TP) to female rats during early postnatal life results in persistent estrus and polyfollicular anovulatory ovaries during adulthood.5–7,11 In one study Wistar female rats were injected with testosterone (T) or 5β-dihydrotestosterone (5βDHT) for the first 5 days of life. Neonatal treatment with 1 mg of T resulted in persistent estrus in all of the animals; however, among the females injected with 1 mg of 5ß-DHT, 74% of rats became sterile at 120 days of age. When the daily dose of 5β-DHT was reduced to 0.1 mg, only 33% of the rats became sterile.32 In another study, the androgenized rats showed hyperprolactinemia 40 days after they were given 1.25 mg TP at 5 days of age. The polycystic ovaries showed hyperthecosis, but no thickening of the tunica albuginea.33

CHAPTER 43 / RAT MODELS OF POLYCYSTIC OVARY SYNDROME

The serum LH, FSH, T, and E2 values in TP androgenized rats were not different from control values, and treatment with FSH did not increase serum E2 concentrations or the ovarian weights. The androgenized rats showed a similar postunilateral ovariectomy increase in serum FSH, and compensatory ovarian hypertrophy was not noted compared with controls. Because of these findings it was suggested that the androgenized rat is not a proper model for the human PCO syndrome.34 Daily treatment with 4 or 20 mg of danazol in the TP androgenized rats caused regression of cystic follicles. Serum levels of LH, T, and E2 were significantly decreased, and the vaginal smears changed to diestrus suggesting inhibition of estrogenic activity.35 Giving an antiandrogen flutamide caused partial normalization of the hormonal and morphological characteristics and recovery of fertility in the animals.36 Long-term persistence of hormone imbalances in androgen-sterilized rats can induce atypical hyperplasia and adenocarcinoma of the uterus after 500 days of age.37 An androgen-sterilized rat (ASR) model that showed obesity was established by subcutaneous injections of 1.25 mg TP to Sprague–Dawley female rats at the age of 9 days. The rats sacrificed around 112 days of age showed polycystic ovaries, anovulation, high food intake, increased body weight, and obesity. High levels of serum E2 and leptin were noted, but serum FSH and LH levels were reduced significantly compared with normal rats. After feeding herbal tea to the animals, the endocrine changes were normalized, ovulation was induced, and the animals also lost weight.38 Another ASR model of obesity and polycystic ovaries was developed in Wistar-Hannover rats that showed a significant increase in body weight starting at the fifth week of life compared with control rats.39 Implanting androstenedione (ADO) Silastic capsules that released 63.2 ± 4.4 µg/24 h ADO into cyclic rats resulted in significant prolongation of the estrous cycles; most rats showed 5day cycles after the first normal cycle. There was a decline in ovulation rate with low serum LH levels and normal FSH on the morning of estrus.40 Giving dehydroepiandrosterone (DHEA) to neonatal female rats results in persistent estrus and polycystic ovaries with or without corpora lutea. Serum FSH and LH levels showed an ovulatory surge on day 30 at the time of precocious ovulation. Serum FSH levels were comparable to control rats, LH levels were low, and serum PRL was raised significantly. The withdrawal of DHEA treatment resulted in restoration of cyclicity in this model.8,9,41,42 In DHEA-induced PCO rats, serum FSH, LH, and PRL levels did not differ significantly compared with controls, and the levels of serum DHEA, T, and ADO were high.43 In another study, the DHEA-treated rats showed normal serum LH and FSH levels, increased DHEAS, ADO, T, and E2, and reduced serum P level.44 With pharmacological doses of androgens given to female rats, the acyclic pattern of gonadotropin secretion is associated with irreversible changes in the hypothalamic cyclic centers normally responsive to steroid feedback. Dose–response studies using various androgens in the neonatal pups to induce persistent estrus during adulthood are limited. According to one study, the majority (99%) of the rats treated with androgens other than DHEA were in persistent estrus by 90 days of age.45 In another dose–response study, female rats were androgenized by giving a single injection of 10 or 100 µg TP at 5 days of age. Treatment with 100 µg TP resulted in persistent estrus while

407

treatment with 10 µg TP resulted in regular estrous cycles followed by persistent estrus vaginal smears.46 Dose–response studies of the reproductive and developmental effects of exposure to prenatal TP and other sex steroids provide unique androgenized rat models.47

ESTROGENS Like androgens, giving estrogens to female rats during early postnatal life results in persistent estrus and polyfollicular anovulatory ovaries during adulthood.5–7,11 Female rats injected with 2 mg estradiol valerate (EV) develop persistent estrus and small polyfollicular ovaries within 2 months. Plasma E2 concentrations are high while LH and FSH concentrations are within the high and low normal range, respectively. This model has been used to study the pathogenesis of chronic anovulation in female rats.48–53 Increased sympathetic activity plays a role in forming polyfollicular ovarian cysts in EV-treated rats. This theory was tested using a combined cold and restraint stress that induced an increase in the ovarian sympathetic nerves.54 The activation of the sympathetic nervous system precedes the induction of polycystic ovaries in EV-treated rats. The mechanism involves both direct and neurogenic components.55,56 There is evidence that persistent estrus in estradiol benzoate (EB)-treated rats is associated with delayed maturation of the tonic GnRH systems and that it affects estrogen receptor synthesis in the uterine tissues during adulthood.57,58

ANTIPROGESTERONE Mifepristone (RU486) is an 11ß-dimethylaminophenyl derivative of norethindrone with a high affinity for P and glucocorticoid receptors. Subcutaneous administration of 4 mg of RU486 to cyclic rats over 8 consecutive days induces persistent estrus and anovulatory cystic ovaries. In this model, serum LH/FSH and T/E2 ratios are high.59 Giving RU486 to cyclic rats on proestrus advances the preovulatory surge of LH, which results in 3-day estrous cycles.60 The mechanism of inducing chronic anovulation in the RU486 rat model is not fully understood. Several studies in RU486 rats have been conducted to elucidate the pathogenesis of chronic anovulation and to show the validity of the model.61 Like the constant light and DHEA rat models, the RU486 rat PCO model is also reversible.

AROMATASE INHIBITORS Giving a third-generation aromatase inhibitor letrozole daily for 21 days induces polycystic ovaries in cyclic female rats. The ovaries a showed high incidence of subcapsular polyfollicular cysts, capsular thickening, incomplete luteinization, and a decreased number of corpora lutea. Serum E2 and P levels were reduced in a dose-dependent manner and serum LH and T levels were high. Serum levels of FSH were increased at higher doses of letrozole (0.5 and 1.0 mg/kg), contrary to a low dose of letrozole (0.1 mg/kg) at which a slight decrease was observed. The low-dose letrozole persistent estrus rat model shows morphological and hormonal findings that mimic the human PCO syndrome.62

DISCUSSION This chapter reviews various methods of producing persistent estrus, chronic anovulation, and PCO in the laboratory rat. The principal validated models are constant light exposure, hypotha-

408

SECTION V / MODELS OF BEHAVIOR

lamic lesions, androgen- and estrogen-induced models, and the mifepristone (RU486) model. The vast literature on other animal models has been reviewed by the author at http://obg.lsuhsc-s. edu/pco/index.html. The main biological systems involved in the development of chronic anovulation and PCO in animal models include the hypothalamus, the pituitary gland, the adrenals, and the ovary with its paracrine, autocrine, and intracrine regulators. The arrested follicular growth, lack of dominant follicles, and thecal and stromal hyperplasias in the polycystic ovaries may represent expressions of several pathogenic pathways including the ovarian paracrine, autocrine, and intracrine regulators.63 The pathogenesis of chronic anovulation, the hallmark feature of the PCO syndrome, has been recently studied using animal models such as the prenatally androgenized sheep and rhesus monkey.1,2 The laboratory rats and mice have key advantages over the androgenized sheep and monkey models to study chronic anovulation and PCO because of their small size, high reproductive index, genetic diversity, and low maintenance costs. The newer animal models and their implications for future research should be considered in the context for replicating the human disorders. For example, the transgenic mice models have been investigated to determine how excess LH leads to cyst formation in the ovary associated with infertility. Targeted overexpression of LH in transgenic mice leads to obesity, infertility, polycystic ovaries, and the formation of ovarian tumors.64,65 The prenatally androgenized transgenic mice show irregular estrous cycles and elevated testosterone and luteinizing hormone levels, suggesting altered hypothalamic–pituitary–gonadal axis function.66 Hormone receptors and knockout mouse models may also provide new insights into the interacting roles of hormones, receptors, and enzymes in chronic anovulation.67–69 Environmental factors that cause significant stress can also trigger chronic anovulation and PCO both in animal models and in women with this syndrome.70–72 Studies in our laboratory have shown, for example, that exposure of adult female Sprague– Dawley rats to continuous noise for more than a month produces persistent estrus and PCO much like the constant-light rats. The ovaries of the noise-exposed rats after 150 days of high-intensity noise showed morphological features similar to the constant-light rats.73 Polycystic ovary syndrome is a complex and heterogeneous disorder characterized by hyperandrogenemia, hyperinsulinemia, insulin resistance, and chronic anovulation. Currently PCO syndrome is considered a polygenic trait that might result from the interactions of susceptible and protective genomic variants and environmental factors during either prenatal or postnatal life. Prenatal androgenization of the female fetus induced by genetic and environmental factors, or the interaction of both, may program differentiating target tissues toward the development of the PCO syndrome phenotype in adult life.1,2,74 Computer models and in vitro cell culture studies have enhanced our understanding of physiologically important phenomena in reproductive biology.75 Animal models are central to making the transition from scientific concepts to understanding the reality of a human disease. Validated animal models can be used for therapeutic screens, in preclinical trials, and for basic research in reproductive biology. However, animal models of chronic anovulation and PCO may not fully reproduce the reproductive events seen in the human syndrome.

REFERENCES 1. Tsilchorozidou T, Overton C, Conway GS. The pathophysiology of polycystic ovary syndrome. Clin Endocrinol (Oxf) 2004;60:1–17. 2. Abbott DH, Dumesic DA, Franks S. Developmental origin of polycystic ovary syndrome—a hypothesis. J Endocrinol 2002;174:1–5. 3. Singh KB, Greenwald GS. Effects of continuous light on the reproductive cycle of the female rat: Induction of ovulation and pituitary gonadotrophins during persistent oestrus. J Endocrinol 1967;38: 389–394. 4. Singh KB. Induction of polycystic ovarian disease in rats by continuous light. I. The reproductive cycle, organ weights, and histology of the ovaries. Am J Obstet Gynecol 1969;103:1078–1083. 5. Singh KB. Persistent estrus: An experimental model of the polycystic ovary syndrome. Obstet Gynecol Surv 1969;24:2–17. 6. Baldissera SF, Motta LD, Almeida, MC, Antunes-Rodrigues J. Proposal of an experimental model for the study of polycystic ovaries. Braz J Med Biol Res 1991;24:747–751. 7. Mahajan DK. Polycystic ovarian disease: Animal models. Endocrinol Metab Clin North Am 1988;17:705–732. 8. Mahesh VB, Costoff A, Mills TM, Bagnell CA. The polycystic ovary syndrome and experimental models for the study of its pathogenesis. Prog Clin Biol Res 1982;112:301–313. 9. Mahesh VB, Mills TM, Bagnell CA, Conway BA. Animal models for study of polycystic ovary and ovarian atresia. Adv Exp Med Biol 1987;219:237–257. 10. Szukiewicz D, Uilenbroek JT. Polycystic ovary syndrome—searching for an animal model. J Med 1998;29:259–275. 11. Singh KB. Persistent estrus rat models of polycystic ovary disease: An update. Fertil Steril 2005;84:1228–1234. 12. Marcondes FK, Bianchi FJ, Tanno AP. Determination of the estrous cycle phases of rats: Some helpful considerations. Braz J Biol 2002;62:609–617. 13. Takahashi M, Ford JJ, Yoshinaga K, Greep RO. Ovulation in lightestrous rats induced by darkness. Endocrinol Jpn 1977;24:89–96. 14. Weber AL, Adler NT. Delay of constant light-induced persistent vaginal estrus by 24-hour time cues in rats. Science 1979;204: 323–325. 15. Watts AG, Fink G. Effects of short-term constant light on the proestrous luteinizing hormone surge and pituitary responsiveness in the female rat. Neuroendocrinology 1981;33:176–180. 16. Rowland DL, van der Schoot P. Effect of constant light on parturition and postpartum reproduction in the rat. Physiol Behav 1995;58: 567–572. 17. Singh KB, Mahajan DK. Ultrastructural basis for continued steroidogenesis in the rat polycystic ovary. J Reprod Med 1990;35: 222–228. 18. Singh KB, Mahajan DK, Tewari RP. Hormonal modulation of the vaginal bacterial flora in experimental polycystic ovarian disease. J Clin Lab Anal 1996;10:233–238. 19. Takeo Y. Influence of continuous illumination on estrous cycle of rats: Time course of changes in levels of gonadotropins and ovarian steroids until occurrence of persistent estrus. Neuroendocrinology 1984;39:97–104. 20. Takeo Y, Kohno J, Hokano M. Ultrastructural evidence for estradiol synthesis in the ovary of persistent-estrous rats exposed to continuous illumination. Acta Anat 1986;127:161–170. 21. Singh KB. Induction of polycystic ovarian disease in rats by continuous light. II. Observations on mating, pregnancy, and the postpartum period. Am J Obstet Gynecol 1969;104:1004–1007. 22. Singh KB. Induction of polycystic ovarian disease in rats by continuous light. III. Mechanism of ovarian compensatory hypertrophy and ovulation after unilateral oophorectomy. Am J Obstet Gynecol 1969;104:1008–1011. 23. Hulse GK, Coleman GJ, Copolov DL, Lee VW. The role of endogenous opioid peptides in the effects of constant illumination on reproductive function in the rat. Pharmacol Biochem Behav 1985;23:535–539. 24. Milosevic V, Trifunovic S, Sekulic M, Sosic-Jurjevic B, Filipovic B, Negic N, et al. Chronic exposure to constant light affects morphology

CHAPTER 43 / RAT MODELS OF POLYCYSTIC OVARY SYNDROME

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40. 41.

42.

43.

44.

and secretion of adrenal zona fasciculata cells in female rats. Gen Physiol Biophys 2005;24:299–309. Trentini GP, Mess B, De Gaetani CF, Ruzsas C. Effect of melatonin on induction of ovulation in the light-induced constant estrousanovulatory syndrome and possible role of the brain serotoninergic system. J Endocrinol Invest 1978;1:305–310. Giammanco S, Ernandes M, La Guardia M. Effects of environmental lighting and tryptophan devoid diet on the rat vaginal cycle. Arch Physiol Biochem 1997;105:445–449. Petersen SL, Ottem EN, Carpenter CD. Direct and indirect regulation of gonadotropin-releasing hormone neurons by estradiol. Biol Reprod 2003;69:1771–1778. Ma YJ, Kelly MJ, Ronnekleiv OK. Pro-gonadotropin-releasing hormone (ProGnRH) and GnRH content in the preoptic area and the basal hypothalamus of anterior medial preoptic nucleus/suprachiasmatic nucleus-lesioned persistent estrous rats. Endocrinology 1990;127:2654–2664. Edwards HE, Burnham WM, Ng MM, Asa S, MacLusky NJ. Limbic seizures alter reproductive function in the female rat. Epilepsia 1999;40:1370–1377. Edwards HE, MacLusky NJ, Burnham, WM. The effect of seizures and kindling on reproductive hormones in the rat. Neurosci Biobehav Rev 2000;24:753–762. Prata Lima MF, Baracat EC, Simoes MJ. Effects of melatonin on the ovarian response to pinealectomy or continuous light in female rats: Similarity with polycystic ovary syndrome. Braz J Med Biol Res 2004;37:987–995. Arai Y, Yamanouchi K, Mizukami S, Yanai R, Shibata K, Nagasawa H. Induction of anovulatory sterility by neonatal treatment with 5 beta-dihydrotestosterone in female rats. Acta Endocrinol (Copenh) 1981;96:439–443. Ota H, Fukushima M, Maki M. Endocrinological and histological aspects of the process of polycystic ovary formation in the rat treated with testosterone propionate. Tohoku J Exp Med 1983;140:121– 131. Jones HM, Vernon MW, Rush ME. Systematic studies invalidate the neonatally androgenized rat as a model for polycystic ovary disease. Biol Reprod 1987;36:1253–1265. Raj SG, Raj MH, Talbert LM, Dy RC. Structural and functional regression of polycystic ovaries by danazol. Fertil Steril 1981;36:392–395. Reznikov AG, Sinitsyn PV, Tarasenko LV, Polyakova LI. Neuroendocrine mechanisms of development of experimental hyperandrogeninduced anovulation. Neurosci Behav Physiol 2003;33:773–776. Morikawa S, Sekiya S, Naitoh M, Iwasawa H, Takeda B, Takamizawa H. Spontaneous occurrence of atypical hyperplasia and adenocarcinoma of the uterus in androgen-sterilized SD rats. J Natl Cancer Inst 1982;69:95–101. Sun F, Yu J. The effect of a special herbal tea on obesity and anovulation in androgen-sterilized rats. Proc Soc Exp Biol Med 2000; 223:295–301. Borges da Silva B, Rocha Gontijo JA, Crete AR, de Jesus Simoes M. Evaluation of body weight in androgenized female rats. Clin Exp Obstet Gynecol 2002;29:97–99. Gnodde,HP, van Dieten JA, Van Look PF. Effect of androstenedione on the estrous cycle of the rat. J Reprod Fertil 1979;56:675–678. Knudsen JF, Costoff A, Mahesh VB. Dehydroepiandrosteroneinduced polycystic ovaries and acyclicity in the rat. Fertil Steril 1975;26:807–817. Ward RC, Costoff A, Mahesh VB. The induction of polycystic ovaries in mature cycling rats by the administration of dehydroepiandrosterone (DHA). Biol Reprod 1978;18:614–623. Anderson E, Lee GY, O’Brien K. Polycystic ovarian condition in the dehydroepiandrosterone-treated rat model: Hyperandrogenism and the resumption of meiosis are major initial events associated with cystogenesis of antral follicles. Anat Rec 1997;249:44–53. Henmi H, Endo T, Nagasawa K, Hayashi T, Chida M, Akutagawa N, et al. Lysyl oxidase and MMP-2 expression in dehydroepiandrosterone-induced polycystic ovary in rats. Biol Reprod 2001;64: 157–162.

409

45. Mallampati RS, Johnson DC. Gonadotropins in female rats androgenized by various treatments: Prolactin as an index to hypothalamic damage. Neuroendocrinology 1974;15:255–266. 46. Handa RJ, Gorski RA. Alterations in the onset of ovulatory failure and gonadotropin secretion following steroid administration to lightly androgenized female rats. Biol Reprod 1985;32:248–256. 47. Wolf CJ, Hotchkiss A, Ostby JS, LeBlanc GA, Gray LE Jr. Effects of prenatal testosterone propionate on the sexual development of male and female rats: A dose-response study. Toxicol Sci 2002; 65:71–86. 48. Brawer JR, Naftolin F, Martin J, Sonnenschein C. Effects of a single injection of estradiol valerate on the hypothalamic arcuate nucleus and on reproductive function in the female rat. Endocrinology 1978;103:501–512. 49. Brawer J, Schipper H, Naftolin F. Ovary-dependent degeneration in the hypothalamic arcuate nucleus. Endocrinology 1980;107:274– 279. 50. Brawer JR., Munoz M, Farookhi R. Development of the polycystic ovarian condition (PCO) in the estradiol valerate-treated rat. Biol Reprod 1986;35:647–655. 51. Grosser PM, McCarthy GF, Robaire B, Farookhi R, Brawer JR. Plasma patterns of LH, FSH and prolactin in rats with a polycystic ovarian condition induced by oestradiol valerate. J Endocrinol 1987;114:33–39. 52. McCarthy GF, Farookhi R, Brawer JR. Plasma gonadotropin patterns characterizing the development of polycystic ovaries in the estradiol valerate treated rat. Can J Physiol Pharmacol 1990;68:28–33. 53. McCarthy GF, Brawer JR. Induction of Stein-Leventhal-like polycystic ovaries (PCO) in the rat: A new model for cystic ovarian disease. Anat Rec 1990;228:137–144. 54. Paredes A, Galvez A, Leyton V, Aravena G, Fiedler JL, Bustamante D, et al. Stress promotes development of ovarian cysts in rats: The possible role of sympathetic nerve activation. Endocrine 1998;8:309–315. 55. Lara HE, Dorfman M, Venegas M, Luza SM, Luna SL, Mayerhofer A, et al. Changes in sympathetic nerve activity of the mammalian ovary during a normal estrous cycle and in polycystic ovary syndrome: Studies on norepinephrine release. Microsc Res Tech 2002; 59:495–502. 56. Stener-Victorin E, Lindholm C. Immunity and beta-endorphin concentrations in hypothalamus and plasma in rats with steroid-induced polycystic ovaries: Effect of low-frequency electroacupuncture. Biol Reprod 2004;70:329–333. 57. Aihara M, Hayashi S. Induction of persistent diestrus followed by persistent estrus is indicative of delayed maturation of tonic gonadotropin-releasing systems in rats. Biol Reprod 1989;40:96–101. 58. Aihara M, Kobayashi H, Kimura T, Hayashi S, Kato J. Changes in uterine estrogen receptor concentrations in persistent estrous and persistent diestrous rats. Endocrinol Jpn 1988;35:57–70. 59. Sanchez-Criado JE, Sanchez A, Ruiz A, Gaytan F. Endocrine and morphological features of cystic ovarian condition in antiprogesterone RU486-treated rats. Acta Endocrinol (Copenh) 1993;129: 237–245. 60. Tebar M, Ruiz A, Gonzalez D, Hernandez G, Alonso R, SanchezCriado JE. Effect of RU486 injected on proestrous morning on LHRH, LH and 17beta-estradiol secretion during the estrous cycle in rat. J Physiol Biochem 1998;54:91–97. 61. Ruiz A, Aguilar R, Tebar M, Gaytan F, Sanchez-Criado JE. RU486treated rats show endocrine and morphological responses to therapies analogous to responses of women with polycystic ovary syndrome treated with similar therapies. Biol Reprod 1996;55:1284–1291. 62. Kafali H, Iriadam M, Ozardali I, Demir N. Letrozole-induced polycystic ovaries in the rat: A new model for cystic ovarian disease. Arch Med Res 2004;35:103–108. 63. Dye RB, Rabinovici J, Jaffe RB. Inhibin and activin in reproductive biology. Obstet Gynecol Surv 1992;47:173–185. 64. Mann RJ, Keri RA, Nilson JH. Consequences of elevated luteinizing hormone on diverse physiological systems: Use of the LHbetaCTP transgenic mouse as a model of ovarian hyperstimulation-induced pathophysiology. Recent Prog Horm Res 2003;58:343–375.

410

SECTION V / MODELS OF BEHAVIOR

65. Risma KA, Clay CM, Nett TM, Wagner T, Yun J, Nilson JH. Targeted overexpression of luteinizing hormone in transgenic mice leads to infertility, polycystic ovaries, and ovarian tumors. Proc Natl Acad Sci USA 1995;92:1322–1326. 66. Sullivan SD, Moenter SM. Prenatal androgens alter GABAergic drive to gonadotropin-releasing hormone neurons: Implications for a common fertility disorder. Proc Natl Acad Sci USA 2004;101: 7129–7134. 67. Couse JF, Yates MM, Sanford R, Nyska A, Nilson JH, Korach KS. Formation of cystic ovarian follicles associated with elevated luteinizing hormone requires estrogen receptor-beta. Endocrinology 2004;145:4693–4702. 68. Rosenfeld CS, Roberts RM, Lubahn DB. Estrogen receptor- and aromatase-deficient mice provide insight into the roles of estrogen within the ovary and uterus. Mol Reprod Dev 2001;59:336–346. 69. Britt KL, Drummond AE, Dyson M, Wreford NG, Jones ME, Simpson ER, Findlay JK. The ovarian phenotype of the aromatase knockout (ArKO) mouse. J Steroid Biochem Mol Biol 2001;79: 181–185.

70. Greiner M, Paredes A, Araya V, Lara HE. Role of stress and sympathetic innervation in the development of polycystic ovary syndrome. Endocrine 2005;28:319–324. 71. Singh KB. Menstrual disorders in college students. Am J Obstet Gynecol 1981;140:299–302. 72. Singh KB: Effects of sound on the female reproductive system. Am J Obstet Gynecol 1972;112:981–991. 73. Singh KB, Rao PS. Studies on the polycystic ovaries of rats under continuous auditory stress. Am J Obstet Gynecol 1970;108: 557–564. 74. Xita N, Tsatsoulis A. Review: Fetal programming of polycystic ovary syndrome by androgen excess: Evidence from experimental, clinical, and genetic association studies. J Clin Endocrinol Metab 2006;91:1660–1666. 75. Washington TM, Blum JJ, Reed MC, Conn PM. A mathematical model for LH release in response to continuous and pulsatile exposure of gonadotrophs to GnRH. Theor Biol Med Model 2004;1:9. Available at http://www.tbiomed.com/content/1/1/9. Accessed May 30, 2006.

44

Murine Models for Reproduction DAMAYANTI CHAKRABORTY AND T. RAJENDRA KUMAR

ABSTRACT

and the information is disseminated into public databases. There is a high degree of sequence similarity between mice and humans in many of the loci, important for reproduction.4 Both gain- and loss-of-function mouse models have been generated that have defects ranging from primordial germ cell specification, migration, sex determination, somatic and germ cell development and function, fertilization, implantation, and embryo development. Furthermore, models with defects in pituitary gonadotrope and hypothalamus development and function and their reproductive consequences have also been characterized. Studies with these random or targeted transgenic and knockout mouse models have also been complemented with analysis of naturally occurring mutant strains. Here, we will present a summary of various ways to manipulate the mouse genome and provide lists of representative models that have reproductive phenotypes.

Recent developments in mouse manipulation techniques have resulted in the generation of various single gene knockout mouse models. It is estimated that approximately 300 novel strains of mice with mutations in loci directly or indirectly affecting reproductive development have already been generated. Furthermore, the ability to spatiotemporally inactivate or activate gene expression in vivo using the “Cre-lox” technology has recently emerged as a powerful approach to understand various developmental processes involved in reproduction in a physiological setting. In parallel with these gene-targeting approaches, large-scale mutagenesis programs have been initiated with the goal of uncovering novel genes important for reproduction. These and other related transgenic approaches are offering unlimited opportunities to systematically analyze the complex process of reproduction. The principles of conventional and conditional gene targeting technologies, the recent advances in functional genomic approaches, and their applications to reproductive biology will be discussed. Key Words: Mice, Transgene, Knockout, Hypothalamus, Pituitary, Gonads.

TRANSGENIC APPROACHES Nearly 25 years ago, transgenic mice expressing human growth hormone were successfully developed.11 This work revolutionized various fields of biology including reproductive biology and provided a basis for many gain-of-function experiments. Several hundreds of transgenic mice related to reproduction have since been produced. In this approach, foreign genes or cDNAs are first injected into fertilized one-cell mouse embryos usually obtained following a superovulation protocol.10 Next, these transgeneinjected embryos are transferred into oviducts of pseudopregnant foster females.10 Subsequently, pups produced are screened for the presence and number of copies of the injected transgene. Independent lines will then be established by breeding the transgene-carrying founder mice. Because the transgene is injected into the mouse genome prior to the first round of replication, the foreign DNA is randomly integrated into multiple tissues.10 Most often, the transgenes are expressed in a tissue/cellspecific manner using either homologous (i.e., mouse) or heterologous (for example, rat, cow, sheep, human, pig) gene regulatory sequences.12,13 Mouse genes or cDNAs appropriately marked with random oligos or engineered with heterologous downstream polyadenylation DNA sequences are also used.12,13 The effects of overexpression of such transgenes on reproductive function are then monitored. In some instances, ectopic expression of the transgene is also achieved by purposefully directing its expression into tissues/cells different from those in which the corresponding mouse gene is normally expressed.12,13

INTRODUCTION Reproduction is a complex process that involves multiple interactions. The mammalian reproductive axis consists of three major centers, namely, the hypothalamus, pituitary, and gonads. Defects in regulation of the reproductive axis may lead to cancers, hyperstimulation syndromes, and infertility.1–3 The majority of the genes and the corresponding proteins they encode are evolutionarily conserved across various mammalian species.1,4 In recent years, the mouse has emerged as a useful model to manipulate the reproductive axis and several murine models of reproduction have been generated.5–9 There are several advantages of using mice to study basic aspects of reproduction and model human reproductive diseases. First, mice are relatively cheap, easy to maintain in a laboratory setting, and have a short gestation time and life span. Second, an exhaustive amount of data exist with regard to their genetic make-up and linkage of genes. Third, techniques to manipulate the mouse genome have been well established.10 Finally, mouse genome sequencing has been completed

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

411

412

SECTION V / MODELS OF BEHAVIOR

MAPPING REGULATORY REGIONS OF GENES THAT CONFER TISSUE/CELL-SPECIFIC EXPRESSION Promoter or other regulatory elements that confer hormone responsiveness and cell-specific expression and dictate combinatorial binding of various transcription factors are routinely mapped using cell culture models.14–20 However, many times these data may not correlate well with the in vivo expression data. In other instances, well-established cell lines may not be available for fine mapping studies of regulatory regions of some genes.21 In these cases, transgenic mice can be used as a powerful in vivo expression system. First, a series of deletion constructs are engineered from a larger piece of the gene that is known to confer cell/specific expression.21 Subsequently, the truncated transgenes are microinjected to produce transgenic mice and as an endpoint, its expression in the selected cell type is monitored at the RNA and/or protein level.21 Similar strategies have also been used in which a known transcription factor-binding site is mutated on a given

promoter driving the expression of a transgene and its functional consequence tested in vivo.22–26 TRANSGENIC MICE AND QUANTIFIABLE PROMOTER ACTIVITY If the promoter elements of a given gene are well mapped, then these can be used to direct the expression of useful reporters that can be assayed quantitatively. Strength or the effects of positive or negative factors that regulate gene expression or mutations in the promoter region that affect transcription factor binding can be quantified by this approach. The most commonly used quantifiable reporters include lacZ from Escherichia coli, chloramphenicol acetyltransferase, and the firefly luciferase.10 Several promoter regions of genes involved in reproduction have been tested using these strategies (Table 44–1). CELL LINEAGE MARKING AND PURIFICATION OF DESIRED CELL TYPES Developmental expression of many genes that have important reproductive function can be tracked using lineage marking and cell fate mapping. Depending on the

Table 44–1 A representative list of various transgenic mouse models related to reproduction Models

Expression/use

Reference

Mouse models for mapping regulatory regions of genes Gene Tissue of expression Oxytocin Hypothalamus FSH-β Pituitary Oct-4 Early germ cells SF-1 Hypothalamus, pituitary, gonads Gdf-9 Ovary, testis SP-10 Testis

119–121 21 122 123–125 126 127–129

Models for quantifiable promoter activity Promoter Reporter used Bovine α-GSU LacZ, CAT Rat LH-β Luciferase Ovine FSH-β Luciferase Mouse Gdf-9 Luciferase

22, 130 131 132, 133 126

Models for lineage marking Promoter Mouse SF-1 Mouse RARE Mouse Oct-4 Mouse GnRH

Reporter used GFP LacZ GFP GFP

134, 135 136 137, 138 139, 140

Models for cell ablation Promoter Human α-GSU Mouse inhibin-α Rat FSH-β

Toxin used Diphtheria toxin Herpes simplex virus thymidine kinase Herpes simplex virus thymidine kinase

23 32 30

Models for cell-specific immortalization Promoter Rat GnRH Human α-GSU Rat LH-β Mouse GnRH-R Human FSH-β Ovine FSH-β Human anti-Mù1llerian hormone Mouse inhibin-α Mouse inhibin-α

Oncogene used SV40 TAg SV40 TAg SV40 TAg SV40 TAg SV40 ts TAg SV40 TAg SV40 TAg SV40 TAg SV40 TAg

Cell immortalized Hypothalamic neurons Gonadotrope Gonadotrope Gonadotrope Gonadotrope Gonadotrope Sertoli cell Leydig cell Granulosa cell

141, 142 39 38 143 36 37 144 145, 146 145, 146

CHAPTER 44 / MURINE MODELS FOR REPRODUCTION

specificity and expressivity of the gene regulatory sequences and the earliest time at which these are activated during embryogenesis, expression of either lacZ, alkaline phosphatase, or various fluorescent reporters (for example, GFP, CFP, YFP or dsRed) can be targeted to specific cell types.10 Tissues/cells are then harvested and the activity of lacZ (formation of a blue product), alkaline phosphatase (formation of a blue or red product), or the visualization of distinct colors under ultraviolet illumination is monitored starting from embryonic stages.10 Tracking through distinct developmental stages will provide a novel way to study the linage specification and differentiation of desired cell types. Because cells expressing fluorescent reporters can be sorted by fluorescence-activated cell sorting, these transgenic mice will provide novel resources to purify desired cell types from a tissue consisting of heterogeneous populations of various other cells.27–29 These can be further used for gene/protein expression profiling under normal physiological conditions. CELL ABLATION BY TARGETED EXPRESSION OF TOXINS Identification of cell/specific regulatory elements has also been useful for selectively ablating cells at desired times and studying the consequences of the loss of hormones secreted from these cells.10 This has been achieved by expressing diphtheria toxin,23 or herpes simplex virus thymidine kinase,30 or viralspecific ion channels.31 In the latter two cases, either an appropriate substrate (gancyclovir) or an ionophore (calcium or sodium channel activator or blocker) is used to produce either a cell-toxic product or changes in ion flux that affect hormone secretion. These approaches have been used to study the consequences of ablation of gonadotropes on gonadal development and reproduction.30 More recently, ablation of Sertoli cells has been achieved to study the consequences on germ cell and consequently male reproduction.32 CELL-SPECIFIC IMMORTALIZATION Targeted expression of viral oncogenes in transgenic mice is a powerful way to immortalize desired cell types in vivo.33 This strategy permits immortalization of rare cell types that are otherwise difficult to obtain in large numbers and good purity for routine cell transfection analyses.34–39 Moreover, novel cell lines are derived from these tumors and established as useful in vitro tools for various studies. Since many cell types within the reproductive axis are postmitotic, this approach has been particularly useful for immortalizing these cell types and establishing novel cell lines.34–39 Many of these cell lines have been used to investigate specific signal transduction pathways, and transcriptional regulation.34–39 In some cases, these tumor-prone mouse models also phenocopy known human cancers and thus have tremendous potential to help in understanding the pathobiology of the human disease.36 Furthermore, these models can also be a useful resource for identifying novel cancer biomarkers. HIGH BASAL LEVELS AND INDUCIBLE EXPRESSION OF TRANSGENES Although expression of a given transgene is dependent on the promoter that drives its expression, the site of the integration, and the copy number, some promoters exhibit high basal activity in many tissues.40,41 This feature has been exploited to ectopically express various hormones in multiple tissues at high basal levels; the consequences of this on reproduction have been analyzed.42–44 Some of these promoters are also inducible, for example, metallothionein-1 is induced by heavy metals such as zinc and cadmium.40,41

413

An alternate strategy to temporally induce transgene expression has been developed. In this approach, the desired protein encoding cDNA or gene is engineered downstream of a conditionally activated “gene switch.”45,46 This “gene switch” consists of DNA sequences that encode a bacterial repressor protein and an operator region that is linked to the gene of interest. The repressor binds to the adjacent operator DNA sequence that is linked to the gene of interest and keeps it inactive. This entire “gene switch” cassette along with the gene of interest is under the regulation of a tissue/cell-specific promoter. When a drug that binds the repressor and prevents it from binding to the operator region is administered to transgenic mice, it allows the transcription of the desired gene at specific times. Based on this principle and using teton,47 ecdysone,45,48 RU486,49 and tamoxifen50 as inducible gene expression models, transgenic mice have been developed. Several representative models illustrating the application of the above strategies to reproduction research are listed in Table 44–1. Many transgenic approaches have also been successfully used in gene targeting experiments to genetically rescue mutant mice, spatiotemporally inactivate genes, cell fate mapping, and gene/protein expression profiling. These gene knockout models will be described in the following sections.

GENE KNOCKOUT APPROACHES Loss-of-function mouse models are commonly produced through site-specific mutations at desired loci in embryonic stem (ES) cells by homologous recombination. ES cells are derived from the inner cell mass of blastocysts at embryonic day 3.5.10 These are propagated in vitro on fibroblast cell feeder layers and in an undifferentiated state by providing selected growth factors. Desired mutations are engineered using considerable length of homologous gene sequences flanking the deletion and an appropriate drug selectable marker.51–54 These engineered DNA sequences are called the targeting vectors and are usually electroporated into ES cells derived from agouti coat color mice.10 Following appropriate drug selection, enrichment and expansion into individual clones, and identification of the correct gene-targeting event (i.e., one allele disrupted), the mutant ES cells are injected into host blastocysts derived from black coat color mice and transferred to uterine horns of pseudopregnant foster mice.10 The injected mutant ES cells mix with the inner cell mass of the host and contribute to all parts of the body, including the germ line, and the resulting chimeric mice have patches of both agouti and black coat colors.10,51 The chimeric mice, mostly males, are bred to normal females and germ line transmission of the mutant allele is achieved resulting in production of heterozygous mice. If viable and fertile, the heterozygous male and female mice are intercrossed to generate the homozygous gene knockout mice.10,51 If the mutation in the homozygous recessive condition does not cause embryonic lethality, then 25% of knockout mice are produced according to Mendelian inheritance. The majority of the murine models of reproduction have been produced by this standard approach (Tables 44–2 to 44–4). More recently, application of several variations of this basic theme have resulted in production of numerous gene knockout models (Table 44–5). These approaches are described in the following sections. TISSUE/CELL-RESTRICTED AND TEMPORALLY REGULATED CONDITIONAL GENE INACTIVATION MODELS Many proteins are expressed throughout the reproductive axis or,

414

SECTION V / MODELS OF BEHAVIOR

Table 44–2 Examples of knockout mice with reproductive defects only in males Mouse model

Major reproductive phenotypes

Reference

Acrosin AMH or Amh-R Bax Bclw Bmp8a Bmp8b Calmegin CREM Dhh Hoxd13 HR6B Hsp70 KLHL10 PC4 RARα RARβ RARγ TEX14

Delayed fertility Infertility in males due to obstruction by uteri development Premeiotic stage block in spermatogenesis; infertility Infertile; progressive loss of Sertoli and germ cells Germ cell degeneration, spermiogenesis defects, epididymis degenerate and progressive infertility Infertility; defects in primordial germ cell proliferation Infertility due to impaired sperm binding to zona pellucida of eggs Infertility; block at first stage of spermiogenesis Defective germ, Sertoli, and Leydig cell development; infertility Defects in male accessory sex glands Infertility; defects in histone polyubiquitination and degradation during spermatogenesis Infertility; increased spermatocyte apoptosis Haploinsufficiency causes asynchronous spermatid development; infertility Impaired fertilizing ability of spermatozoa; infertility Seminiferous tubule degeneration leading to male infertility Germ cell maturation defects and infertility Squamous metaplasia of seminal vesicles and prostate and male infertility Infertility; failure to form intracellular bridges after spermatogonia differentiate

147 96, 148 149 150 151 152 153, 154 155, 156 157–159 160 161 162, 163 164 165 166 167 168 169

additionally, in other tissues during distinct developmental stages.55–57 A deletion of the corresponding gene throughout the body may often result in embryonic lethality.56,57 Thus, it precludes analyzing the roles in adult physiology, for example, in gonad development and function. This commonly encountered problem is circumvented by a conditional or tissue/cell-specific gene deletion approach. In this approach, first, within the gene of interest, typically in introns flanking the exon to be deleted, two loxP (a 34-bp sequence that consists of two 13-bp inverted repeats and an 8-base pair asymmetric spacer region) sites, one on each

side, are engineered in the same orientation.58–60 A “floxed” mouse is generated in a heterozygous or homozygous condition by standard gene targeting approaches, and this introduction of loxP sites does not interfere with the normal expression of the gene of interest. Second, a transgenic line of mice is produced in which cre, a phage P1 recombinase, is selectively expressed in desired tissue/ cells.58–60 When the flox mice are intercrossed with the cre line, in desired cells, the DNA between the loxP sites is deleted by cre-mediated recombination, leaving one loxP site intact at the locus.58–60

Table 44–3 Examples of knockout mice with reproductive defects only in females Mouse model

Major reproductive phenotypes

Reference

Activin/inhibin βB Caspase-2 C/EBPβ c-mos Connexin 37 Connexin 43 Cox2 Foxo3a Gdf9 IL-11 R NGFI-A Nobox Npm2 PR PRL Prostaglandin F-R Ptx3 Srd5a1 Stat5a/5b Zar1 ZP3

Large litters but delayed parturition and nursing defects Excess number of germ cells in ovaries; oocytes resistant to cell death following drug treatment Infertility; defects in ovulation and corpora lutea formation Ovarian cysts and teratoma formation, decreased fertility Infertility; defects in late stage ovarian folliculogenesis Defects in primordial germ cells, block at the primary follicle stage Infertility; defects in ovulation and corpora lutea formation Oocyte death, early depletion of functional ovarian follicles, and secondary infertility Defects at one layered follicle stage; infertility Infertility; implantation defects Infertility; suppressed luteinizing hormone and ovulatory defects Infertility; postnatal oocyte loss, arrest in folliculogenesis at primordial stage Nucleolar defects in oocytes and preimplantation embryos, subfertility Infertility; defects in ovary, pituitary, and hypothalamus Infertility, irregular estrus cycles Infertility; lack of induction of oxytocin receptor Subfertility due to defects in the integrity of the cumulus cell–oocyte complexes Reduced litter size; parturition defects Infertility; absence of corpora lutea and implantation failure Infertility; developmental arrest of one-cell embryos Infertility; no zona pellucida formation

170 171 172 173, 174 175 176 177 178 179 180 181, 182 183 184 185 186 187 188 189 190 191 192, 193

CHAPTER 44 / MURINE MODELS FOR REPRODUCTION

415

Table 44–4 Examples of knockout mice with reproductive defects in both males and females Mouse model

Major reproductive phenotypes

Reference

α-Inhibin Acvr2a

Infertility in both sexes, granulosa/Sertoli cell tumors Hypogonadism in both sexes, infertility in females, subfertility and male sexual behavioral defects in males Infertile; hypogonadism and hypothyroidism Male and female infertility; absence of germ cells Male infertility; pachytene arrest of spermatogenesis; nursing defects in females due to impaired mammary gland development Male and female infertility due to abnormal glycoprotein hormone glycosylation Females infertile; block in folliculogenesis at the preantral stage; hypogonadism in males Male and female infertility; loss of germ cells Accelerated degeneration of Wolffian duct and mesonephric tubules without formation of Mù1llerian duct Uterine/ovarian defects in females; small testes, reduced sperm number Hypogonadism, males fertile with reduced sperm number and motility; females infertile, preantral stage block in folliculogenesis Hypogonadal and infertile; preantral block in folliculogenesis; defects in Leydig cell development Hypogonadism and infertility in both sexes; spermiogenesis block, reduced testosterone levels, reduced estrogen, and an ovulation defect in females Male and female infertility; pachytene stage defects in males; meiosis II defects in females Males infertile; females fertile only in the presence of males; hypothalamic defects Female infertility; corpus luteum defects; males exhibit macroorchidism Females infertile due to irregular estrus cycles and implantation defects, idiopathic infertility in males

194 55, 195

α-GSU Atm A-myb β1,4-Galactosyltransferase Cyclin D2 Dazla Emx2 ERα FSH-β IGF-1 LH/LH-R Mlh1 Nhlh2 p27kip1 PRL-R

196 197, 198 199 200 201 202 203 204, 205 206 207, 208 209–212 213, 214 215 216–218 219–221

Table 44–5 Examples of various mutagenesis approaches Approach

Mouse model

Reference

Activin βB knockin to the βA locus Cre knockin to the Amhr locus lacZ knockin to the ROSA locus

69 222, 223 224, 225

Sertoli cell-specific deletion of androgen receptor Granulosa cell-specific deletions of follistatin, SMAD4 Gonadotrope-specific deletions of GATA2, SF-1 Hypothalamus-specific deletion of SF-1

226–228 223, 229 230, 231 232, 233

Down’s syndrome Padre–Willi syndrome

70 70

LRG 4-R GnRH-R

234, 235 236

Inhibin/AMH Acvr2a/FSH-β Inhibin/FSH-β ERα/ERβ Protamines 1 and 2 Transition proteins 1 and 2 Cyclins D1, D2, and D3

237 92 43 90 238 239 240

FSH-β null mice with human FSH-β transgenes Dax1 null mice with a human DAX1 cDNA SF-1 null mice with a rat YAC containing SF-1 genomic sequences

97 241, 242 124, 243

Knockin

Cre/lox

Long-range deletions

Large-scale mutagenesis Gene trap ENU Multiple mutations

Genetic rescue

416

SECTION V / MODELS OF BEHAVIOR

Although the above approach provides spatially restricted gene deletion, temporal control is not possible with this cre/lox-based system. Recently, inducible strategies similar to those previously described have been developed in which cre enzyme expression is induced in desired cells at distinct times.58,61–68 A combination of spatially and temporally regulated gene inactivation offers limitless opportunities to study cell-specific gene expression and function throughout the course of development without affecting the same gene in other tissues. GENE KNOCKIN STRATEGIES A gene targeting approach can eliminate many of the drawbacks with transgene-based approaches. For example, reporters can be knocked-in to the desired loci (lacZ or GFP or Cre); thus random integration or effects of site of integration can be avoided.10 In this way, transgenes can be expressed as a single copy from the same endogenous locus. Moreover, bicistronic cassettes can be introduced into endogenous loci and thus gene disruption along with expression of two markers from the same disrupted locus can be achieved.10 If a known gene is already disrupted in ES cells, variants or mutant forms of the same gene can be knocked-in at the same locus and the effects monitored directly in vivo.10 The other advantage of the knockin strategy is that genes encoding related family members with overlapping patterns of expression are replaced with one another.69 Thus while disruption of one member is achieved, the other member is transcribed from the disrupted locus using the same regulatory sequences.69 Most importantly, knockin of cre gene sequences into desired loci and subsequent generation of mice provide a powerful genetic tool for conditional gene deletion purposes.58,61–68 LONG-RANGE DELETIONS AND CHROMOSOME ENGINEERING The advent of cre-lox-mediated recombination has led to the emergence of another genetic approach, called chromosome engineering.70 In this strategy, long-range deletions of desired DNA sequences are achieved in ES cells, although at a low frequency, by selectively engineering loxP sites over megabase intervals on a given chromosome.70 By altering the orientation of the loxP sites, various chromosomal events including duplication and inversion are also achieved, similar to those originally pioneered in classical Drosophila genetics.70–72 Mice bearing these global chromosomal rearrangements are often maintained as balancer stocks.70–72 These models coupled with other genetic approaches provide an in vivo platform for systematic functional analysis of all the genes that lie within the long intervals on chromosomes. LARGE-SCALE RANDOM MUTAGENESIS SCHEMES TO GENERATE MULTIPLE DOMINANT OR RECESSIVE MUTANT MODELS It is estimated that ∼30,000 genes are expressed in the genome, and about a third of these are believed to be important for reproductive function.4 Conventional gene targeting approaches are relatively time consuming and mostly aim at mutations in single genes. To rapidly and randomly generate hundreds of mutations in ES cells, several approaches have been used. In one approach, special vectors designated gene trap vectors are randomly introduced into ES cells, and individual vector integrated ES cells are identified and used to produce mutant mice.73 These vectors are usually designed to disrupt either exons or promoter regions and carry a lacZ reporter expressed from the inserted locus. This facilitates tracking the expression of the gene and eventually cloning the gene itself.73 In another approach, N-ethyl-N-nitrosourea (ENU), an alkylating agent, is injected into wild-type male mice. ENU is a powerful

mutagen in mouse spermatogonial stem cells and the goal is to saturate the mouse chromosomes with point mutations.74–79 The mutagenized males are bred to propagate scores of these mutations into subsequent generations.74–79 By designing appropriate phenotypic screens, mutants with reproductive defects are further characterized. With the aid of additional genetic tools, the transmission of the mutant allele can be followed visually by coat color and the gene of interest cloned.74–79 One advantage of this random mutagenesis scheme is that it is possible to recover many types of mutations: null, hypomorphic, and dominant or recessive. Thus, an allelic series of mutations in a given locus can be generated by this approach.74–79 ENU mutagenesis in ES cells has also been achieved, and methodologies have been developed to identify the mutant alleles in these mutagenized ES cells. The ES cells with desired point mutations are subsequently used to derive mutant strains of mice.80,81 Finally, transposon-mediated recombination is also being used to create random mutations in mice.82–85 Transposons are mobile genetic elements that can integrate into DNA, disrupt the gene, and move over to another region within the same locus or genomewide.82–85 This genetic event is facilitated by transposase, the enzyme that recognizes specific sequences to carry out this process, called transposition. Initially, two strains of transgenic mice are generated, one that carries the transposon vector along with a green fluorescent marker between the transposon recognition sequences and the other, transposase. These two lines are bred together and double transgene-positive mice are produced. They are then mated to wild-type mice and several progeny are screened for transposition events that took place in the germ line. By this approach, various mutations clustered within the 3–4 megabase range and outside this region have been produced.86,87 Although considered not as efficient as the ENU method, transposon-mediated mutagenesis appears mostly useful for region-specific mutagenesis schemes. COMBINATION OF MULTIPLE MUTATIONS The availability of mutations in desired loci permits a simple genetic intercross strategy to combining these mutations. This is possible if the targeted mutations are on distinct chromosomes or sufficiently apart on the same chromosome.88–96 Multiple mutations in different loci have been combined for various studies. This approach is useful to analyze the synergistic and/or redundant roles of related family members in a given pathway, or to understand the effect of several modifier factors that regulate a tumorigenesis pathway.88–96 Similarly, targeted or ectopic expression of transgenes in the null genetic background results in a genetic rescue.97 If the rescue can be achieved conditionally, then it will provide a useful tool to developmentally follow the effect of the transgene at desired times in the absence of the corresponding endogenous mouse gene.

RESOURCES Although we have summarized various principles of generating mouse mutations, the reader is encouraged to browse through excellent mouse and mouse genome resources available. The Jackson Labs in Barr Harbor, ME and the mouse genome informatics are great resources for all the mouse genome-based information. This is tightly linked to the National Center for Biotechnology Information (NCBI) that provides various mouse genomics tools. Similarly, the ENSEMBL website maintained by

CHAPTER 44 / MURINE MODELS FOR REPRODUCTION

417

Table 44–6 Web links to some useful resources International Gene Trap Consortium Baygenomics The Jackson Laboratory (General site) Federation of International Mouse Resources European Mouse Mutant Archive Ensembl Mouse Mouse Genome Informatics Trans-NIH Mouse Initiative Lexicon Genetics Endocrine Reviews Baylor College of Medicine (ENU project) Cornell University (ENU project) The Jackson Laboratory (infertility website) The Sloan-Kettering Mouse Project

http://www.genetrap.org/ http://baygenomics.ucsf.edu/ http://jax.org/ http://www.fimre.org/ http://www.emma.rm.cnr.it/ http://www.ensembl.org/ Mus_musculus/index.html http://www.informatics.jax.org/ http://nih.gov/science/models/mouse/resources/index.html http://www.lexicon-genetics.com/index.php edrv.endojournals.org/cgi http://www.mouse-genome.bcm.tmc.edu/ http://www.vertebrategenomics.cornell.edu/ http://jaxmice.jax.org/library/notes/496c.html http://mouse.ski.mskcc.org/

the Sanger Institute in Cambridge, UK has numerous genetic tools related to mouse and human as well as other species. Two authoritative reference collections of knockout mouse models are available through The American Endocrine Society and The BioMed Central. A number of institution-based websites list and periodically update mutations obtained with their ENU schemes or provide information particularly focused on reproductive phenotypes. Some of these useful sites are listed in Table 44–6. Finally, ready-to-use practical manuals are also available that describe details of transgenic and ES cell technologies.10,98,99

ACKNOWLEDGMENTS We wish to emphasize that for simplicity’s sake, in preparing this chapter, we have chosen only representative examples to illustrate the principles and applications of transgenic and knockout mice. We acknowledge the financial support received from the National Institutes of Health, The Moran Foundation, Houston, TX, and The Hall Family Foundation, Kansas City, MO. D.C. is a graduate student in and is supported by the Integrated Graduate Program in Biomedical Sciences, University of Kansas Medical Center.

CONCLUSIONS AND FUTURE DIRECTIONS There are now several mouse manipulation approaches available to study the in vivo roles of genes at a whole organism level. These include global or conditional (cell-specific or temporally inducible) gain- or loss-of-function mouse models. These models are powerful genetic tools and when used in combination with genomics and proteomics, they offer limitless opportunities to study reproduction.100–103 New methods of rapid germline modification can be anticipated using spermatogonial stem cells instead of ES cells.104–106 Recent advances in RNA-interference, and micro-RNA-regulated pathways are readily feasible in vivo using transgenic/ES cell-based techniques.107–114 These novel in vivo approaches will enable us to achieve more tightly controlled regulation of gene expression in vivo. Large-scale mutagenesis programs coupled with long-range deletions can be used to mimic microdeletions on the Y-chromosome that commonly occur in several cases of male infertility.115,116 Moreover, the genetic tools will facilitate identification of candidate genes responsible for many naturally occurring mouse mutations that have not yet been characterized.117 Finally, several new targets for either fertility control or enhancement may also be identified by these genetic approaches.100,118

REFERENCES 1. Nishimori K, Matzuk MM. Transgenic mice in the analysis of reproductive development and function. Rev Reprod 1996;1: 203–212. 2. Seminara SB, Crowley WF Jr. Perspective: The importance of genetic defects in humans in elucidating the complexities of the hypothalamic-pituitary-gonadal axis. Endocrinology2001;142:2173– 2177. 3. Seminara SB, Crowley WF Jr. Genetic approaches to unraveling reproductive disorders: Examples of bedside to bench research in the genomic era. Endocr Rev 2002;23:382–392. 4. Matzuk MM, Lamb DJ. Genetic dissection of mammalian fertility pathways. Nat Cell Biol 2002;4(Suppl.):s41–49. 5. Burns KH, Matzuk MM. Minireview: Genetic models for the study of gonadotropin actions. Endocrinology 2002;143:2823–2835. 6. Furnes B, Schimenti JC. Fast forward to new genes in mammalian reproduction. J Physiol 2006;578(Pt. 1):25–32. 7. Jorgez CJ, Lin YN, Matzuk MM. Genetic manipulations to study reproduction. Mol Cell Endocrinol 2005;234:127–135. 8. Roy A, Matzuk MM. Deconstructing mammalian reproduction: Using knockouts to define fertility pathways. Reproduction 2006;131:207–219. 9. Wang H, Dey SK. Roadmap to embryo implantation: Clues from mouse models. Nat Rev Genet 2006;7:185–199.

418

SECTION V / MODELS OF BEHAVIOR

10. Nagy A, Gertsenstein M, Vintersten K, Behringer R. Manipulating the Mouse Embryo: A Laboratory Manual, 3rd ed. Woodbury, NY: Cold Spring Harbor Laboratory Press, 2003. 11. Palmiter RD, Brinster RL, Hammer RE, Trumbauer ME, Rosenfeld MG, Birnberg NC, Evans RM. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 1982;300:611–615. 12. Brinster RL, Palmiter RD. Introduction of genes into the germ line of animals. Harvey Lect 1984;80:1–38. 13. Palmiter RD, Brinster RL. Germ-line transformation of mice. Annu Rev Genet 1986;20:465–499. 14. Bailey JS, Rave-Harel N, McGillivray SM, Coss D, Mellon PL. Activin regulation of the follicle-stimulating hormone beta-subunit gene involves Smads and the TALE homeodomain proteins Pbx1 and Prep1. Mol Endocrinol 2004;18:1158–1170. 15. Barnhart KM, Mellon PL. The orphan nuclear receptor, steroidogenic factor-1, regulates the glycoprotein hormone alpha-subunit gene in pituitary gonadotropes. Mol Endocrinol 1994;8:878– 885. 16. Harris AN, Mellon PL. The basic helix-loop-helix, leucine zipper transcription factor, USF (upstream stimulatory factor), is a key regulator of SF-1 (steroidogenic factor-1) gene expression in pituitary gonadotrope and steroidogenic cells. Mol Endocrinol 1998;12: 714–726. 17. Liu F, Austin DA, Mellon PL, Olefsky JM, Webster NJ. GnRH activates ERK1/2 leading to the induction of c-fos and LHbeta protein expression in LbetaT2 cells. Mol Endocrinol 2002;16: 419–434. 18. Schoderbek WE, Kim KE, Ridgway EC, Mellon PL, Maurer RA. Analysis of DNA sequences required for pituitary-specific expression of the glycoprotein hormone alpha-subunit gene. Mol Endocrinol 1992;6:893–903. 19. Vasilyev VV, Lawson MA, Dipaolo D, Webster NJ, Mellon PL. Different signaling pathways control acute induction versus longterm repression of LHbeta transcription by GnRH. Endocrinology 2002;143:3414–3426. 20. Vasilyev VV, Pernasetti F, Rosenberg SB, Barsoum MJ, Austin DA, Webster NJ, Mellon PL. Transcriptional activation of the ovine follicle-stimulating hormone-beta gene by gonadotropin-releasing hormone involves multiple signal transduction pathways. Endocrinology 2002;143:1651–1659. 21. Kumar TR, Schuff KG, Nusser KD, Low MJ. Gonadotroph-specific expression of the human follicle stimulating hormone beta gene in transgenic mice. Mol Cell Endocrinol 2006;247:103–115. 22. Hamernik DL, Keri RA, Clay CM, Clay JN, Sherman GB, Sawyer HR Jr, Nett TM, Nilson JH. Gonadotrope- and thyrotrope-specific expression of the human and bovine glycoprotein hormone alphasubunit genes is regulated by distinct cis-acting elements. Mol Endocrinol 1992;6:1745–1755. 23. Kendall SK, Saunders TL, Jin L, Lloyd RV, Glode LM, Nett TM, Keri RA, Nilson JH, Camper SA. Targeted ablation of pituitary gonadotropes in transgenic mice. Mol Endocrinol 1991;5:2025– 2036. 24. Keri RA, Bachmann DJ, Behrooz A, Herr BD, Ameduri RK, Quirk CC, Nilson JH. An NF-Y binding site is important for basal, but not gonadotropin-releasing hormone-stimulated, expression of the luteinizing hormone beta subunit gene. J Biol Chem 2000;275: 13082–13088. 25. Keri RA, Nilson JH. A steroidogenic factor-1 binding site is required for activity of the luteinizing hormone beta subunit promoter in gonadotropes of transgenic mice. J Biol Chem 1996;271:10782– 10785. 26. Quirk CC, Lozada KL, Keri RA, Nilson JH. A single Pitx1 binding site is essential for activity of the LHbeta promoter in transgenic mice. Mol Endocrinol 2001;15:734–746. 27. Balthasar N, Mery PF, Magoulas CB, Mathers KE, Martin A, Mollard P, Robinson IC. Growth hormone-releasing hormone (GHRH) neurons in GHRH-enhanced green fluorescent protein transgenic mice: A ventral hypothalamic network. Endocrinology 2003;144:2728–2740.

28. Bonnefont X, Lacampagne A, Sanchez-Hormigo A, Fino E, Creff A, Mathieu MN, Smallwood S, Carmignac D, Fontanaud P, Travo P, Alonso G, Courtois-Coutry N, Pincus SM, Robinson IC, Mollard P. Revealing the large-scale network organization of growth hormone-secreting cells. Proc Natl Acad Sci USA 2005;102: 16880–16885. 29. Magoulas C, McGuinness L, Balthasar N, Carmignac DF, Sesay AK, Mathers KE, Christian H, Candeil L, Bonnefont X, Mollard P, Robinson IC. A secreted fluorescent reporter targeted to pituitary growth hormone cells in transgenic mice. Endocrinology 2000;141:4681–4689. 30. Markkula M, Kananen K, Klemi P, Huhtaniemi I. Pituitary and ovarian expression of the endogenous follicle-stimulating hormone (FSH) subunit genes and an FSH beta-subunit promoter-driven herpes simplex virus thymidine kinase gene in transgenic mice; specific partial ablation of FSH-producing cells by antiherpes treatment. J Endocrinol 1996;150:265–273. 31. Le Tissier PR, Carmignac DF, Lilley S, Sesay AK, Phelps CJ, Houston P, Mathers K, Magoulas C, Ogden D, Robinson IC. Hypothalamic growth hormone-releasing hormone (GHRH) deficiency: Targeted ablation of GHRH neurons in mice using a viral ion channel transgene. Mol Endocrinol 2005;19:1251–1262. 32. Ahtiainen M, Toppari J, Poutanen M, Huhtaniemi I. Indirect Sertoli cell-mediated ablation of germ cells in mice expressing the inhibinalpha promoter/herpes simplex virus thymidine kinase transgene. Biol Reprod 2004;71:1545–1550. 33. Hanahan D. Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes. Nature 1985;315:115–122. 34. Alarid ET, Holley S, Hayakawa M, Mellon PL. Discrete stages of anterior pituitary differentiation recapitulated in immortalized cell lines. Mol Cell Endocrinol 1998;140:25–30. 35. Alarid ET, Windle JJ, Whyte DB, Mellon PL. Immortalization of pituitary cells at discrete stages of development by directed oncogenesis in transgenic mice. Development 1996;122:3319–3329. 36. Kumar TR, Graham KE, Asa SL, Low MJ. Simian virus 40 T antigen-induced gonadotroph adenomas: A model of human null cell adenomas. Endocrinology 1998;139:3342–3351. 37. Pernasetti F, Spady TJ, Hall SB, Rosenberg SB, Givens ML, Anderson S, Paulus M, Miller WL, Mellon PL. Pituitary tumorigenesis targeted by the ovine follicle-stimulating hormone beta-subunit gene regulatory region in transgenic mice. Mol Cell Endocrinol 2003;203:169–183. 38. Thomas P, Mellon PL, Turgeon J, Waring DW. The L beta T2 clonal gonadotrope: A model for single cell studies of endocrine cell secretion. Endocrinology 1996;137:2979–2989. 39. Windle JJ, Weiner RI, Mellon PL. Cell lines of the pituitary gonadotrope lineage derived by targeted oncogenesis in transgenic mice. Mol Endocrinol 1990;4:597–603. 40. Palmiter RD. Molecular biology of metallothionein gene expression. Experientia Suppl 1987;52:63–80. 41. Palmiter RD, Norstedt G, Gelinas RE, Hammer RE, Brinster RL. Metallothionein-human GH fusion genes stimulate growth of mice. Science 1983;222:809–814. 42. Guo Q, Kumar TR, Woodruff T, Hadsell LA, DeMayo FJ, Matzuk MM. Overexpression of mouse follistatin causes reproductive defects in transgenic mice. Mol Endocrinol 1998;12:96–106. 43. Kumar TR, Palapattu G, Wang P, Woodruff TK, Boime I, Byrne MC, Matzuk MM. Transgenic models to study gonadotropin function: The role of follicle-stimulating hormone in gonadal growth and tumorigenesis. Mol Endocrinol 1999;13:851–865. 44. Matzuk MM, DeMayo FJ, Hadsell LA, Kumar TR. Overexpression of human chorionic gonadotropin causes multiple reproductive defects in transgenic mice. Biol Reprod 2003;69:338–346. 45. Ryding AD, Sharp MG, Mullins JJ. Conditional transgenic technologies. J Endocrinol 2001;171:1–14. 46. Schnutgen F, Doerflinger N, Calleja C, Wendling O, Chambon P, Ghyselinck NB. A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse. Nat Biotechnol 2003;21:562–565.

CHAPTER 44 / MURINE MODELS FOR REPRODUCTION

47. Zhu Z, Zheng T, Lee CG, Homer RJ, Elias JA. Tetracyclinecontrolled transcriptional regulation systems: Advances and application in transgenic animal modeling. Semin Cell Dev Biol 2002;13: 121–128. 48. Karzenowski D, Potter DW, Padidam M. Inducible control of transgene expression with ecdysone receptor: Gene switches with high sensitivity, robust expression, and reduced size. Biotechniques 2005;39:191–192, 194, 196 passim. 49. Pierson TM, Wang Y, DeMayo FJ, Matzuk MM, Tsai SY, Omalley BW. Regulable expression of inhibin A in wild-type and inhibin alpha null mice. Mol Endocrinol 2000;14:1075–1085. 50. Albanese C, Hulit J, Sakamaki T, Pestell RG. Recent advances in inducible expression in transgenic mice. Semin Cell Dev Biol 2002;13:129–141. 51. Bradley A, Hasty P, Davis A, Ramirez-Solis R. Modifying the mouse: Design and desire. Biotechnology (NY) 1992;10:534–539. 52. Hasty P, Rivera-Perez J, Bradley A. The length of homology required for gene targeting in embryonic stem cells. Mol Cell Biol 1991;11:5586–5591. 53. Hasty P, Rivera-Perez J, Chang C, Bradley A. Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells. Mol Cell Biol 1991;11:4509–4517. 54. Zhang H, Hasty P, Bradley A. Targeting frequency for deletion vectors in embryonic stem cells. Mol Cell Biol 1994;14:2404–2410. 55. Matzuk MM, Kumar TR, Bradley A. Different phenotypes for mice deficient in either activins or activin receptor type II. Nature 1995;374:356–360. 56. Matzuk MM, Kumar TR, Vassalli A, Bickenbach JR, Roop DR, Jaenisch R, Bradley A. Functional analysis of activins during mammalian development. Nature 1995;374:354–356. 57. Matzuk MM, Lu N, Vogel H, Sellheyer K, Roop DR, Bradley A. Multiple defects and perinatal death in mice deficient in follistatin. Nature 1995;374:360–363. 58. Le Y, Sauer B. Conditional gene knockout using Cre recombinase. Mol Biotechnol 2001;17:269–275. 59. Sauer B, Henderson N. Cre-stimulated recombination at loxP-containing DNA sequences placed into the mammalian genome. Nucleic Acids Res 1989;17:147–161. 60. Sauer B, Henderson N. Targeted insertion of exogenous DNA into the eukaryotic genome by the Cre recombinase. New Biol 1990; 2:441–449. 61. Belteki G, Haigh J, Kabacs N, Haigh K, Sison K, Costantini F, Whitsett J, Quaggin SE, Nagy A. Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction. Nucleic Acids Res 2005;33:e51. 62. Buch T, Heppner FL, Tertilt C, Heinen TJ, Kremer M, Wunderlich FT, Jung S, Waisman A. A Cre-inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration. Nat Methods 2005;2:419–426. 63. Chow LM, Tian Y, Weber T, Corbett M, Zuo J, Baker SJ. Inducible Cre recombinase activity in mouse cerebellar granule cell precursors and inner ear hair cells. Dev Dyn 2006;235:2991–2998. 64. Hayashi S, McMahon AP. Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: A tool for temporally regulated gene activation/inactivation in the mouse. Dev Biol 2002; 244:305–318. 65. Hunter NL, Awatramani RB, Farley FW, Dymecki SM. Ligandactivated Flpe for temporally regulated gene modifications. Genesis 2005;41:99–109. 66. Tannour-Louet M, Porteu A, Vaulont S, Kahn A, Vasseur-Cognet M. A tamoxifen-inducible chimeric Cre recombinase specifically effective in the fetal and adult mouse liver. Hepatology 2002;35: 1072–1081. 67. Weber P, Schuler M, Gerard C, Mark M, Metzger D, Chambon P. Temporally controlled site-specific mutagenesis in the germ cell lineage of the mouse testis. Biol Reprod 2003;68:553–559. 68. Zhao J, Nassar MA, Gavazzi I, Wood JN. Tamoxifen-inducible NaV1.8-CreERT2 recombinase activity in nociceptive neurons of dorsal root ganglia. Genesis 2006;44:364–371.

419

69. Brown CW, Houston-Hawkins DE, Woodruff TK, Matzuk MM. Insertion of Inhbb into the Inhba locus rescues the Inhba-null phenotype and reveals new activin functions. Nat Genet 2000;25: 453–457. 70. Bradley A, van der Weyden L. Mouse: Chromosome engineering for modeling human disease. Annu Rev Genomics Hum Genet 2006;7 [Epub ahead of print]. 71. Zheng B, Mills AA, Bradley A. Introducing defined chromosomal rearrangements into the mouse genome. Methods 2001;24:81–94. 72. Zheng B, Sage M, Cai WW, Thompson DM, Tavsanli BC, Cheah YC, Bradley A. Engineering a mouse balancer chromosome. Nat Genet 1999;22:375–378. 73. Chen YT, Liu P, Bradley A. Inducible gene trapping with drugselectable markers and Cre/loxP to identify developmentally regulated genes. Mol Cell Biol 2004;24:9930–9941. 74. Clark AT, Goldowitz D, Takahashi JS, Vitaterna MH, Siepka SM, Peters LL, Frankel WN, Carlson GA, Rossant J, Nadeau JH, Justice MJ. Implementing large-scale ENU mutagenesis screens in North America. Genetica 2004;122:51–64. 75. Herron BJ, Lu W, Rao C, Liu S, Peters H, Bronson RT, Justice MJ, McDonald JD, Beier DR. Efficient generation and mapping of recessive developmental mutations using ENU mutagenesis. Nat Genet 2002;30:185–189. 76. Justice MJ. From the atomic age to the Genome Project. Genetica 2004;122:3–7. 77. Justice MJ, Carpenter DA, Favor J, Neuhauser-Klaus A, Hrabe de Angelis M, Soewarto D, Moser A, Cordes S, Miller D, Chapman V, Weber JS, Rinchik EM, Hunsicker PR, Russell WL, Bode VC. Effects of ENU dosage on mouse strains. Mamm Genome 2000;11:484–488. 78. Kile BT, Hentges KE, Clark AT, Nakamura H, Salinger AP, Liu B, Box N, Stockton DW, Johnson RL, Behringer RR, Bradley A, Justice MJ. Functional genetic analysis of mouse chromosome 11. Nature 2003;425:81–86. 79. Noveroske JK, Weber JS, Justice MJ. The mutagenic action of Nethyl-N-nitrosourea in the mouse. Mamm Genome 2000;11: 478–483. 80. Chen Y, Vivian JL, Magnuson T. Gene-based chemical mutagenesis in mouse embryonic stem cells. Methods Enzymol 2003;365: 406–415. 81. Vivian JL, Chen Y, Yee D, Schneider E, Magnuson T. An allelic series of mutations in Smad2 and Smad4 identified in a genotype-based screen of N-ethyl-N-nitrosourea-mutagenized mouse embryonic stem cells. Proc Natl Acad Sci USA 2002;99:15542– 15547. 82. Luo G, Ivics Z, Izsvak Z, Bradley A. Chromosomal transposition of a Tc1/mariner-like element in mouse embryonic stem cells. Proc Natl Acad Sci USA 1998;95:10769–10773. 83. Score PR, Belur LR, Frandsen JL, Guerts JL, Yamaguchi T, Somia NV, Hackett PB, Largaespada DA, McIvor RS. Sleeping Beautymediated transposition and long-term expression in vivo: Use of the LoxP/Cre recombinase system to distinguish transposition-specific expression. Mol Ther 2006;13:617–624. 84. Collier LS, Largaespada DA. Hopping around the tumor genome: Transposons for cancer gene discovery. Cancer Res 2005;65: 9607–9610. 85. Gueguen E, Rousseau P, Duval-Valentin G, Chandler M. The transpososome: Control of transposition at the level of catalysis. Trends Microbiol 2005;13:543–549. 86. Yae K, Keng VW, Koike M, Yusa K, Kouno M, Uno Y, Kondoh G, Gotow T, Uchiyama Y, Horie K, Takeda J. Sleeping beauty transposon-based phenotypic analysis of mice: Lack of Arpc3 results in defective trophoblast outgrowth. Mol Cell Biol 2006;26:6185–6196. 87. Yant SR, Wu X, Huang Y, Garrison B, Burgess SM, Kay MA. High-resolution genome-wide mapping of transposon integration in mammals. Mol Cell Biol 2005;25:2085–2094. 88. Su YQ, Wu X, O’Brien MJ, Pendola FL, Denegre JN, Matzuk MM, Eppig JJ. Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: Genetic

420

89.

90.

91.

92.

93.

94.

95.

96.

97.

98. 99.

100.

101. 102. 103.

104.

105.

106.

107. 108. 109.

SECTION V / MODELS OF BEHAVIOR

evidence for an oocyte-granulosa cell regulatory loop. Dev Biol 2004;276:64–73. Kumar TR, Agno J, Janovick JA, Conn PM, Matzuk MM. Regulation of FSHbeta and GnRH receptor gene expression in activin receptor II knockout male mice. Mol Cell Endocrinol 2003;212: 19–27. Burns KH, Agno JE, Chen L, Haupt B, Ogbonna SC, Korach KS, Matzuk MM. Sexually dimorphic roles of steroid hormone receptor signaling in gonadal tumorigenesis. Mol Endocrinol 2003;17: 2039–2052. Burns KH, Agno JE, Sicinski P, Matzuk MM. Cyclin D2 and p27 are tissue-specific regulators of tumorigenesis in inhibin alpha knockout mice. Mol Endocrinol 2003;17:2053–2069. Kumar TR, Varani S, Wreford NG, Telfer NM, de Kretser DM, Matzuk MM. Male reproductive phenotypes in double mutant mice lacking both FSHbeta and activin receptor IIA. Endocrinology 2001;142:3512–3518. Cipriano SC, Chen L, Burns KH, Koff A, Matzuk MM. Inhibin and p27 interact to regulate gonadal tumorigenesis. Mol Endocrinol 2001;15:985–996. Yan C, Wang P, DeMayo J, DeMayo FJ, Elvin JA, Carino C, Prasad SV, Skinner SS, Dunbar BS, Dube JL, Celeste AJ, Matzuk MM. Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function. Mol Endocrinol 2001;15:854–866. Lau AL, Kumar TR, Nishimori K, Bonadio J, Matzuk MM. Activin betaC and betaE genes are not essential for mouse liver growth, differentiation, and regeneration. Mol Cell Biol 2000;20:6127– 6137. Mishina Y, Rey R, Finegold MJ, Matzuk MM, Josso N, Cate RL, Behringer RR. Genetic analysis of the Mullerian-inhibiting substance signal transduction pathway in mammalian sexual differentiation. Genes Dev 1996;10:2577–2587. Kumar TR, Low MJ, Matzuk MM. Genetic rescue of follicle-stimulating hormone beta-deficient mice. Endocrinology 1998;139: 3289–3295. Joyner AL. Gene Targeting: A Practical Approach, 2nd ed. New York: Oxford University Press, 2001. Hofker MH, Van Deursen J. Transgenic Mouse: Methods and Protocols, Methods in Molecular Biology, Vol. 209. Totowa, NJ: Humana Press, 2003. Chu DS, Liu H, Nix P, Wu TF, Ralston EJ, Yates JR 3rd, Meyer BJ. Sperm chromatin proteomics identifies evolutionarily conserved fertility factors. Nature 2006;443:101–105. Lin YN, Matzuk MM. High-throughput discovery of germ-cellspecific genes. Semin Reprod Med 2005;23:201–212. Rockett JC. Genomic and proteomic techniques applied to reproductive biology. Genome Biol 2001;2:REPORTS4020. Sirard MA, Dufort I, Coenen K, Tremblay K, Massicotte L, Robert C. The use of genomics and proteomics to understand oocyte and early embryo functions in farm animals. Reprod Suppl 2003;61:117–129. Shinohara T, Kato M, Takehashi M, Lee J, Chuma S, Nakatsuji N, Kanatsu-Shinohara M, Hirabayashi M. Rats produced by interspecies spermatogonial transplantation in mice and in vitro microinsemination. Proc Natl Acad Sci USA 2006;103:13624–13628. Kanatsu-Shinohara M, Ikawa M, Takehashi M, Ogonuki N, Miki H, Inoue K, Kazuki Y, Lee J, Toyokuni S, Oshimura M, Ogura A, Shinohara T. Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells. Proc Natl Acad Sci USA 2006;103:8018–8023. Kanatsu-Shinohara M, Toyokuni S, Shinohara T. Transgenic mice produced by retroviral transduction of male germ line stem cells in vivo. Biol Reprod 2004;71:1202–1207. Glaser S, Anastassiadis K, Stewart AF. Current issues in mouse genome engineering. Nat Genet 2005;37:1187–1193. Lin SL, Chang DC, Ying SY. Isolation and identification of genespecific microRNAs. Methods Mol Biol 2006;342:313–320. Lin SL, Chang SJ, Ying SY. Transgene-like animal models using intronic microRNAs. Methods Mol Biol 2006;342:321–334.

110. Lin SL, Ying SY. Gene silencing in vitro and in vivo using intronic microRNAs. Methods Mol Biol 2006;342:295–312. 111. Muljo SA, Kanellopoulou C. Mouse embryonic stem cells as a model genetic system to dissect and exploit the RNA interference machinery. Methods Mol Biol 2006;342:57–72. 112. Prawitt D, Brixel L, Spangenberg C, Eshkind L, Heck R, Oesch F, Zabel B, Bockamp E. RNAi knock-down mice: An emerging technology for post-genomic functional genetics. Cytogenet Genome Res 2004;105:412–421. 113. Ristevski S. Making better transgenic models: Conditional, temporal, and spatial approaches. Mol Biotechnol 2005;29:153–163. 114. Ying SY, Chang DC, Miller JD, Lin SL. The microRNA: Overview of the RNA gene that modulates gene functions. Methods Mol Biol 2006;342:1–18. 115. Affara NA, Mitchell MJ. The role of human and mouse Y chromosome genes in male infertility. J Endocrinol Invest 2000;23: 630–645. 116. Burgoyne PS. The role of Y-encoded genes in mammalian spermatogenesis. Semin Cell Dev Biol 1998;9:423–432. 117. Lyon MF, Rastan S, Brown SDM, Eds. Genetic Variants and Strains of the Laboratory Mouse, 3rd ed. New York: Oxford University Press, 1995. 118. Heck S, Qian X, Velleca M. Genetically engineered mouse models for drug discovery: New chemical genetic approaches. Curr Drug Discov Technol 2004;1:13–26. 119. Gainer H. Cell-specific gene expression in oxytocin and vasopressin magnocellular neurons. Adv Exp Med Biol 1998;449:15–27. 120. Jeong SW, Castel M, Zhang BJ, Fields RL, Paras P, Arnheiter H, Chin H, Gainer H. Cell-specific expression and subcellular localization of neurophysin-CAT-fusion proteins expressed from oxytocin and vasopressin gene promoter-driven constructs in transgenic mice. Exp Neurol 2001;171:255–271. 121. Ratty AK, Jeong SW, Nagle JW, Chin H, Gainer H, Murphy D, Venkatesh B. A systematic survey of the intergenic region between the murine oxytocin- and vasopressin-encoding genes. Gene 1996;174:71–78. 122. Yeom YI, Fuhrmann G, Ovitt CE, Brehm A, Ohbo K, Gross M, Hubner K, Scholer HR. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 1996;122:881–894. 123. Daggett MA, Rice DA, Heckert LL. Expression of steroidogenic factor 1 in the testis requires an E box and CCAAT box in its promoter proximal region. Biol Reprod 2000;62:670–679. 124. Karpova T, Presley J, Manimaran RR, Scherrer SP, Tejada L, Peterson KR, Heckert LL. A FTZ-F1-containing yeast artificial chromosome recapitulates expression of steroidogenic factor 1 in vivo. Mol Endocrinol 2005;19:2549–2563. 125. Scherrer SP, Rice DA, Heckert LL. Expression of steroidogenic factor 1 in the testis requires an interactive array of elements within its proximal promoter. Biol Reprod 2002;67:1509–1521. 126. Yan C, Elvin JA, Lin YN, Hadsell LA, Wang J, DeMayo FJ, Matzuk MM. Regulation of growth differentiation factor 9 expression in oocytes in vivo: A key role of the E-box. Biol Reprod 2006;74:999–1006. 127. Acharya KK, Govind CK, Shore AN, Stoler MH, Reddi PP. Cisrequirement for the maintenance of round spermatid-specific transcription. Dev Biol 2006;295:781–790. 128. Reddi PP, Flickinger CJ, Herr JC. Round spermatid-specific transcription of the mouse SP-10 gene is mediated by a 294-base pair proximal promoter. Biol Reprod 1999;61:1256–1266. 129. Reddi PP, Shore AN, Shapiro JA, Anderson A, Stoler MH, Acharya KK. Spermatid-specific promoter of the SP-10 gene functions as an insulator in somatic cells. Dev Biol 2003;262:173–182. 130. Keri RA, Andersen B, Kennedy GC, Hamernik DL, Clay CM, Brace AD, Nett TM, Notides AC, Nilson JH. Estradiol inhibits transcription of the human glycoprotein hormone alpha-subunit gene despite the absence of a high affinity binding site for estrogen receptor. Mol Endocrinol 1991;5:725–733. 131. Fallest PC, Trader GL, Darrow JM, Shupnik MA. Regulation of rat luteinizing hormone beta gene expression in transgenic mice by

CHAPTER 44 / MURINE MODELS FOR REPRODUCTION

132.

133.

134.

135.

136.

137.

138.

139.

140.

141.

142.

143.

144.

145.

146.

147.

148.

steroids and a gonadotropin-releasing hormone antagonist. Biol Reprod 1995;53:103–109. Huang HJ, Sebastian J, Strahl BD, Wu JC, Miller WL. Transcriptional regulation of the ovine follicle-stimulating hormone-beta gene by activin and gonadotropin-releasing hormone (GnRH): Involvement of two proximal activator protein-1 sites for GnRH stimulation. Endocrinology 2001;142:2267–2274. Huang HJ, Sebastian J, Strahl BD, Wu JC, Miller WL. The promoter for the ovine follicle-stimulating hormone-beta gene (FSHbeta) confers FSHbeta-like expression on luciferase in transgenic mice: Regulatory studies in vivo and in vitro. Endocrinology 2001;142: 2260–2266. Stallings NR, Hanley NA, Majdic G, Zhao L, Bakke M, Parker KL. Development of a transgenic green fluorescent protein lineage marker for steroidogenic factor 1. Endocr Res 2002;28:497– 504. Stallings NR, Hanley NA, Majdic G, Zhao L, Bakke M, Parker KL. Development of a transgenic green fluorescent protein lineage marker for steroidogenic factor 1. Mol Endocrinol 2002;16: 2360–2370. Bowles J, Knight D, Smith C, Wilhelm D, Richman J, Mamiya S, Yashiro K, Chawengsaksophak K, Wilson MJ, Rossant J, Hamada H, Koopman P. Retinoid signaling determines germ cell fate in mice. Science 2006;312:596–600. Boiani M, Kehler J, Scholer HR. Activity of the germline-specific Oct4-GFP transgene in normal and clone mouse embryos. Methods Mol Biol 2004;254:1–34. Keiser JT, Jobst PM, Garst AS, Boone JT, Geyer CB, Phelps C, Ayares DL, Page RL. Preimplantation screening for transgenesis using an embryonic specific promoter and green fluorescent protein. Cloning 2001;3:23–30. Suter KJ, Song WJ, Sampson TL, Wuarin JP, Saunders JT, Dudek FE, Moenter SM. Genetic targeting of green fluorescent protein to gonadotropin-releasing hormone neurons: Characterization of whole-cell electrophysiological properties and morphology. Endocrinology 2000;141:412–419. Suter KJ, Wuarin JP, Smith BN, Dudek FE, Moenter SM. Wholecell recordings from preoptic/hypothalamic slices reveal burst firing in gonadotropin-releasing hormone neurons identified with green fluorescent protein in transgenic mice. Endocrinology 2000;141: 3731–3736. Mellon PL, Windle JJ, Goldsmith PC, Padula CA, Roberts JL, Weiner RI. Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 1990;5:1–10. Weiner RI, Wetsel W, Goldsmith P, Martinez de la Escalera G, Windle J, Padula C, Choi A, Negro-Vilar A, Mellon P. Gonadotropin-releasing hormone neuronal cell lines. Front Neuroendocrinol 1992;13:95–119. Albarracin CT, Frosch MP, Chin WW. The gonadotropin-releasing hormone receptor gene promoter directs pituitary-specific oncogene expression in transgenic mice. Endocrinology 1999;140:2415– 2421. Peschon JJ, Behringer RR, Cate RL, Harwood KA, Idzerda RL, Brinster RL, Palmiter RD. Directed expression of an oncogene to Sertoli cells in transgenic mice using Mullerian inhibiting substance regulatory sequences. Mol Endocrinol 1992;6:1403–1411. Kananen K, Markkula M, el-Hefnawy T, Zhang FP, Paukku T, Su JG, Hsueh AJ, Huhtaniemi I. The mouse inhibin alpha-subunit promoter directs SV40 T-antigen to Leydig cells in transgenic mice. Mol Cell Endocrinol 1996;119:135–146. Rahman NA, Kananen Rilianawati K, Paukku T, Mikola M, Markkula M, Hamalainen T, Huhtaniemi IT. Transgenic mouse models for gonadal tumorigenesis. Mol Cell Endocrinol 1998;145: 167–174. Adham IM, Nayernia K, Engel W. Spermatozoa lacking acrosin protein show delayed fertilization. Mol Reprod Dev 1997;46: 370–376. Jamin SP, Arango NA, Mishina Y, Hanks MC, Behringer RR. Genetic studies of the AMH/MIS signaling pathway for Mullerian duct regression. Mol Cell Endocrinol 2003;211:15–19.

421

149. Knudson CM, Tung KS, Tourtellotte WG, Brown GA, Korsmeyer SJ. Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science 1995;270:96–99. 150. Ross AJ, Waymire KG, Moss JE, Parlow AF, Skinner MK, Russell LD, MacGregor GR. Testicular degeneration in Bclw-deficient mice. Nat Genet 1998;18:251–256. 151. Zhao GQ, Liaw L, Hogan BL. Bone morphogenetic protein 8A plays a role in the maintenance of spermatogenesis and the integrity of the epididymis. Development 1998;125:1103–1112. 152. Zhao GQ, Deng K, Labosky PA, Liaw L, Hogan BL. The gene encoding bone morphogenetic protein 8B is required for the initiation and maintenance of spermatogenesis in the mouse. Genes Dev 1996;10:1657–1669. 153. Ikawa M, Nakanishi T, Yamada S, Wada I, Kominami K, Tanaka H, Nozaki M, Nishimune Y, Okabe M. Calmegin is required for fertilin alpha/beta heterodimerization and sperm fertility. Dev Biol 2001;240:254–261. 154. Ikawa M, Wada I, Kominami K, Watanabe D, Toshimori K, Nishimune Y, Okabe M. The putative chaperone calmegin is required for sperm fertility. Nature 1997;387:607–611. 155. Blendy JA, Kaestner KH, Weinbauer GF, Nieschlag E, Schutz G. Severe impairment of spermatogenesis in mice lacking the CREM gene. Nature 1996;380:162–165. 156. Nantel F, Monaco L, Foulkes NS, Masquilier D, LeMeur M, Henriksen K, Dierich A, Parvinen M, Sassone-Corsi P. Spermiogenesis deficiency and germ-cell apoptosis in CREM-mutant mice. Nature 1996;380:159–162. 157. Bitgood MJ, Shen L, McMahon AP. Sertoli cell signaling by Desert hedgehog regulates the male germline. Curr Biol 1996;6:298– 304. 158. Clark AM, Garland KK, Russell LD. Desert hedgehog (Dhh) gene is required in the mouse testis for formation of adult-type Leydig cells and normal development of peritubular cells and seminiferous tubules. Biol Reprod 2000;63:1825–1838. 159. Pierucci-Alves F, Clark AM, Russell LD. A developmental study of the Desert hedgehog-null mouse testis. Biol Reprod 2001;65: 1392–1402. 160. Podlasek CA, Duboule D, Bushman W. Male accessory sex organ morphogenesis is altered by loss of function of Hoxd-13. Dev Dyn 1997;208:454–465. 161. Roest HP, van Klaveren J, de Wit J, van Gurp CG, Koken MH, Vermey M, van Roijen JH, Hoogerbrugge JW, Vreeburg JT, Baarends WM, Bootsma D, Grootegoed JA, Hoeijmakers JH. Inactivation of the HR6B ubiquitin-conjugating DNA repair enzyme in mice causes male sterility associated with chromatin modification. Cell 1996;86:799–810. 162. Dix DJ, Allen JW, Collins BW, Mori C, Nakamura N, PoormanAllen P, Goulding EH, Eddy EM. Targeted gene disruption of Hsp70–2 results in failed meiosis, germ cell apoptosis, and male infertility. Proc Natl Acad Sci USA 1996;93:3264–3268. 163. Dix DJ, Allen JW, Collins BW, Poorman-Allen P, Mori C, Blizard DR, Brown PR, Goulding EH, Strong BD, Eddy EM. HSP70–2 is required for desynapsis of synaptonemal complexes during meiotic prophase in juvenile and adult mouse spermatocytes. Development 1997;124:4595–4603. 164. Yan W, Ma L, Burns KH, Matzuk MM. Haploinsufficiency of kelch-like protein homolog 10 causes infertility in male mice. Proc Natl Acad Sci USA 2004;101:7793–7798. 165. Mbikay M, Tadros H, Ishida N, Lerner CP, De Lamirande E, Chen A, El-Alfy M, Clermont Y, Seidah NG, Chretien M, Gagnon C, Simpson EM. Impaired fertility in mice deficient for the testicular germ-cell protease PC4. Proc Natl Acad Sci USA 1997;94: 6842–6846. 166. Lufkin T, Lohnes D, Mark M, Dierich A, Gorry P, Gaub MP, LeMeur M, Chambon P. High postnatal lethality and testis degeneration in retinoic acid receptor alpha mutant mice. Proc Natl Acad Sci USA 1993;90:7225–7229. 167. Kastner P, Mark M, Leid M, Gansmuller A, Chin W, Grondona JM, Decimo D, Krezel W, Dierich A, Chambon P. Abnormal spermatogenesis in RXR beta mutant mice. Genes Dev 1996;10:80–92.

422

SECTION V / MODELS OF BEHAVIOR

168. Lohnes D, Kastner P, Dierich A, Mark M, LeMeur M, Chambon P. Function of retinoic acid receptor gamma in the mouse. Cell 1993;73:643–658. 169. Greenbaum MP, Yan W, Wu MH, Lin YN, Agno JE, Sharma M, Braun RE, Rajkovic A, Matzuk MM. TEX14 is essential for intercellular bridges and fertility in male mice. Proc Natl Acad Sci USA 2006;103:4982–4987. 170. Vassalli A, Matzuk MM, Gardner HA, Lee KF, Jaenisch R. Activin/ inhibin beta B subunit gene disruption leads to defects in eyelid development and female reproduction. Genes Dev 1994;8: 414–427. 171. Bergeron L, Perez GI, Macdonald G, Shi L, Sun Y, Jurisicova A, Varmuza S, Latham KE, Flaws JA, Salter JC, Hara H, Moskowitz MA, Li E, Greenberg A, Tilly JL, Yuan J. Defects in regulation of apoptosis in caspase-2-deficient mice. Genes Dev 1998;12: 1304–1314. 172. Sterneck E, Tessarollo L, Johnson PF. An essential role for C/ EBPbeta in female reproduction. Genes Dev 1997;11:2153–2162. 173. Furuta Y, Shigetani Y, Takeda N, Iwasaki K, Ikawa Y, Aizawa S. Ovarian teratomas in mice lacking the protooncogene c-mos. Jpn J Cancer Res 1995;86:540–545. 174. Hashimoto N, Watanabe N, Furuta Y, Tamemoto H, Sagata N, Yokoyama M, Okazaki K, Nagayoshi M, Takeda N, Ikawa Y, et al. Parthenogenetic activation of oocytes in c-mos-deficient mice. Nature 1994;370:68–71. 175. Simon AM, Goodenough DA, Li E, Paul DL. Female infertility in mice lacking connexin 37. Nature 1997;385:525–529. 176. Juneja SC, Barr KJ, Enders GC, Kidder GM. Defects in the germ line and gonads of mice lacking connexin43. Biol Reprod 1999;60:1263–1270. 177. Lim H, Paria BC, Das SK, Dinchuk JE, Langenbach R, Trzaskos JM, Dey SK. Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 1997;91:197–208. 178. Castrillon DH, Miao L, Kollipara R, Horner JW, DePinho RA. Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science 2003;301:215–218. 179. Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM. Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 1996;383:531–535. 180. Robb L, Li R, Hartley L, Nandurkar HH, Koentgen F, Begley CG. Infertility in female mice lacking the receptor for interleukin 11 is due to a defective uterine response to implantation. Nat Med 1998;4:303–308. 181. Lee SL, Sadovsky Y, Swirnoff AH, Polish JA, Goda P, Gavrilina G, Milbrandt J. Luteinizing hormone deficiency and female infertility in mice lacking the transcription factor NGFI-A (Egr-1). Science 1996;273:1219–1221. 182. Topilko P, Schneider-Maunoury S, Levi G, Trembleau A, Gourdji D, Driancourt MA, Rao CV, Charnay P. Multiple pituitary and ovarian defects in Krox-24 (NGFI-A, Egr-1)-targeted mice. Mol Endocrinol 1998;12:107–122. 183. Rajkovic A, Pangas SA, Ballow D, Suzumori N, Matzuk MM. NOBOX deficiency disrupts early folliculogenesis and oocyte-specific gene expression. Science 2004;305:1157–1159. 184. Burns KH, Viveiros MM, Ren Y, Wang P, DeMayo FJ, Frail DE, Eppig JJ, Matzuk MM. Roles of NPM2 in chromatin and nucleolar organization in oocytes and embryos. Science 2003;300:633– 636. 185. Lydon JP, DeMayo FJ, Funk CR, Mani SK, Hughes AR, Montgomery CA Jr, Shyamala G, Conneely OM, O’Malley BW. Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes Dev 1995;9:2266–2278. 186. Horseman ND, Zhao W, Montecino-Rodriguez E, Tanaka M, Nakashima K, Engle SJ, Smith F, Markoff E, Dorshkind K. Defective mammopoiesis, but normal hematopoiesis, in mice with a targeted disruption of the prolactin gene. EMBO J 1997;16: 6926–6935. 187. Sugimoto Y, Yamasaki A, Segi E, Tsuboi K, Aze Y, Nishimura T, Oida H, Yoshida N, Tanaka T, Katsuyama M, Hasumoto K, Murata T, Hirata M, Ushikubi F, Negishi M, Ichikawa A, Narumiya S.

188.

189.

190.

191.

192.

193.

194.

195.

196.

197.

198.

199.

200.

201.

202.

203.

204.

205.

206.

Failure of parturition in mice lacking the prostaglandin F receptor. Science 1997;277:681–683. Varani S, Elvin JA, Yan C, DeMayo J, DeMayo FJ, Horton HF, Byrne MC, Matzuk MM. Knockout of pentraxin 3, a downstream target of growth differentiation factor-9, causes female subfertility. Mol Endocrinol 2002;16:1154–1167. Mahendroo MS, Cala KM, Russell DW. 5 Alpha-reduced androgens play a key role in murine parturition. Mol Endocrinol 1996;10: 380–392. Teglund S, McKay C, Schuetz E, van Deursen JM, Stravopodis D, Wang D, Brown M, Bodner S, Grosveld G, Ihle JN. Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses. Cell 1998;93:841–850. Wu X, Viveiros MM, Eppig JJ, Bai Y, Fitzpatrick SL, Matzuk MM. Zygote arrest 1 (Zar1) is a novel maternal-effect gene critical for the oocyte-to-embryo transition. Nat Genet 2003;33:187–191. Dean J. Oocyte-specific genes regulate follicle formation, fertility and early mouse development. J Reprod Immunol 2002;53: 171–180. Rankin TL, O’Brien M, Lee E, Wigglesworth K, Eppig J, Dean J. Defective zonae pellucidae in Zp2-null mice disrupt folliculogenesis, fertility and development. Development 2001;128:1119– 1126. Matzuk MM, Finegold MJ, Su JG, Hsueh AJ, Bradley A. Alphainhibin is a tumour-suppressor gene with gonadal specificity in mice. Nature 1992;360:313–319. Ma X, Reyna A, Mani SK, Matzuk MM, Kumar TR. Impaired male sexual behavior in activin receptor type II knockout mice. Biol Reprod 2005;73:1182–1190. Kendall SK, Samuelson LC, Saunders TL, Wood RI, Camper SA. Targeted disruption of the pituitary glycoprotein hormone alphasubunit produces hypogonadal and hypothyroid mice. Genes Dev 1995;9:2007–2019. Barlow C, Hirotsune S, Paylor R, Liyanage M, Eckhaus M, Collins F, Shiloh Y, Crawley JN, Ried T, Tagle D, Wynshaw-Boris A. Atm-deficient mice: A paradigm of ataxia telangiectasia. Cell 1996;86:159–171. Xu Y, Ashley T, Brainerd EE, Bronson RT, Meyn MS, Baltimore D. Targeted disruption of ATM leads to growth retardation, chromosomal fragmentation during meiosis, immune defects, and thymic lymphoma. Genes Dev 1996;10:2411–2422. Toscani A, Mettus RV, Coupland R, Simpkins H, Litvin J, Orth J, Hatton KS, Reddy EP. Arrest of spermatogenesis and defective breast development in mice lacking A-myb. Nature 1997;386: 713–717. Lu Q, Hasty P, Shur BD. Targeted mutation in beta1,4-galactosyltransferase leads to pituitary insufficiency and neonatal lethality. Dev Biol 1997;181:257–267. Sicinski P, Donaher JL, Geng Y, Parker SB, Gardner H, Park MY, Robker RL, Richards JS, McGinnis LK, Biggers JD, Eppig JJ, Bronson RT, Elledge SJ, Weinberg RA. Cyclin D2 is an FSHresponsive gene involved in gonadal cell proliferation and oncogenesis. Nature 1996;384:470–474. Ruggiu M, Speed R, Taggart M, McKay SJ, Kilanowski F, Saunders P, Dorin J, Cooke HJ. The mouse Dazla gene encodes a cytoplasmic protein essential for gametogenesis. Nature 1997;389:73–77. Miyamoto N, Yoshida M, Kuratani S, Matsuo I, Aizawa S. Defects of urogenital development in mice lacking Emx2. Development 1997;124:1653–1664. Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M, Korach KS, Gustafsson JA, Smithies O. Generation and reproductive phenotypes of mice lacking estrogen receptor beta. Proc Natl Acad Sci USA 1998;95:15677–15682. Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O. Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci USA 1993;90:11162–11166. Kumar TR, Wang Y, Lu N, Matzuk MM. Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nat Genet 1997;15:201–204.

CHAPTER 44 / MURINE MODELS FOR REPRODUCTION

207. Baker J, Hardy MP, Zhou J, Bondy C, Lupu F, Bellve AR, Efstratiadis A. Effects of an Igf1 gene null mutation on mouse reproduction. Mol Endocrinol 1996;10:903–918. 208. Baker J, Liu JP, Robertson EJ, Efstratiadis A. Role of insulin-like growth factors in embryonic and postnatal growth. Cell 1993; 75:73–82. 209. Lei ZM, Mishra S, Zou W, Xu B, Foltz M, Li X, Rao CV. Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene. Mol Endocrinol 2001;15:184–200. 210. Ma X, Dong Y, Matzuk MM, Kumar TR. Targeted disruption of luteinizing hormone beta-subunit leads to hypogonadism, defects in gonadal steroidogenesis, and infertility. Proc Natl Acad Sci USA 2004;101:17294–17299. 211. Rao CV, Lei ZM. Consequences of targeted inactivation of LH receptors. Mol Cell Endocrinol 2002;187:57–67. 212. Zhang FP, Poutanen M, Wilbertz J, Huhtaniemi I. Normal prenatal but arrested postnatal sexual development of luteinizing hormone receptor knockout (LuRKO) mice. Mol Endocrinol 2001;15:172–183. 213. Edelmann W, Cohen PE, Kane M, Lau K, Morrow B, Bennett S, Umar A, Kunkel T, Cattoretti G, Chaganti R, Pollard JW, Kolodner RD, Kucherlapati R. Meiotic pachytene arrest in MLH1-deficient mice. Cell 1996;85:1125–1134. 214. Edelmann W, Cohen PE, Kneitz B, Winand N, Lia M, Heyer J, Kolodner R, Pollard JW, Kucherlapati R. Mammalian MutS homologue 5 is required for chromosome pairing in meiosis. Nat Genet 1999;21:123–127. 215. Good DJ, Porter FD, Mahon KA, Parlow AF, Westphal H, Kirsch IR. Hypogonadism and obesity in mice with a targeted deletion of the Nhlh2 gene. Nat Genet 1997;15:397–401. 216. Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E, Polyak K, Tsai LH, Broudy V, Perlmutter RM, Kaushansky K, Roberts JM. A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice. Cell 1996;85:733–744. 217. Kiyokawa H, Kineman RD, Manova-Todorova KO, Soares VC, Hoffman ES, Ono M, Khanam D, Hayday AC, Frohman LA, Koff A. Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27(Kip1). Cell 1996;85:721–732. 218. Nakayama K, Ishida N, Shirane M, Inomata A, Inoue T, Shishido N, Horii I, Loh DY, Nakayama K. Mice lacking p27(Kip1) display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell 1996;85:707–720. 219. Goffin V, Binart N, Clement-Lacroix P, Bouchard B, Bole-Feysot C, Edery M, Lucas BK, Touraine P, Pezet A, Maaskant R, Pichard C, Helloco C, Baran N, Favre H, Bernichtein S, Allamando A, Ormandy C, Kelly PA. From the molecular biology of prolactin and its receptor to the lessons learned from knockout mice models. Genet Anal 1999;15:189–201. 220. Grosdemouge I, Bachelot A, Lucas A, Baran N, Kelly PA, Binart N. Effects of deletion of the prolactin receptor on ovarian gene expression. Reprod Biol Endocrinol 2003;1:12. 221. Kelly PA, Binart N, Lucas B, Bouchard B, Goffin V. Implications of multiple phenotypes observed in prolactin receptor knockout mice. Front Neuroendocrinol 2001;22:140–145. 222. Jeyasuria P, Ikeda Y, Jamin SP, Zhao L, De Rooij DG, Themmen AP, Behringer RR, Parker KL. Cell-specific knockout of steroidogenic factor 1 reveals its essential roles in gonadal function. Mol Endocrinol 2004;18:1610–1619. 223. Jorgez CJ, Klysik M, Jamin SP, Behringer RR, Matzuk MM. Granulosa cell-specific inactivation of follistatin causes female fertility defects. Mol Endocrinol 2004;18:953–967. 224. Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 1999;21:70–71. 225. Zambrowicz BP, Imamoto A, Fiering S, Herzenberg LA, Kerr WG, Soriano P. Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of betagalactosidase in mouse embryos and hematopoietic cells. Proc Natl Acad Sci USA 1997;94:3789–3794. 226. Chang C, Chen YT, Yeh SD, Xu Q, Wang RS, Guillou F, Lardy H, Yeh S. Infertility with defective spermatogenesis and hypotestos-

227.

228.

229.

230.

231.

232.

233.

234.

235.

236.

237.

238.

239.

240.

241.

242.

243.

423

teronemia in male mice lacking the androgen receptor in Sertoli cells. Proc Natl Acad Sci USA 2004;101:6876–6881. De Gendt K, Swinnen JV, Saunders PT, Schoonjans L, Dewerchin M, Devos A, Tan K, Atanassova N, Claessens F, Lecureuil C, Heyns W, Carmeliet P, Guillou F, Sharpe RM, Verhoeven G. A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis. Proc Natl Acad Sci USA 2004;101: 1327–1332. Holdcraft RW, Braun RE. Androgen receptor function is required in Sertoli cells for the terminal differentiation of haploid spermatids. Development 2004;131:459–467. Pangas SA, Li X, Robertson EJ, Matzuk MM. Premature luteinization and cumulus cell defects in ovarian-specific Smad4 knockout mice. Mol Endocrinol 2006;20:1406–1422. Charles MA, Saunders TL, Wood WM, Owens K, Parlow AF, Camper SA, Ridgway EC, Gordon DF. Pituitary-specific Gata2 knockout: Effects on gonadotrope and thyrotrope function. Mol Endocrinol 2006;20:1366–1377. Zhao L, Bakke M, Krimkevich Y, Cushman LJ, Parlow AF, Camper SA, Parker KL. Steroidogenic factor 1 (SF1) is essential for pituitary gonadotrope function. Development 2001;128:147–154. Davis AM, Seney ML, Stallings NR, Zhao L, Parker KL, Tobet SA. Loss of steroidogenic factor 1 alters cellular topography in the mouse ventromedial nucleus of the hypothalamus. J Neurobiol 2004;60:424–436. Dellovade TL, Young M, Ross EP, Henderson R, Caron K, Parker K, Tobet SA. Disruption of the gene encoding SF-1 alters the distribution of hypothalamic neuronal phenotypes. J Comp Neurol 2000;423:579–589. Nord AS, Chang PJ, Conklin BR, Cox AV, Harper CA, Hicks GG, Huang CC, Johns SJ, Kawamoto M, Liu S, Meng EC, Morris JH, Rossant J, Ruiz P, Skarnes WC, Soriano P, Stanford WL, Stryke D, von Melchner H, Wurst W, Yamamura K, Young SG, Babbitt PC, Ferrin TE. The International Gene Trap Consortium Website: A portal to all publicly available gene trap cell lines in mouse. Nucleic Acids Res 2006;34:D642–648. Skarnes WC, von Melchner H, Wurst W, Hicks G, Nord AS, Cox T, Young SG, Ruiz P, Soriano P, Tessier-Lavigne M, Conklin BR, Stanford WL, Rossant J. A public gene trap resource for mouse functional genomics. Nat Genet 2004;36:543–544. Pask AJ, Kanasaki H, Kaiser UB, Conn PM, Janovick JA, Stockton DW, Hess DL, Justice MJ, Behringer RR. A novel mouse model of hypogonadotrophic hypogonadism: N-ethyl-N-nitrosourea-induced gonadotropin-releasing hormone receptor gene mutation. Mol Endocrinol 2005;19:972–981. Matzuk MM, Finegold MJ, Mishina Y, Bradley A, Behringer RR. Synergistic effects of inhibins and Mullerian-inhibiting substance on testicular tumorigenesis. Mol Endocrinol 1995;9:1337– 1345. Cho C, Willis WD, Goulding EH, Jung-Ha H, Choi YC, Hecht NB, Eddy EM. Haploinsufficiency of protamine-1 or -2 causes infertility in mice. Nat Genet 2001;28:82–86. Shirley CR, Hayashi S, Mounsey S, Yanagimachi R, Meistrich ML. Abnormalities and reduced reproductive potential of sperm from Tnp1- and Tnp2-null double mutant mice. Biol Reprod 2004;71:1220–1229. Kozar K, Ciemerych MA, Rebel VI, Shigematsu H, Zagozdzon A, Sicinska E, Geng Y, Yu Q, Bhattacharya S, Bronson RT, Akashi K, Sicinski P. Mouse development and cell proliferation in the absence of D-cyclins. Cell 2004;118:477–491. Jeffs B, Ito M, Yu RN, Martinson FA, Wang ZJ, Doglio LT, Jameson JL. Sertoli cell-specific rescue of fertility, but not testicular pathology, in Dax1 (Ahch)-deficient male mice. Endocrinology 2001;142:2481–2488. Meeks JJ, Russell TA, Jeffs B, Huhtaniemi I, Weiss J, Jameson JL. Leydig cell-specific expression of DAX1 improves fertility of the Dax1-deficient mouse. Biol Reprod 2003;69:154–160. Karpova T, Maran RR, Presley J, Scherrer SP, Tejada L, Heckert LL. Transgenic rescue of SF-1-null mice. Ann NY Acad Sci 2005;1061:55–64.

45

Pig Model to Study Dynamics of Steroids During Ovarian Follicular Growth and Maturation DAMODAR K. MAHAJAN

ABSTRACT

Key Words: Gilts, Steroids, Ovary, Follicle, Follicular fluid, Perifollicular fluid, Follicular growth, Progesterone, Androgens, Estrogens.

The pig is a polyovular farm animal and its prolificacy differs in different breeds. The female pig has an estrous cycle of 20–21 days with a short follicular phase of only 3 days and a long luteal phase. The luteal phase starts at day 1 after ovulation to day 15 of the estrous cycle and it overlaps the early follicular phase on day 16–17. The follicular phase lasts only 3–4 days. The early follicular growth is initiated while a high concentration of progesterone (P) is secreted by active corpora lutea, circulating it in the peripheral blood. In this study, P, androstenedione (A), testosterone (T), estrone (E1), and estradiol-17β (E2) were measured in peripheral blood, ovarian venous blood, follicular fluid, and perifollicular blood at various stages of the pig estrous cycle. Concentrations of P in peripheral blood, ovarian venous blood, follicular fluid, and perifollicular blood (blood from the vasculature surrounding the follicle) during the active luteal phase of gilts were 19.0 ± 2.4, 59.2 ± 12.7, and 3937 ± 710 ng/ml, respectively. Interestingly, the high concentration of P in perifollicular blood did not equilibrate with the follicular fluid and it appears that there is a blood barrier between the two. This level of P was significantly lower in the late luteal, follicular, and estrous stages. The follicular fluid in the follicular phase contained the highest concentrations of A (163 ± 37.9 ng/ml) of all the stages of follicular growth. During this stage the perifollicular blood also contained a concentration of A higher than that observed at any other stage of the estrous cycle. On the day of estrus the amounts of both A and T in the follicular fluid as well as in ovarian venous blood were at their lowest. Concentrations of T in perifollicular blood were higher in the luteal phase than in the follicular phase. The high levels of E1 (22.94 ± 2.95 ng/ml) and E2 (18.96 ± 1.80 ng/ml) in the follicular fluid during the follicular phase may be due to aromatization of A and T, which are also present in high concentrations in follicular fluid and are in contact with granulosa cells. On the day of estrus E1 and E2 in the follicular fluid were 7.42 ± 1.45 and 6.93 ng/ml, respectively, which may precede the onset of the preovulatory surge. The lower levels of E1 and E2 in perifollicular blood than in follicular fluid suggest that the main site of aromatization could be the granulosa cells in the follicles.

INTRODUCTION The pig is an ideal animal model to study reproductive endocrinology and, in particular, to understand ovarian physiology. The ovarian morphology, tissue differentiation, and actions of individual cell types have been extensively studied. The ease of obtaining ovaries from abattoir facilitated the initial basic investigation of ovarian function, though the background of the gilts or sows were in question. However, availability of defined breeds, knowledge of their ages, nutrition, and any hormonal treatment and assessment of the estrous period made it easy to study carefully defined protocols of investigation. The pig is polyovular and the important limiting factor affecting sow productivity is prolificacy, which is defined as the number of viable piglets born per litter. Chinese Meishan farrow have an average of three to five more piglets per litter than the European breed White Composite. Other mixed breeds vary with a minimum of 12 to 20 piglets.1,2 The ovulation rate is a major determinant of reproductive efficacy, which is genetically controlled within a particular breed. After recruitment of the primordial follicles, the preovulatory follicles are selected, which then become dominant and progress toward ovulation. Growth, maturation, and ovulation are dependent on endocrine and paracrine control. The sequential effect of folliclestimulating hormone (FSH) and leutinizing hormone (LH) on the development of the follicles is controlled by a hypothalamic–pituitary–ovarian feedback mechanism. Binding of insulin to various follicular cells as well as the action of insulin-like growth factors IGF-I and IGF-II and their binding proteins,3,4 such as inhibin and activin,5 produced within the follicle exert metabolic actions directly or indirectly on the follicular cells and overall follicular growth. Follicular angiogenesis is promoted by vascular endothelial growth factor (VEGF), which is produced by the granulosa cells within the follicle. Steroids produced by interstitial, theca, and granulosa cells are distributed within and out of the ovary through the basement membrane and the follicular vasculature of the follicle to reach various organs to exert or promote metabolic actions. Synthesis of various steroids in the ovary and their secretion into the circulation are a complex process because of the ovarian–

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

425

426

SECTION V / MODELS OF BEHAVIOR

pituitary feedback system. The two-cell type theory of steroid synthesis in ovarian tissue postulating that the theca interna cells of the follicle produce mainly androgens and estrogens, while the luteinized granulosa cells secrete progestins6 was suggested by Short in 1962, and it was based on steroid concentrations in the follicular fluid and luteal tissue. In the following year it was reported that equine corpora lutea have a low level of 17αhydroxylase (P45017α) and 17α-hydroxyprogesterone-20-lyase (P450c17) enzyme activity.7 Thus, the large amount of progesterone (P) produced by the corpus luteum was not converted to C19 steroids within the luteal tissue. The theca cells of the Graafian follicle are able to produce P and contain all the necessary enzymes to convert P to androgens and estrogens.7–9 The granulosa cells appear to have lower P450c17 activity than theca cells but possess very active aromatase (P450arom), which converts ∆4-androstenedione (A) and testosterone (T) to estrogens.10–12 When steroid synthesis by theca and granulosa cells in culture was studied, it was observed that the combined cell culture more efficiently synthesized estradiol-17β (E2) than either of the cell types alone.13–15 From these in vitro studies and the data on steroid concentrations in follicular fluid at various stages of follicular growth, it is postulated that P, A, T, estrone (E1), and E2 are synthesized in different compartments of the ovary and that the ability of these different tissues to produce steroids changes during various stages of follicular growth. These compartments do not appear to be isolated and there appears to be an active exchange of steroids between different types of steroid-producing cells. Since the corpus luteum, stroma, and theca are vascularized, steroids produced by these cells would be readily mixed into the follicular circulation and may accumulate in the follicular fluid. There is sufficient evidence to indicate that substances in the follicular fluid constantly equilibrate with the ovarian blood,6,16 although no correlation was found between the concentration of steroids in follicular fluid and the corresponding steroid level in peripheral plasma of the same pig during the estrous cycle.17 Based on the observation that very high amounts of ovarian steroids were present in the follicular fluid of preovulatory follicles, it was theorized that follicular fluid serves as a storage for steroids, particularly E2 and P, which are required by the uterus for implantation. In the present investigation P, A, T, E1, and E2 were measured in peripheral blood, ovarian venous blood, follicular fluid, and perifollicular blood during the entire estrous cycle of gilts; there is speculation as to whether they correlate and whether the active corpus luteum could be a source of progesterone to theca and granulosa cells for conversion into C19 and C18 steroids and serve as one of the regulators of steroidogenesis in the pig. The quantitative determination of steroidogenic enzyme mRNAs and certain steroids in follicular tissue and follicular fluid was carried out during either the regumate or altrenogestsynchronized preovulatory period of follicular growth of the gilts.18–20 Day 1 was considered the prefollicular phase, day 3 the early follicular phase, day 5 the mid-follicular phase, and day 7 the late follicular phase. It was concluded that the P450arom enzyme mRNA increased successively from day 1 to day 5, dropped significantly on day 7 to a level less than day 1, and correlated only partially with the E2 in follicular fluid. A similar poor correlation was true for P45017α mRNA to testosterone and cholesterol side chain cleavage (P450scc) to progesterone. In animal husbandry when the pigs are grown for the meat industry, all animals are

treated with progestational compound or progestational compound plus estrogenic compounds to arrest ovulation or continuation of cycling and also to increase their weight. By rules and regulations, steroid treatment is stopped at least 2 weeks before they are brought to abattoirs. Therefore ovaries collected at the abattoir have small, immature follicles of 2–5 mm or so, and the results of studies conducted on these ovaries could be different from studies conducted on naturally cycling gilts. In the cycling gilts after ovulation, days 3–5 are early luteal phase with active corpora lutea producing a large amount of progesterone. At this stage the surface of the ovary is covered with corpora lutea and the small immature follicles are situated among the corpora lutea in close proximity. It is reported that follicular fluid contains a large amount of progesterone,21 which may not be produced by the follicular tissue, but may be accumulated from the vasculature of the corpora lutea. This progesterone may inhibit some enzyme activity, specifically P45017α and P450c17,7 and at the same time it may act as a precursor for steroid-producing enzymes. Hence, in such situations the follicular steroid enzymatic mRNA may not correlate with steroids present in the follicular fluid of these small follicles, some of which may be destined to become atretic follicles.

VARIOUS BLOOD SAMPLES AND FOLLICULAR FLUID COLLECTION Ovarian follicular fluid, perifollicular blood, and peripheral blood samples were collected from 40 gilts at the swine research farm at Ohio Agricultural Research and Development Center, Wooster, OH. Gilts 1–2 years of age were observed for regularity of their estrous cycles according to a method previously described22 and only those having three consistent consecutive estrous cycles of 19–21 days were chosen for the study. The first day of estrous behavior was designated day 0 of the cycle. For each gilt on the appropriate day of the estrous cycle, surgical anesthesia was induced with 1.5–2 g of sodium thiamylal (10% solution) intravenously via an ear vein, and surgical anesthesia was maintained by methoxyflurane and oxygen inhalation with a closed circuit anesthesia machine. First, a 20-ml peripheral blood sample was withdrawn from the anterior vena cava and allowed to clot. Laparotomy was performed and the ovaries were exposed. Follicular fluid from an individual “healthy-looking” follicle (a follicle full of fluid, reddish in color, and transparent without white deposit in it) was withdrawn with a tuberculin syringe fitted with a 26-gauge hypodermic needle until the follicle had completely collapsed. Then, within a few seconds, the empty follicle was filled with blood, which was then withdrawn over a 1- min period with 1-min intervals between samples. These samples were considered to be perifollicular blood.17,23 Five such consecutive samples of perifollicular blood were collected. Sometimes external pressure was applied on the surrounding side of the follicle to fill the empty antrum with blood. Follicular fluid and perifollicular blood samples from two or three follicles were collected from both ovaries. Blood samples less than 25 µl were discarded. The remaining follicles on each ovary were aspirated, and that fluid was pooled for the right or left ovary. The volume of the follicular fluid or blood sample was measured immediately by a capillary pipette or by a narrow graduated tube. The individual blood volume varied from 30 µl to 600 µl and steroids were measured in the whole blood of perifollicular blood samples. Fluid from nonovulated follicular cysts of the previous estrous cycle were not

CHAPTER 45 / STEROIDS DURING OVARIAN FOLLICULAR GROWTH AND MATURATION

included in the pool of follicular fluid of an individual ovary, but were processed separately. The follicles up to day 5 of the estrous cycle were too small in size ( +/− /− > −/+ Transgene +/+/+ > +/+/− +/+ > +/− +/− > −/− +/+ > +/− +/+ > +/− +/+ > +/− +/+ > +/− +/+ > +/− +/+ > +/− +/+ > +/− +/− > −/− +/− > +/+ +/+/+/+ > +/+/+/− +/+/+/+ > +/+/+/− +/+/+/+ > +/+/+/− +/O > −/O +/O > −/O +/O > −/O +/O > −/O +/O > −/O −/O > +/O +/O > −/O +/O > −/O +/O > −/O +/O > −/O +/−* > +/− +/−* > −/−* +/−* > +/− +/−* > −/−* Transgene Transgene +/+ > +/− +/− > −/− +/+ > +/− +/+ > +/− +/+ > +/− +/− > +/+ +/+ > +/−

HeLa cells HT1080 cells HT1080 cells HT1080 cells HT1080 cells HT1080 cells + RAD51 o/e JB and LB cells + oligos HT1080 cells HepG2 cells HEC1A cells HEC1A cells EJ cells Hu11 cells RPE/RPK cells HT1080 cells JM cells CEM cells HT1080 cells HT1080 cells + CDC2 o/e CFE HT29-18-C1 HT29-18-C1 HT29-18-C1 PLB-985 cells HT1080 cells HT1080 cells HT1080 cells HT1080 cells HT1080 cells ± BLM RNAi HT1080 cells HT1080 cells HT1080 cells + RAD51 o/e HT1080 cells DLD-1 cells DLD-1 cells HEC1A cells HEC1A cells EJ cells HT1080 cells LF1 cells LF1 cells LF1 cells LF1 cells LF1 cells SNB-19 cells + oligos ES cells

Reference 125 125 126 127 51 51 128 53 81 129 129 74 74 76 56 57 58 82 82 130 75 75 75 131 53 53 53 56 56 50 51 51 49 83 83 129 129 132 133 78 78 77 77 77 128 54

+: Number of correctly targeted clones identified. #: Total number of clones analyzed. c Frequency: Number of correctly targeted clones (+) divided by the total number of clones (#) analyzed. d Genotype: (+) designates wild type; (−) designates mutated; (−*) designates a preexisting mutated allele; (O) designates a hemizygous configuration. e Miscellaneous: (+RAD51 o/e) designates that the experiment was performed in cells overexpressing hRAD51; (+oligos) designates a study done solely by oligonucleotide transfections without a targeting vector; (+CDC2 o/e) designates that the experiment was performed in cells overexpressing hCDC2; (±BLM RNAi) designates a study done in the presence and absence, respectively, of RNAi directed against hBLM1. a b

CHAPTER 53 / GENE TARGETING IN HUMAN SOMATIC CELLS

with targeting frequencies as high as 28%51 and as low as 0.01%53 having been reported for HT1080 cells. Parameters such as chromatin or accessibility differences, frequency of repetitive DNA within a given locus, variable expression of HR gene products between cell lines, use or nonuse of “isogenic” DNA,8 choice of delivery for the vector, length of homology on the donor DNA, to mention just a few, have all been invoked to explain differences in targeting frequency. Unfortunately, it is not known how important any of these parameters really are. Thus, there currently does not exist an a priori method to determine whether a particular gene can be correctly targeted and, if so, at what frequency. A comprehensive investigation of why some genes “target better” than other genes would greatly benefit the field.

517

The HCT116 Cell Line In the early 1990s, Shirasawa et al. described successful gene targeting of the Ki-Ras gene in two human colon carcinoma cell lines, DLD-1 and HCT116.83 The laboratories of Drs. Bert Vogelstein and Kenneth Kinzler, which already had an established record in the investigation of colon cancer and genetic instability,84,85 apparently took this report to heart and initiated their own series of studies in which they disrupted genes thought to be involved in DNA Repair/chromosome stability in the HCT116 cell line.59,77,86–89 The impressive success of these studies caught the attention of the DNA Repair community and many other laboratories have subsequently followed in their footsteps. Overall, 22 genes have been disrupted in this cell line in the intervening 13 years (Table 53–2). Some of these genes

Table 53–2 Gene targeting studies in the HCT116 cell line Gene 14-3-3σ 14-3-3σ ATR ATR Bax β-Catenin β-Catenin β-Catenin β-Catenin β-Catenin β-Catenin BLM BLM DNMT1 DNMT1 DNMT3b DNMT3b DNMT3b DNMT3b EME1 HPRT HPRT Ki-Ras Ki-Ras Ku86 Ku86 Ku86 Ku86 Ku86 Ku86 Ku86 Mus81 Mus81 ORC2 ORC2 p21 p21 p21 p53 p53 p53 p53

+a

#b

Frequencyc

2 26

∼5,000 216

∼0.0004 0.1204

5

576

0.0087

1 12 2 4 4 9

190 106 5,250 9,700 12,000 93

0.0053 0.1132 0.0004 0.0004 0.0003 0.0968

0 2 3 2 2 1 1 2 2

500 354 487 487 229 54 279 744 2,380

0 37 5 1 1 1 8

232 100 20 600 940 1,141 527

0.0000 0.0056 0.0062 0.0041 0.0087 0.0185 0.0036 0.0027 0.0008 ∼0.0250 ∼0.0880 0.0000 0.3700 0.2500 0.0017 0.0011 0.0009 0.0152

Genotyped +/+ > +/− +/− > −/− +/+ > +/− +/− > −/− +/− > −/− +/−* > +/− +/−* > −/−* +/−* > +/− +/−* > −/−* +/−* > +/− +/−* > −/−* +/+ > +/− +/− > −/− +/+ > +/− +/− > −/− +/+ > +/− +/− > −/− +/+ > +/− +/+ > +/− +/+ > +/− +/O > −/O +/O > −/O +/−* > +/− +/−* > −/−* +/+ > +/− +/+ > +/− +/− > +/− −/+ > −/− +/+ > +/− +/+ > +/− +/− > +/− +/+ > +/− +/− > −/− +/+ > +/− +/− > −/− +/+ > +/− +/+ > +/− +/+ > +/− +/+ > +/− +/− > −/− +/+ > +/− +/+ > +/−

Miscellaneouse

In a BLM−/− bkg In a p53−/− bkg

HSV TK selection DT selection Retargeting In a p53−/− bkg Retargeting

Promoter + Promoterless

In a BLM−/− bkg

Reference 86 86 73 73 98 134 134 99 99 65 65 72 72 87 87 89 89 72 72 135 52 52 83 83 64 64 64 64 62 62 62 135 135 136 136 59 59 59 77 77 72 72 continued

518

SECTION V / MODELS OF BEHAVIOR

Table 53–2 (continued) Gene RAD51C RAD51C RAD51L2 RAD51L2 RAD51L2 RAD51L2 RAD51L2 RAD54B RAD54B RAD54B RAD54B RAD54B Securin Securin Smad4 Smad4 XRCC2 XRCC2 XRCC2 XRCC2 XRCC2 XRCC3 XRCC3 Totals

+a

#b

Frequencyc

5 3 6 6 7 1 0 2 9 2 7 6

1,930 1,361 7,197 5,948 6,509 10,382 7,398 25 61 78 283 349

0.0026 0.0022 0.0008 0.0010 0.0011 0.0001 0.0000 0.0800 0.1475 0.0256 0.0247 0.0172

4 2 6 7 14 1 0

300 254 94 93 166 173 120

0.0133 0.0079 0.0638 0.0753 0.0843 0.0058 0.0058

5 226

193 85,119

0.0259 0.0027

Genotyped +/+ > +/− +/+ > +/− +/+ > +/− +/+ > +/− +/+ > +/− +/+ > +/− +/+ > +/− +/+/+ > +/+/− +/+/− > +/−/− +/−/− > −/−/− +/+/+ > +/+/− +/+/+ > +/+/− +/+ > +/− +/− > −/− +/+ > +/− +/− > −/− +/+ > +/− +/+ > +/− +/+ > +/− +/+ > +/− +/+ > +/− +/+ > +/− +/− > −/−

Miscellaneouse

Reference

In a XRCC3−/− bkg In a RAD54B+/+/+ bkg In a RAD54B+/+/− bkg In a RAD54B+/−/− bkg In a RAD54B−/−/− bkg In a RAD54B−/−/− bkg

In a XRCC3−/− bkg

In a RAD54B+/+/+ In a RAD54B+/+/− In a RAD54B+/−/− In a RAD54B−/−/− In a RAD54B−/−/−

bkg bkg bkg bkg bkg

90 90 61 61 61 61 61 61 61 61 90 90 88 88 137 137 61 61 61 61 61 90 90

a−d

As defined in Table 53–1. Miscellaneous: (In a BLM−/−, p53−/−, XRCC3−/−, RAD54+/+/+, RAD54+/+/−, RAD54+/−/−, RAD54−/−/−, bkg) designates studies in which the gene targeting was done in the indicated respective backgrounds; (HSV TK selection) designates a study using herpes simplex virus thymidine kinase gene as the negative selection; (DT selection) designates a study using the diphtheria toxin gene as the negative selection; (Retargeting) designates events in which the targeting vector has integrated in the previously disrupted allele; (Promoter +) designates a study done with a vector using a selectable marker driven by a promoter; (Promoterless) designates a study done with a vector using a promoterless selectable marker. e

are identical to those that have been disrupted in other cell lines (e.g., p21, p53, and HPRT; compare Table 53–1 with Table 53–2), but many of them are novel. In particular, the HCT116 studies are noteworthy because of the large number of times that two rounds of successful gene targeting have been carried out, allowing for an assessment of the null phenotype (Table 53–2). Moreover, HCT116 is the only cell line, with the recent exception of NALM6 cells (see below), in which compound knockout strategies—in which two or more genes are disrupted to investigate their genetic interaction with one another— have succeeded.52,61,62,72,90 Overall, 226 correctly targeted integrations have been reported for 85,119 drug-resistant clones analyzed (Table 53–2). This represents a rather pathetic targeting frequency of 0.27%, which is an order of magnitude lower than that reported for the non-HCT116, nonNALM-6 cell lines (Table 53–1). At this frequency, the work required to identify a correctly targeted clone becomes a significant issue and is thus usually undertaken only by laboratories with sufficient manpower. This frequency does, however, include a study in which the RAD54B gene was first disrupted in HCT116 cells and then (mostly unsuccessful) attempts were made to target the RAD51L2 and XRCC2 loci, thus validating the requirement for RAD54 in HR/gene targeting (Figure 53–2). Even if this large, negative study (two positive clones from 17,973 clones analyzed) and other studies in which the HCT116 genetic background was altered prior to use for gene targeting are omitted from analysis, the targeting frequency in a wild-type HCT116 background is still

a paltry 0.39%. This frequency is low enough to daunt all but the most committed of researchers. Still, it should be noted that there is great variability in the targeting frequency in HCT116 between loci, with frequencies as high as 37% being reported for the p21 locus59 and many instances of targeting frequencies ranging from 1 to 10% (Table 53–2). Again, unfortunately, there is no a priori way to know ahead of time how well a particular gene may or may not target. In summary, for the past 5–10 years, the HCT116 cell line has been the gold standard for performing gene knockouts in human somatic cells. Although it is an immortalized, transformed colorectal carcinoma cell line, its utility stems from its relatively stable karyotype91–93 and because it is diploid and wild-type for almost all of the relevant DNA Repair, DNA checkpoint, and chromosome stability genes59,77,88 with the exception of meiotic recombination defective 1194 (Mre11). Nonetheless, HCT116 is not a “normal” cell line. (1) It is mutated in the Mut L homolog 1 (MLH1) gene, which makes it a mismatch repair95,96 as well as a transcription-coupled repair97 defective cell line. As such, it has an overall microsatellite instability phenotype. In addition, genes that have homopolymeric tracts within them, such as Bax and Mre11, often spontaneously mutate one or both alleles.94,98 (2) HCT116 cells have gains of parts of chromosomes 8q (accounting for the triploid nature of the RAD54B gene61), 10q, 16q, and 17q and also contain two derivative chromosomes carrying two translocations, der(16)t(8;16) and der(18)t(17;18).91,92 (3) Finally,

CHAPTER 53 / GENE TARGETING IN HUMAN SOMATIC CELLS

519

Table 53–3 Gene targeting studies in the NALM-6 cell line Gene

+a

#b

APRT BLM BLM HPRT Ku70 Ku86 LIGIV LIGIV LIGIV LIGIV p53 p53 p53 p53 TDP1 XRCC3 XRCC3 Totals

5 14 5

134 168 274

0.0373 0.0833 0.0182

3 7 2 1

80 84 124 20

1 9 1 4 2 1 1 56

9 116 1 42 7 6 6 1,071

0.0375 0.0833 0.0161 0.0500 ∼0.0340 ∼0.0085 0.1111 0.0776 1.0000 0.0952 0.2857 0.1667 0.1667 0.0523

Frequencyc

Genotyped

Miscellaneouse

+/+ > +/− +/+ > +/− +/− > −/− −/O > +/O +/+ > +/− +/+ > +/− +/+ > +/− +/− > −/− +/+ > +/− +/− > −/− +/+ > +/− +/− > −/− +/+ > +/− +/− > −/− +/+ > +/− +/+ > +/− +/+ > +/−

±BLM RNAi Rescue ± BLM RNAi

In a BLM−/− bkg In a BLM−/− bkg

In a LIGIV−/− bkg

Reference 56 101 101 56 100 100 60 60 66 66 10 10 10 10 10 10 10

a−d

As defined in Table 53–1. Miscellaneous: (±BLM RNAi) designates a study in which the gene targeting was carried out in the presence (+) and absence (−), respectively, of RNAi directed against the Bloom’s syndrome helicase; (Rescue ± BLM RNAi) designates a study in which the correcting gene targeting a knockin experiment was carried out in the presence (+) and absence (−), respectively, of RNAi directed against the Bloom’s syndrome helicase; (In a BLM−/− bkg and LIGIV−/− bkg) designates studies in which the gene targeting was done in the indicated respective genetic backgrounds. e

HCT116 cells also contain a dominant gain-of-function mutation in the β-catenin gene, a potent transcription factor that drives c-myc expression.99 As a word of caution, it should be noted that the aberrancies described above are the ones that are known and reported. It is quite likely that other genes are mutated or misexpressed relative to a normal, nonimmortalized, nontransformed cell in HCT116 cells and the impact of such differences on the phenotypes of a knockout of your favorite gene is hard to assess. In the same cautionary vein, recent reports of phenotypic differences between a knockout of the Ku86 gene in HCT116 cells64,93 and NALM-6 cells100 suggest that investigators need to take care in generalizing their results to other cell lines and other systems. The NALM-6 Cell Line In 1998, Grawunder et al. described the successful targeting of both alleles of the DNA LIGIV gene in the human pre-B acute lymphoblastic leukemia (ALL) cell line, NALM-6.60 A total of three correctly targeted clones were obtained from 144 analyzed for a relatively robust 2.08% targeting frequency. This cell line then lay (scientifically) dormant for the next 5 years until a report appeared in 2004 from the laboratory of Dr. Koyama describing the disruption of the BLM gene.101 In the subsequent 2 years, the Koyama laboratory published a series of papers10,56,66,100 describing the disruption of an additional eight genes in this cell line (Table 53–3). Impressively, 56 correct targeting events from a total of 1071 drug-resistant clones analyzed have been reported, for an overall frequency of 5.23% (Table 53–3). Thus, this cell line appears to carry out gene targeting at least twice as well as most other human cell lines (Table 53–1) and 20 times better than HCT116 cells (Table 53–2). If these frequencies can be reproduced in other laboratories, it is likely that the NALM-6 cell line will supplant HCT116 as a model system for human somatic cell knockouts.

It must be reiterated, however, that NALM-6, like HCT116, cells are not “normal.” This line was established in culture some three decades ago from the peripheral blood of a 19-year-old man suffering from ALL.102 Although the cell line is diploid, it carries at least one derivative chromosome containing a translocation [der t(5;12)(q33.2;p13.2)].103 This translocation has fused the plateletderived growth factor receptor-β (PDGFRB) gene (a tyrosine kinase involved in cell proliferation) to the translocation-ETSleukemia (TEL) transcription factor gene,104 resulting in a dominant gain-of-function chimeric kinase. Coincidentally, NALM-6, like HCT116, is also defective in mismatch repair. In the case of NALM-6 neither mutS homolog 2 (MSH2) nor mutS homolog 6 (MSH6) is expressed.104 Thus, although it has not been directly demonstrated, it seems highly likely that NALM-6 cells will show microsatelite instability and high mutability of genes that have homopolymeric tracts within them. In addition, it can be anticipated that the more popular this cell line becomes, the greater is the likelihood that other genes that are mutated or misexpressed relative to a normal, nonimmortalized, nontransformed cell will be uncovered. Lastly, the same cautionary note concerning the recent reports of phenotypic differences between a knockout of the Ku86 gene in HCT116 cells64,93 and NALM-6 cells100 suggests that investigators utilizing NALM-6 cells—just like those utilizing HCT116 cells—need to take care in generalizing their results to other cell lines and other systems.

THE FUTURE OF GENE TARGETING: RECOMBINANT ADENO-ASSOCIATED VIRUS Adeno-associated virus (AAV) is a human parvovirus that has both latent and lytic life cycles.9,105,106 During the latent infection, AAV integrates stably into the human genome, preferentially, but

520

SECTION V / MODELS OF BEHAVIOR

not exclusively, at a site on chromosome 19.107,108 In an ironic twist, it was this characteristic of site-specific integration that first drew the attention of the gene therapy field to AAV, since the use of AAV as a gene delivery vector would not have the serious problem of random insertional mutagenesis attendant with almost every other viral vector delivery system. While this has turned out to be true109 and clinical trials using rAAV are underway,110 the use of AAV as a gene targeting vector has far outpaced its use as a gene therapy delivery vector. The ability to undergo a latent infection is a necessary part of the AAV life cycle since AAV requires the presence of a helper virus (generally adenovirus) for a productive lytic infection. Thus, AAV has been detected only in individuals undergoing the symptoms of an adenoviral105,111 or herpes simplex virus112 infection. Approximately 85% of the human population is seropositive for AAV, although there is no pathology or disease that has been attributed to an AAV infection.106 AAV is a single-stranded DNA virus of 4.68 kb. Either strand of the integrated AAV DNA can be packaged113 and both are equally infectious.114 Multiple serotypes of the virus are known, but only the AAV-2 serotype is routinely utilized in the laboratory. AAV encodes two genes, Rep (replication) and Cap (capsid). Rep is required for viral replication, needed for viral integration, and can potentiate viral gene expression.106 Cap encodes the viral capsid proteins, of which three isoforms, VP1, VP2, and VP3, exist.115,116 These genes are flanked by identical inverted terminal repeats (ITRs) of 145 bp that form T-shaped hairpins, due to three palindromic sequences residing within the ITRs.117 The ITRs are essentially the only cis-acting sequences necessary for viral replication,118 packaging, and integration.119 This fact greatly facilitated the development of rAAV as a gene delivery and genetargeting vector. Thus, if Rep and Cap are provided in trans, then the internal 4.39 kb of AAV can consist of recombinant DNA of the investigator’s choosing.120 Similarly, the adenoviral gene products E1A, E1B, E4, E2A, and VA are required for AAV replication.106 Originally, these were provided by coinfecting with live adenovirus, which, of course, precluded the use of the resulting AAV stock for gene therapy experiments. This significant detriment was overcome once again simply by providing recombinant adenoviral functions in trans.121 In summary, AAV has been known to the research community for some four decades and the life cycle of the virus is quite well understood. The virus was subsequently conscripted in the 1980s by investigators who were interested in viral vectors for use in gene therapy. Recombinant molecular biological methodologies were then used to develop an rAAV system for the production of virus stocks that could productively infect every human (and many other mammalian) cell line tested to date. This story took a very unexpected and surprising turn in 1998, when Russell and Hirata demonstrated that rAAV vectors could be used for gene targeting.122 Impressively, they used rAAV not just to inactivate genes, but to introduce defined modifications into homologous chromosomal sequences at very high frequency. They demonstrated this first with a Neo reporter construct, but also showed that they could modify an endogenous locus (HPRT) with similar efficiency.122 In the intervening 8 years, the Russell laboratory and many others have confirmed and extended these original observations. Thus, 15 endogenous loci have been inactivated in a wide variety of human cell lines, ranging from investigator favorites such as HCT116 and NALM-6 to more biologically relevant normal diploid fibroblastic or stem cell lines

(Table 53–4). The most spectacular use of rAAV to date was the correction of a dominant negative mutation in the COL1A1 gene in stem cells derived from patients afflicted with osteogenesis imperfecta.123 Overall, 234 correctly targeted events have been recorded from a total of 6305 drug-resistant colonies examined (Table 53–4). This is an overall targeting frequency of 3.71%, which is much better than the traditional transfection-based approaches using HCT116 cells (compare Table 53–2 to Table 53–4) and comparable to the targeting frequencies reported for non-HCT116, non-NALM-6 (compare Table 53–1 to Table 53–4), and NALM-6 (Compare Table 53–3 to Table 53–4) cells. Why rAAV works so well for gene targeting is not clear. A priori, the virus, because of its ITRs, has at least 145 nt of nonhomology at either end of the donor DNA and this is generally a deterrent to homologous integration. Moreover, because the drug selection cassettes that are usually present on a targeting vector are 2 to 2.5 kb in length, this restricts the left and right homology arms to 0.9 to 1.0 kb each, such that the resulting recombinant virus can be packaged properly. This length of homology for the arms is extremely short in comparison to traditional approaches using transfection of linear dsDNA, where arms of 3–8 kb are standard. That rAAV gene targeting does work, however, is indisputable. Since invasion of a single-stranded donor DNA into the recipient chromosome is an essential feature of gene targeting (Figure 53–2), it is probable that rAAV—an ssDNA virus— readily becomes an HR intermediate.9 Needless to say, other factors, such as the likely higher concentration of DNA in the nucleus provided by a viral infection as compared to DNA transfection, probably also work in its favor. Research into other parameters, such as multiplicity of infection, DNA sequence of the arms, cell cycle effects, and promoterless selection schemes, are currently all under investigation and it is likely that the rAAV gene targeting frequency can be substantially optimized. What must be emphasized, however, is the ease of working with rAAV in comparison to the traditional methodology. Thus, historically, an investigator needed to spend 6 months or more assembling a targeting vector (although see Iiizumi et al.66 for a faster alternative) and then spend another 6 months to a year laboriously transfecting their targeting construct into cells and screening the resulting drug-resistant clones by Southern blot analysis. With rAAV methodology, the entire experiment starting from the idea, “I’d like to knockout gene X,” to having a cell line in hand can take as little as 2 months (B. Ruis, S. Oh, and E.A. Hendrickson, unpublished data). Importantly, Kohli et al. did the field a major service by developing a protocol, driven almost exclusively by PCR, for the development of targeting vectors and viral stocks.124 Since the viral vectors and helper plasmids (expressing the necessary AAV and adenoviral gene products) are all commercially available (Stratagene), anyone can adopt this methodology into their laboratory. Additionally, in an ironic twist, because the rAAV targeting arms are so short, it becomes technically feasible to screen the resulting clones not by Southern blot analysis, but by PCR, thus once again expediting the targeting process.124 It should be pointed out that almost all of the rAAV studies carried out to date have used a simple positive selection, in which the rAAV vector carries a drug selection marker constitutively expressed using a strong viral promoter. Recently, a promoterless version of an rAAV vector has been described and targeting frequencies of ∼20% were reported.65 If the use of this

CHAPTER 53 / GENE TARGETING IN HUMAN SOMATIC CELLS

521

Table 53–4 Gene targeting studies using rAAV vectors Gene ALKP ALKP β-Catenin β-Catenin β-Catenin β-Catenin CCR5 CCR5 CDX2 CDX2 CDX2 CDX2 CDX2 CDX2 COL1A1 COL1A1 DNA-PKcs FHIT GFP GFP HAUSP HAUSP HAUSP HAUSP HAUSP HAUSP HPRT HPRT HPRT HPRT HPRT HPRT Ku70 Ku70 Ku86 Ku86 LIGIV NEO NEO NEO NEO p53 p53 SCO2 XIAP XIAP XIAP XIAP Totals a−d

+a

14 3 3 20 12 13

#b

60 741 170 109 173 244

Frequencyc

0.2333 0.0400 0.0176 0.1835 0.0694 0.0533

∼0.0100 67 35 19 2 2

2,000 52 27 55 92

0.0335 0.6731 0.7037 0.0364 0.0217 ∼0.0100

3 2 2 2 2

437 121 90 379 176

0.0069 0.0165 0.0222 0.0053 0.0114 ∼0.0014 ∼0.0008 ∼0.0010

4 26 3

95 94 1,200

0.0421 0.2766 0.0025

234

6,305

0.0371

Genotyped

Miscellaneouse

Reference

Transgene Transgene +/−* > +/− or −/−* +/−* > −/−* +/−* > −/−* +/+ > +/− +/+ > +/− +/+ > +/− +/+ > +/− +/− > −/− +/+ > +/− +/− > −/− +/+ > +/− +/− > −/− +/−* > +/− or −/−* +/+ > +/− +/+ > +/− +/+ > +/− Transgene Transgene +/+ > +/− +/− > −/− +/+ > +/− +/− > +/− +/+ > +/− +/− > +/− +/O > −/O +/O > −/O +/O > −/O +/O > −/O +/O > −/O +/O > −/O +/+ > +/− +/+ > +/− +/+ > +/− +/+ > +/− +/+ > +/− Transgene Transgene Transgene Transgene +/+ > +/− +/+ > +/− +/+ > +/− +/+ > +/− +/− > −/− +/+ > +/− +/− > −/−

MHF2 cells MHF2 cells HCT116 cells HCT116 cells; knockin HCT116 cells; promoterless DLD1 cells; knockin hTERT-RPE cells HCT116 cells MKN5 cells MKN5 cells LOVO cells LOVO cells SW48 cells SW48 cells Stem cells; knockin MHF2 cells HCT116 cells HCT116 cells 293 cells; ±ISceI 293 cells HCT116 cells HCT116 cells SW48 cells SW48 cells; retargeting hTERT-RPE cells hTERT-RPE cells; retargeting HT1080 cells MHF1 cells; knockin MHF2 cells; knockin MHF3 cells; knockin MHF2 cells MHF2 cells HCT116 cells NALM-6 cells HCT116 cells NALM-6 cells HCT116 cells HT1080 cells MHF2 cells ± hTERT HeLa cells HeLa cells HCT116 cells HCT116 cells; promoterless HCT116 cells HCT116 cells HCT116 cells DLD-1 cells DLD-1 cells

138 139 65 65 65 65 124 124 140 140 141 141 141 141 123 123 BR and EAH, unpublishedf 124 142 143 144 144 144 144 144 144 122 122 122 122 139 145 RF and EAH, unpublishedf RF and EAH, unpublishedf BR and EAH, unpublishedf BR and EAH, unpublishedf SO and EAH, unpublishedf 146 146 146 122 65 65 147 148 148 148 148

As defined in Table 53–1. Miscellaneous: For each study, the cell line in which the gene targeting was performed is indicated; (knockin) designates a study in which a subtle mutation was introduced into an exon as opposed to the complete ablation of the exon; (Promoterless) designates a gene targeting study in which the positive selection marker relied upon an endogenous promoter for expression; (±IScel) designates a study in which the gene targeting was carried out in the presence (+) and absence (−), respectively, of the endonuclease, I(ntron)-Saccharomyces cerevisiae I (I-Scel); (Retargeting) designates a study in which the gene targeting event occurred on a previously targeted allele; (±hTERT) designates a study in which the gene targeting was done in a cell line in the presence (+) and absence (−), respectively, of h(uman) telomeric reverse transcriptase (hTERT). f EAH, Eric A. Hendrickson; BR, Brian Ruis; RF, Riaz Fattah; SO, Sehyun OH. e

522

SECTION V / MODELS OF BEHAVIOR

vector becomes generally applicable, there will be few obstacles to targeting any gene of interest. Lastly, it should be noted that one obvious improvement might involve using rAAV methodology with the NALM-6 cell line, which appears to be so proficient in standard gene targeting. However, in our unpublished studies, rAAV seems to target no better in NALM-6 than in HCT116 cells (Table 53–4).125–148

CONCLUSIONS In the 21 years that have elapsed since the first report of a gene-targeting study in human somatic cells,74 a total 46 endogenous genes have been disrupted or modified. While modestly impressive in its own right, this number pales in comparison to the number of knockout mice strains that have been generated in the same time frame. Some of this difference is simply a reflection of the choice of model systems and the biological questions being addressed. Thus, for example, questions of development cannot be addressed in somatic cells in culture. Some of the difference, however, has been due to the fact that carrying out knockouts in human cells in culture is inherently more difficult than in murine ES cell lines. All in all, a total of 704 correctly targeted events in human somatic cells have been recorded from 100,791 clones examined, for a less than robust 0.7% success rate. Importantly, however, the identification of new cell lines such as NALM-6 and, most importantly, the development of the novel rAAV technology portend great improvements in this success rate in the foreseeable future. Moreover, since the rAAV methodology is still relatively in its infancy, we can expect that modifications will be developed to improve this already promising technique. While it is unlikely that gene targeting in human somatic cells will ever be as facile as gene replacement in yeast, it is clear that powerful methodology already exists to make such studies practical. Moreover, if the promise of the rAAV system comes to fruition, researchers working on human somatic cells in culture will have a powerful and facile weapon at their scientific disposal.

ACKNOWLEDGMENTS I thank Dr. Anja-Katrin Bielinsky (University of Minnesota Medical School) for her comments and helpful discussions. I thank the past and current members of my laboratory who have contributed to the development of this methodology. This work has been supported in part by Grants HL079559 and GM069576 from the NIH to E.A.H.

REFERENCES 1. Sedivy JM, Joyner A. Gene Targeting. New York: W.H. Freeman and Company, 1992. 2. Sorrell DA, Kolb AF. Targeted modification of mammalian genomes. Biotechnol Adv 2005;23:431–469. 3. Yu Y, Bradley A. Engineering chromosomal rearrangements in mice. Nat Rev Genet 2001;2:780–790. 4. Park F, Gow KW. Gene therapy: Future or flop. Pediatr Clin North Am 2006;53:621–638. 5. Cahill D, Connor B, Carney JP. Mechanisms of eukaryotic DNA double strand break repair. Front Biosci 2006;11:1958–1976. 6. van Veelen L, Wesoly J, Kanaar R. Biochemical and cellular aspects of homologous recombination. In: Seide W, Kow YW, Doetsch P, Eds. DNA Damage Recognition. New York: Taylor & Francis Group, 2006:581–607. 7. Hefferin ML, Tomkinson AE. Mechanism of DNA double-strand break repair by nonhomologous end joining. DNA Repair 2005;4:639–648.

8. Sedivy JM, Vogelstein B, Liber HL, Hendrickson EA, Rosmarin A. Gene targeting in human cells without isogenic DNA. Science 1999;283:9a. 9. Vasileva A, Jessberger R. Precise hit: Adeno-associated virus in gene targeting. Nat Rev Microbiol 2005;3:837–847. 10. Adachi N, So S, Iiizumi S, et al. The human pre-B cell line Nalm-6 is highly proficient in gene targeting by homologous recombination. DNA Cell Biol 2006;25:19–24. 11. Sonoda E, Hochegger H, Saberi A, Taniguchi Y, Takeda S. Differential usage of nonhomologous end-joining and homologous recombination in double strand break repair. DNA Repair 2006;5: 1021–1029. 12. Roth DB, Wilson JH. Relative rates of homologous and nonhomologous recombination in transfected DNA. Proc Natl Acad Sci USA 1985;82:3355–3359. 13. Lieber MR, Ma Y, Kefei Y, Pannicke U, Schwarz K. The mechanism of vertebrate nonhomologous DNA end joining and its role in immune system gene rearrangements. In: Seide W, Kow YW, Doetsch P, Eds. DNA Damage Recognition. New York: Taylor & Francis Group, 2006:609–627. 14. Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 1987;51:503– 512. 15. Lee SE, Mitchell RA, Cheng A, Hendrickson EA. Evidence for DNA-PK-dependent and -independent DNA double-strand break repair pathways in mammalian cells as a function of the cell cycle. Mol Cell Biol 1997;17:1425–1433. 16. Takata M, Sasaki MS, Sonoda E, et al. Homologous recombination and non-homologous end-joining pathways of DNA doublestrand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J 1998;17:5497– 5508. 17. Hendrickson EA, Huffman JL, Tainer JA. Structural aspects of Ku and the DNA-dependent protein kinase complex. In: Seide W, Kow YW, Doetsch P, Eds. DNA Damage Recognition. New York: Taylor & Francis Group, 2006:629–684. 18. Getts RC, Stamato TD. Absence of a Ku-like DNA end binding activity in the xrs double-strand DNA Repair-deficient mutant. J Biol Chem 1994;269:15981–15984. 19. Liang F, Romanienko PJ, Weaver DT, Jeggo PA, Jasin M. Chromosomal double-strand break repair in Ku80-deficient cells. Proc Natl Acad Sci USA 1996;93:8929–8933. 20. Inamdar KV, Yu Y, Povirk LF. Resistance of 3’-phosphoglycolate DNA ends to digestion by mammalian DNase III. Radiat Res 2002;157:306–311. 21. Gottlieb TM, Jackson SP. The DNA-dependent protein kinase: Requirement for DNA ends and association with Ku antigen. Cell 1993;72:131–142. 22. Suwa A, Hirakata M, Takeda Y, Jesch SA, Mimori T, Hardin JA. DNA-dependent protein kinase (Ku protein-p350 complex) assembles on double-stranded DNA. Proc Natl Acad Sci USA 1994;91:6904–6908. 23. DeFazio LG, Stansel RM, Griffith JD, Chu G. Synapsis of DNA ends by DNA-dependent protein kinase. EMBO J 2002;21:3192– 3200. 24. Weterings E, Verkaik NS, Bruggenwirth HT, Hoeijmakers JH, van Gent DC. The role of DNA dependent protein kinase in synapsis of DNA ends. Nucleic Acids Res 2003;31:7238–7246. 25. Spagnolo L, Rivera-Calzada A, Pearl LH, Llorca O. Three-dimensional structure of the human DNA-PKcs/Ku70/Ku80 complex assembled on DNA and its implications for DNA DSB repair. Mol Cell 2006;22(4):511–519. 26. Moshous D, Callebaut I, de Chasseval R, et al. Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency. Cell 2001;105:177–186. 27. Ma Y, Pannicke U, Schwarz K, Lieber MR. Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination. Cell 2002;108:781–794.

CHAPTER 53 / GENE TARGETING IN HUMAN SOMATIC CELLS

28. Moshous D, Callebaut I, de Chasseval R, et al. The V(D)J recombination/DNA Repair factor Artemis belongs to the metallo-betalactamase family and constitutes a critical developmental checkpoint of the lymphoid system. Ann NY Acad Sci 2003;987:150–157. 29. Critchlow SE, Bowater RP, Jackson SP. Mammalian DNA doublestrand break repair protein XRCC4 interacts with DNA ligase IV. Curr Biol 1997;7:588–598. 30. McElhinny SAN, Snowden CM, McCarville J, Ramsden DA. Ku recruits the XRCC4 ligase IV complex to DNA ends. Mol Cell Biol 2000;20:2996–3003. 31. Teo SH, Jackson SP. Lif1p targets the DNA ligase Lig4p to sites of DNA double-strand breaks. Curr Biol 2000;10:165–168. 32. Li Z, Otevrel T, Gao Y, et al. The XRCC-4 gene encodes a novel protein involved in DNA double-strand break repair and V(D)J recombination. Cell 1995;83:1079–1089. 33. Gao Y, Frank KM, Dikkes P, et al. A critical role for DNA endjoining proteins in both lymphogenesis and neurogenesis. Cell 1998;95:891–902. 34. Ahnesorg P, Smith P, Jackson SP. XLF interacts with the XRCC4DNA ligase IV complex to promote DNA nonhomologous end-joining. Cell 2006;124:301–313. 35. Buck D, Malivert L, de Chasseval R, et al. Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly. Cell 2006;124:287–299. 36. Hopfner K-P. The Mre11/Rad50/Nbs1 complex. In: Seide W, Kow YW, Doetsch P, Eds. DNA Damage Recognition. New York: Taylor & Francis Group, 2006:705–721. 37. Moreau S, Morgan EA, Symington LS. Overlapping functions of the Saccharomyces cerevisiae Mre11, Exo1 and Rad27 nucleases in DNA metabolism. Genetics 2001;159:1423–1433. 38. Iftode C, Daniely Y, Borowiec JA. Replication protein A (RPA): The eukaryotic SSB. Crit Rev Biochem Mol Biol 1999;34:141– 180. 39. Kawabata M, Kawabata T, Nishibori M. Role of recA/RAD51 family proteins in mammals. Acta Med Okayama 2005;59:1–9. 40. Sung P, Trujillo KM, Van Komen S. Recombination factors of Saccharomyces cerevisiae. Mutat Res 2000;451:257–275. 41. Thacker J. The RAD51 gene family, genetic instability and cancer. Cancer Lett 2005;219:125–135. 42. Sugiyama T, Kowalczykowski SC. Rad52 protein associates with replication protein A (RPA)-single-stranded DNA to accelerate Rad51-mediated displacement of RPA and presynaptic complex formation. J Biol Chem 2002;277:31663–31672. 43. Heyer WD, Li X, Rolfsmeier M, Zhang XP. Rad54: The Swiss Army knife of homologous recombination? Nucleic Acids Res 2006;34:4115–4125. 44. Solinger JA, Kiianitsa K, Heyer WD. Rad54, a Swi2/Snf2like recombinational repair protein, disassembles Rad51 : dsDNA filaments. Mol Cell 2002;10:1175–1188. 45. Hastings PJ, McGill C, Shafer B, Strathern JN. Ends-in vs. ends-out recombination in yeast. Genetics 1993;135:973–980. 46. Sharma S, Doherty KM, Brosh RM Jr. Mechanisms of RecQ helicases in pathways of DNA metabolism and maintenance of genomic stability. Biochem J 2006;398:319–337. 47. Liu Y, Masson JY, Shah R, O’Regan P, West SC. RAD51C is required for Holliday junction processing in mammalian cells. Science 2004;303:243–246. 48. Liu Y, West SC. Happy Hollidays: 40th anniversary of the Holliday junction. Nat Rev Mol Cell Biol 2004;5:937–944. 49. Zheng H, Hasty P, Brenneman MA, et al. Fidelity of targeted recombination in human fibroblasts and murine embryonic stem cells. Proc Natl Acad Sci USA 1991;88:8067–8071. 50. Yanez RJ, Porter AC. Influence of DNA delivery method on gene targeting frequencies in human cells. Somat Cell Mol Genet 1999;25:27–31. 51. Yanez RJ, Porter AC. Gene targeting is enhanced in human cells overexpressing hRAD51. Gene Ther 1999;6:1282–1290. 52. Bunz F, Fauth C, Speicher MR, et al. Targeted inactivation of p53 in human cells does not result in aneuploidy. Cancer Res 2002; 62:1129–1133.

523

53. Hendrie PC, Hirata RK, Russell DW. Chromosomal integration and homologous gene targeting by replication-incompetent vectors based on the autonomous parvovirus minute virus of mice. J Virol 2003;77:13136–13145. 54. Zwaka TP, Thomson JA. Homologous recombination in human embryonic stem cells. Nat Biotechnol 2003;21:319–321. 55. Trobridge G, Hirata RK, Russell DW. Gene targeting by adenoassociated virus vectors is cell-cycle dependent. Hum Gene Ther 2005;16:522–526. 56. So S, Nomura Y, Adachi N, et al. Enhanced gene targeting efficiency by siRNA that silences the expression of the Bloom syndrome gene in human cells. Genes Cells 2006;11:363– 371. 57. Jasin M, Elledge SJ, Davis RW, Berg P. Gene targeting at the human CD4 locus by epitope addition. Genes Dev 1990;4:157– 166. 58. Manjunath N, Johnson RS, Staunton DE, Pasqualini R, Ardman B. Targeted disruption of CD43 gene enhances T lymphocyte adhesion. J Immunol 1993;151:1528–1534. 59. Waldman T, Kinzler KW, Vogelstein B. p21 is necessary for the p53-mediated G1 arrest in human cancer cells. Cancer Res 1995; 55:5187–5190. 60. Grawunder U, Zimmer D, Fugmann S, Schwarz K, Lieber MR. DNA ligase IV is essential for V(D)J recombination and DNA double-strand break repair in human precursor lymphocytes. Mol Cell 1998;2:477–484. 61. Miyagawa K, Tsuruga T, Kinomura A, et al. A role for RAD54B in homologous recombination in human cells. EMBO J 2002;21: 175–180. 62. Ghosh G, Li G, Myung K, Hendrickson EA. The lethality of Ku86 loss-of-function mutations in human cells is p53-independent. Radiat Res 2007;167(1):66–79. 63. Gu H, Marth JD, Orban PC, Mossmann H, Rajewsky K. Deletion of a DNA polymerase beta gene segment in T cells using cell typespecific gene targeting. Science 1994;265:103–106. 64. Li G, Nelsen C, Hendrickson EA. Ku86 is essential in human somatic cells. Proc Natl Acad Sci USA 2002;99:832–837. 65. Topaloglu O, Hurley PJ, Yildirim O, Civin CI, Bunz F. Improved methods for the generation of human gene knockout and knockin cell lines. Nucleic Acids Res 2005;33:e158. 66. Iiizumi S, Nomura Y, So S, et al. Simple one-week method to construct gene-targeting vectors: Application to production of human knockout cell lines. Biotechniques 2006;41:311–316. 67. Sauer B, Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci USA 1988;85:5166–5170. 68. Kolb AF. Genome engineering using site-specific recombinases. Cloning Stem Cells 2002;4:65–80. 69. Austin S, Ziese M, Sternberg N. A novel role for site-specific recombination in maintenance of bacterial replicons. Cell 1981;25: 729–736. 70. Couedel C, Mills KD, Barchi M, et al. Collaboration of homologous recombination and nonhomologous end-joining factors for the survival and integrity of mice and cells. Genes Dev 2004;18:1293– 1304. 71. Celli GB, Denchi EL, de Lange T. Ku70 stimulates fusion of dysfunctional telomeres yet protects chromosome ends from homologous recombination. Nat Cell Biol 2006;8:885–890. 72. Traverso G, Bettegowda C, Kraus J, et al. Hyper-combination and genetic instability in BLM-deficient epithelial cells. Cancer Res 2003;63:8578–8581. 73. Cortez D, Guntuku S, Qin J, Elledge SJ. ATR and ATRIP: Partners in checkpoint signaling. Science 2001;294:1713–1716. 74. Smithies O, Gregg RG, Boggs SS, Koralewski MA, Kucherlapati RS. Insertion of DNA sequences into the human chromosomal betaglobin locus by homologous recombination. Nature 1985;317:230– 234. 75. Montrose-Rafizadeh C, Kole J, Bartkowski LM, et al. Gene targeting of a CFTR allele in HT29 human epithelial cells. J Cell Physiol 1997;170:299–308.

524

SECTION V / MODELS OF BEHAVIOR

76. Williams SR, Ousley FC, Vitez LJ, DuBridge RB. Rapid detection of homologous recombinants in nontransformed human cells. Proc Natl Acad Sci USA 1994;91:11943–11947. 77. Bunz F, Dutriaux A, Lengauer C, et al. Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 1998;282: 1497–1501. 78. Brown JP, Wei W, Sedivy JM. Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts. Science 1997;277:831–834. 79. Rasheed S, Nelson-Rees WA, Toth EM, Arnstein P, Gardner MB. Characterization of a newly derived human sarcoma cell line (HT-1080). Cancer 1974;33:1027–1033. 80. Geiser AG, Anderson MJ, Stanbridge EJ. Suppression of tumorigenicity in human cell hybrids derived from cell lines expressing different activated ras oncogenes. Cancer Res 1989;49:1572– 1577. 81. Farese RV Jr, Flynn LM, Young SG. Modification of the apolipoprotein B gene in HepG2 cells by gene targeting. J Clin Invest 1992;90:256–261. 82. Itzhaki JE, Gilbert CS, Porter AC. Construction by gene targeting in human cells of a “conditional” CDC2 mutant that rereplicates its DNA. Nat Genet 1997;15:258–265. 83. Shirasawa S, Furuse M, Yokoyama N, Sasazuki T. Altered growth of human colon cancer cell lines disrupted at activated Ki-ras. Science 1993;260:85–88. 84. Powell SM, Zilz N, Beazer-Barclay Y, et al. APC mutations occur early during colorectal tumorigenesis. Nature 1992;359:235–237. 85. Vogelstein B, Kinzler KW. The multistep nature of cancer. Trends Genet 1993;9:138–141. 86. Chan TA, Hermeking H, Lengauer C, Kinzler KW, Vogelstein B. 14–3-3s is required to prevent mitotic catastrophe after DNA damage. Nature 1999;401:616–620. 87. Rhee I, Jair KW, Yen RW, et al. CpG methylation is maintained in human cancer cells lacking DNMT1. Nature 2000;404:1003– 1007. 88. Jallepalli PV, Waizenegger IC, Bunz F, et al. Securin is required for chromosomal stability in human cells. Cell 2001;105:445– 457. 89. Rhee I, Bachman KE, Park BH, et al. DNMT1 and DNMT3b cooperate to silence genes in human cancer cells. Nature 2002;416: 552–556. 90. Yoshihara T, Ishida M, Kinomura A, et al. XRCC3 deficiency results in a defect in recombination and increased endoreduplication in human cells. EMBO J 2004;23:670–680. 91. Masramon L, Ribas M, Cifuentes P, et al. Cytogenetic characterization of two colon cell lines by using conventional G-banding, comparative genomic hybridization, and whole chromosome painting. Cancer Genet Cytogenet 2000;121:17–21. 92. Abdel-Rahman WM, Katsura K, Rens W, et al. Spectral karyotyping suggests additional subsets of colorectal cancers characterized by pattern of chromosome rearrangement. Proc Natl Acad Sci USA 2001;98:2538–2543. 93. Myung K, Ghosh G, Fattah FJ, et al. Regulation of telomere length and suppression of genomic instability in human somatic cells by Ku86. Mol Cell Biol 2004;24:5050–5059. 94. Giannini G, Ristori E, Cerignoli F, et al. Human MRE11 is inactivated in mismatch repair-deficient cancers. EMBO Rep 2002;3:248–254. 95. Papadopoulos N, Nicolaides NC, Wei YF, et al. Mutation of a mutL homolog in hereditary colon cancer. Science 1994;263:1625– 1629. 96. Liu B, Nicolaides NC, Markowitz S, et al. Mismatch repair gene defects in sporadic colorectal cancers with microsatellite instability. Nat Genet 1995;9:48–55. 97. Mellon I, Rajpal DK, Koi M, Boland CR, Champe GN. Transcription-coupled repair deficiency and mutations in human mismatch repair genes. Science 1996;272:557–560. 98. Zhang L, Yu J, Park BH, Kinzler KW, Vogelstein B. Role of BAX in the apoptotic response to anticancer agents. Science 2000;290: 989–992.

99. Chan TA, Wang Z, Dang LH, Vogelstein B, Kinzler KW. Targeted inactivation of CTNNB1 reveals unexpected effects of b-catenin mutation. Proc Natl Acad Sci USA 2002;99:8265–8270. 100. Uegaki K, Adachi N, So S, Iiizumi S, Koyama H. Heterozygous inactivation of human Ku70/Ku86 heterodimer does not affect cell growth, double-strand break repair, or genome integrity. DNA Repair 2006;5:303–311. 101. So S, Adachi N, Lieber MR, Koyama H. Genetic interactions between BLM and DNA ligase IV in human cells. J Biol Chem 2004;279:55433–55442. 102. Hurwitz R, Hozier J, LeBien T, et al. Characterization of a leukemic cell line of the pre-B phenotype. Int J Cancer 1979;23:174–180. 103. Wlodarska I, Aventin A, Ingles-Esteve J, et al. A new subtype of pre-B acute lymphoblastic leukemia with t(5;12)(q31q33;p12), molecularly and cytogenetically distinct from t(5;12) in chronic myelomonocytic leukemia. Blood 1997;89:1716–1722. 104. Matheson EC, Hall AG. Assessment of mismatch repair function in leukaemic cell lines and blasts from children with acute lymphoblastic leukaemia. Carcinogenesis 2003;24:31–38. 105. Atchison RW, Casto BC, Hammon WM. Adenovirus-associated defective virus particles. Science 1965;149:754–756. 106. Muzyczka N. Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr Top Microbiol Immunol 1992;158:97–129. 107. Kotin RM, Siniscalco M, Samulski RJ, et al. Site-specific integration by adeno-associated virus. Proc Natl Acad Sci USA 1990; 87:2211–2215. 108. Samulski RJ, Zhu X, Xiao X, et al. Targeted integration of adenoassociated virus (AAV) into human chromosome 19. EMBO J 1991;10:3941–3950. 109. Lu Y. Recombinant adeno-associated virus as delivery vector for gene therapy—a review. Stem Cells Dev 2004;13:133–145. 110. Grieger JC, Samulski RJ. Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications. Adv Biochem Eng Biotechnol 2005;99:119–145. 111. Blacklow NR, Hoggan MD, Rowe WP. Serologic evidence for human infection with adenovirus-associated viruses. J Natl Cancer Inst 1968;40:319–327. 112. Buller RM, Janik JE, Sebring ED, Rose JA. Herpes simplex virus types 1 and 2 completely help adenovirus-associated virus replication. J Virol 1981;40:241–247. 113. Rose JA, Berns KI, Hoggan MD, Koczot FJ. Evidence for a singlestranded adenovirus-associated virus genome: Formation of a DNA density hybrid on release of viral DNA. Proc Natl Acad Sci USA 1969;64:863–869. 114. Samulski RJ, Chang LS, Shenk T. A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication. J Virol 1987;61: 3096–3101. 115. Johnson FB, Ozer HL, Hoggan MD. Structural proteins of adenovirus-associated virus type 3. J Virol 1971;8:860–863. 116. Rose JA, Maizel JV Jr, Inman JK, Shatkin AJ. Structural proteins of adenovirus-associated viruses. J Virol 1971;8:766–770. 117. Lusby E, Fife KH, Berns KI. Nucleotide sequence of the inverted terminal repetition in adeno-associated virus DNA. J Virol 1980;34: 402–409. 118. Xiao X, Xiao W, Li J, Samulski RJ. A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle. J Virol 1997;71:941–948. 119. Samulski RJ, Chang LS, Shenk T. Helper-free stocks of recombinant adeno-associated viruses: Normal integration does not require viral gene expression. J Virol 1989;63:3822–3888. 120. Ferrari FK, Xiao X, McCarty D, Samulski RJ. New developments in the generation of Ad-free, high-titer rAAV gene therapy vectors. Nat Med 1997;3:1295–1297. 121. Xiao X, Li J, Samulski RJ. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol 1998;72:2224–2232. 122. Russwll DW, Hirata RK. Human gene targeting by viral vectors. Nat Genet 1998;18:325–330.

CHAPTER 53 / GENE TARGETING IN HUMAN SOMATIC CELLS

123. Chamberlain JR, Schwarze U, Wang PR, et al. Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 2004;303:1198–1201. 124. Kohli M, Rago C, Lengauer C, Kinzler KW, Vogelstein B. Facile methods for generating human somatic cell gene knockouts using recombinant adeno-associated viruses. Nucleic Acids Res 2004;32: e3. 125. Itzhaki JE, Porter AC. Targeted disruption of a human interferoninducible gene detected by secretion of human growth hormone. Nucleic Acids Res 1991;19:3835–3842. 126. Itzhaki JE, Barnett MA, MacCarthy AB, Buckle VJ, Brown WR, Porter AC. Targeted breakage of a human chromosome mediated by cloned human telomeric DNA. Nat Genet 1992;2:283–287. 127. Porter AC, Itzhaki JE. Gene targeting in human somatic cells. Complete inactivation of an interferon-inducible gene. Eur J Biochem 1993;218:273–281. 128. Culver KW, Hsieh WT, Huyen Y, et al. Correction of chromosomal point mutations in human cells with bifunctional oligonucleotides. Nat Biotechnol 1999;17:989–993. 129. Kim JS, Lee C, Foxworth A, Waldman T. B-Raf is dispensable for K-Ras-mediated oncogenesis in human cancer cells. Cancer Res 2004;64:1932–1937. 130. Kunzelmann K, Legendre JY, Knoell DL, Escobar LC, Xu Z, Gruenert DC. Gene targeting of CFTR DNA in CF epithelial cells. Gene Ther 1996;3:859–867. 131. Zhen L, King AA, Xiao Y, Chanock SJ, Orkin SH, Dinauer MC. Gene targeting of X chromosome-linked chronic granulomatous disease locus in a human myeloid leukemia cell line and rescue by expression of recombinant gp91phox. Proc Natl Acad Sci USA 1993;90:9832–9836. 132. Song KY, Schwartz F, Maeda N, Smithies O, Kucherlapati R. Accurate modification of a chromosomal plasmid by homologous recombination in human cells. Proc Natl Acad Sci USA 1987;84: 6820–6824. 133. Thyagarajan B, Johnson BL, Campbell C. The effect of target site transcription on gene targeting in human cells in vitro. Nucleic Acids Res 1995;23:2784–2790. 134. Kim JS, Crooks H, Dracheva T, et al. Oncogenic beta-catenin is required for bone morphogenetic protein 4 expression in human cancer cells. Cancer Res 2002;62:2744–2748. 135. Hiyama T, Katsura M, Yoshihara T, et al. Haploinsufficiency of the Mus81-Eme1 endonuclease activates the intra-S-phase and G2/M

136.

137.

138. 139.

140.

141.

142.

143.

144.

145.

146.

147. 148.

525

checkpoints and promotes rereplication in human cells. Nucleic Acids Res 2006;34:880–892. Dhar SK, Yoshida K, Machida Y, et al. Replication from oriP of Epstein-Barr virus requires human ORC and is inhibited by geminin. Cell 2001;106:287–296. Zhou S, Buckhaults P, Zawel L, et al. Targeted deletion of Smad4 shows it is required for transforming growth factor b and activin signaling in colorectal cancer cells. Proc Natl Acad Sci USA 1998;95:2412–2416. Hirata RK, Russell DW. Design and packaging of adeno-associated virus gene targeting vectors. J Virol 2000;74:4612–4620. Inoue N, Dong R, Hirata RK, Russell DW. Introduction of single base substitutions at homologous chromosomal sequences by adenoassociated virus vectors. Mol Ther 2001;3:526–530. Dang LH, Chen F, Knock SA, et al. CDX2 does not suppress tumorigenicity in the human gastric cancer cell line MKN45. Oncogene 2006;25:2048–2059. Dang LH, Chen F, Ying C, et al. CDX2 has tumorigenic potential in the human colon cancer cell lines LOVO and SW48. Oncogene 2006;25:2264–2272. Porteus MH, Cathomen T, Weitzman MD, Baltimore D. Efficient gene targeting mediated by adeno-associated virus and DNA doublestrand breaks. Mol Cell Biol 2003;23:3558–3565. Liu X, Yan Z, Luo M, et al. Targeted correction of single-base-pair mutations with adeno-associated virus vectors under nonselective conditions. J Virol 2004;78:4165–4175. Cummins JM, Rago C, Kohli M, Kinzler KW, Lengauer C, Vogelstein B. Tumour suppression: Disruption of HAUSP gene stabilizes p53. Nature 2004;428:1. Hirata R, Chamberlain J, Dong R, Russell DW. Targeted transgene insertion into human chromosomes by adeno-associated virus vectors. Nat Biotechnol 2002;20:735–738. Inoue N, Hirata RK, Russell DW. High-fidelity correction of mutations at multiple chromosomal positions by adeno-associated virus vectors. J Virol 1999;73:7376–7380. Matoba S, Kang JG, Patino WD, et al. p53 regulates mitochondrial respiration. Science 2006;312:1650–1653. Cummins JM, Kohli M, Rago C, Kinzler KW, Vogelstein B, Bunz F. X-linked inhibitor of apoptosis protein (XIAP) is a nonredundant modulator of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human cancer cells. Cancer Res 2004;64:3006–3008.

54

Animal Models for Investigating the Causes and Mechanisms of Mammalian Germ Cell Aneuploidy JOHN B. MAILHES

AND

MARY ANN HANDEL

ABSTRACT

with maternal age4,7,8 and its more frequent occurrence during female meiosis I for certain chromosomes.9–12 Although the relationship between human aneuploidy and maternal age remains elusive, data suggest that such an association is linked to decreased frequencies of chiasmata formation,13 abnormally placed chiasmata,14 progressive losses of cohesion proteins,15,16 and spindle checkpoint proteins.17–19 It is now generally accepted that both nondisjunction and premature centromere separation (PCS) represent major events leading to human germ cell aneuploidy,15,20,21 and that variation exists among chromosomes for the likelihood of missegregation.22,23 Due the multitude of potential mechanisms, it is not surprising that a unique precept about the etiology of aneuploidy remains elusive. Recent information about the molecular mechanisms of chromosome segregation in various species is providing a foundation for research designed to investigate the causes and mechanisms of human aneuploidy. While animal models for aneuploidy (e.g., trisomy models) have been useful for studying the consequences of aneuploidy, most animal models for the genesis of aneuploidy have not been specifically evaluated for their relevance to humans. Such comparisons are difficult because of ethical and sample size limitations and the biological variability inherent among human gametes. Thus, animal models are the only approach to obtain fundamental information about potential genetic hazards when humans are exposed to certain chemicals and other aneuploidy-inducing agents. It is important not only to identify those agents that significantly increase the incidence of aneuploidy in germ cells, but also to know if aneuploid germ cells are transmitted to progeny. However, the value of animal models goes far beyond the fact that they provide an experimentally malleable system for testing various agents. In fact, their greatest value to research on the etiology of aneuploidy may lie in the numerous genetic models that are available. These models enable determination of the roles of specific molecules in processes of meiotic chromosome dynamics and chromosome segregation that lead to gametic aneuploidy. Here we review first, the basic processes of meiosis that, when they go awry, can lead to gametic aneuploidy, second, a sampling of the wealth of genetic models available for dissecting processes of meiosis and the genesis of gametic aneuploidy, and finally, what we have learned from analysis of environmental agents that cause aneuploidy. Our focus is on the laboratory mouse.

Aneuploidy is a condition whereby the chromosome number of a cell or an organism is not an exact multiple of the haploid number for the species. It represents the most prevalent genetic abnormality of mankind and is associated with failed reproduction, mental and physical retardation, and cancer. Although research involving various organisms is providing information about the interactions between the biochemical pathways and the organelles responsible for chromosome segregation, we are still far removed from understanding the numerous potential molecular mechanisms of aneuploidy, especially in mammalian germ cells. Furthermore, the mechanisms of aneuploidy need to be studied in both male and female germ cells because of the inherent differences between male and female gametogenesis. The mouse aneuploid models presented here include those relevant to unique human disorders (Down syndrome) and those involving knockdown and knockout of genes needed for chromosome segregation. Also, models resulting from RNA interference technology and the exposure of cells to chemicals that specifically disrupt specific biochemical pathways or cellular organelles during gametogenesis are presented. Key Words: Animal models, Aneuploidy, Female germ cells, Male germ cells, Molecular mechanisms.

SIGNIFICANCE OF ANEUPLOIDY FOR HUMAN HEALTH AND REPRODUCTION Aneuploidy represents the greatest genetic affliction of humans. However, little is known about the causes and even less about the underlying molecular mechanisms of aneuploidy, especially in mammalian germ cells, where most aneuploidy is thought to arise. Human aneuploidy is linked with embryonic loss, mental and physical anomalies, and cancer. Approximately 10–30% of fertilized human eggs,1 10% of all human conceptuses,2,3 50% of spontaneous abortuses,4,5 and 0.31% (204/64887) of human newborns6 have an abnormal number of chromosomes. Even though numerous hypotheses have been proposed for the etiology of human aneuploidy, the only consistent findings are its positive correlation

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

527

528

SECTION V / MODELS OF BEHAVIOR

OVERVIEW OF MAMMALIAN GAMETOGENESIS One interesting feature about human aneuploidy is its sexually dimorphic aspects, namely, it is highly correlated with maternal age and occurs more frequently in maternal meiosis for some chromosomes. Thus, much of the value of animal models lies in the exploitation of the sexual dimorphism of gametogenesis, which we highlight here in this overview of gametogenic processes and mechanisms, the context in which the origins of aneuploidy must be considered. Fundamental to the origins of aneuploidy is the process of meiosis. This specialized cell division process is defining of gametogenesis, as it occurs only in germ cells. In brief, the stages of meiosis include a premeiotic S-phase (often temporally longer than a mitotic S-phase), an extended prophase of meiosis I, during which chromosome pairing, synapsis, and recombination occur, and, finally, the two meiotic divisions that establish germ cell haploidy. For convenience, the extended meiosis I prophase is described in substages: during leptonema, chromosomes condense and begin to pair; during zygonema, chromosomes are paired and their intimate synapsis is initiated; during the pachytene stage, synapsis is completed and recombination (crossing over) occurs; and during the diplotene stage, desynapsis occurs. The intimate chromosome synapsis required for recombination is mediated by the synaptonemal complex (SC). The crossovers, visually apparent as chiasmata, serve to ensure the stable bipolar orientation of homologous pairs of chromosomes, a prerequisite to their correct segregation during the division process. Thus, although the primary defect leading to gametic aneuploidy may lie in prophase events, the nondisjunction that gives rise to incorrect chromosome segregation occurs in the meiotic division phases. The first meiotic division is a reductional one in which the homologous chromosomes are segregated into daughter cells; during the second, equational, division, sister chromatids are separated. OOGENESIS Mammalian meiosis begins in the fetal ovary and is later arrested postpartum at the diplotene (dictyate) stage of meiosis I. Unless oocytes undergo atresia, they remain in diplotene until meiosis resumes in response to the luteinizing hormone (LH) surge prompting ovulation. Oocyte maturation (OM) is critical in setting up a correct meiotic division and encompasses the transition from diplotene to metaphase II (MII) and involves both nuclear and cytoplasmic remodeling.24–27 In most mammals, metaphase II (MII) oocytes are ovulated and primed for fertilization, which initiates anaphase II. Although differences in the biochemical pathways of OM exist among marine invertebrates, amphibians, fish, and mammals, the initiation and orderly temporal sequence of events during OM are influenced by species-dependent protein modifications by kinases and phosphatases.28 In mammals, the intraoocyte titer of cyclic adenosine monophosphate (cAMP) plays a major role in initiating OM. Elevated levels of cAMP favor cAMP-dependent kinase activity and retention of oocytes in the diplotene stage of meiotic prophase. Conversely, low cAMP levels shift the equilibrium toward cAMP-dependent phosphatase activity, which is needed for activating maturation promoting factor (MPF).24–27,29,30 MPF is composed of a 34-kDa catalytic subunit (CDC2A, sometimes referred to as p34cdc2) that exhibits serine-threonine kinase activity and a 45-kDa cyclin B regulatory subunit (CCNB1). MPF is activated when CDC2A is dephosphorylated at the tyrosine 15 residue and coupled with cyclin B1, whereas, it becomes deacti-

vated when tyrosine is phosphorylated.31–33 MPF activity oscillates; it is highest during metaphase, but decreases at anaphase34,35 and following fertilization36–39 or parthenogenetic activation.38,39 Besides MPF, other kinases and phosphatases are also involved in the nuclear and cytoplasmic changes that occur during OM. Mitogen-activated protein kinases (MAPKs) are serine-threonine protein kinases that phosphorylate many of the same sites as active MPF.40,41 These kinases mediate intracellular signal transmission in response to external stimuli, participate in assembling the first meiotic spindle, and prevent rodent oocytes from entering interphase during the interval between meiosis I and II.42,43 Unlike MPF, MAPK activity remains high throughout OM. Another kinase that has important functions during OM is the Mos protooncogene product MOS. This serine-threonine kinase is detected throughout OM44–46 and exerts numerous speciesdependent functions during OM including activation of the MAPK pathway.47 MOS functions as a cytostatic factor by preventing premature exit from MII in mouse oocytes.48,49 Oocytes from Mosdeficient mice are capable of completing OM, but fail to arrest at MII and subsequently undergo spontaneous parthenogenetic activation.49–51 In addition to their roles during OM, the kinases MPF, MAPKs, and MOS also have important roles in the spindle assembly checkpoint (SAC), anaphase-promoting complex (APC) activation, and initiating the metaphase–anaphase transition (MAT).40,47,52–54 Upon completing OM, mouse oocytes remain in MII for a limited time period until fertilization, spontaneous activation, or atresia. Fertilization or parthenogenesis leads to elevated intraoocyte calcium levels that enable the formation of a calcium– calmodulin complex and calcium-dependent protein kinase II activation.55 This kinase then initiates a cascade of kinasephosphatase reactions that ultimately leads to unique dephosphorylation events, proteolysis of specific proteins, and inactivation of MPF, MAPKs, and MOS. Considering that the synchronous interaction between unique kinases and phosphatases with their target compounds is needed for the normal temporal progression of OM and the MAT, it seems reasonable to suspect that alteration of their normal activities may lead to downstream effects manifested by chromosome missegregation. Based on their antagonistic effects relative to the degree of tyrosine CDC2A phosphorylation,56 certain kinase and phosphatase inhibitors can alter the rate of OM and induce spindle abnormalities and aneuploidy in rodent oocytes. Okadaic acid (OA) specifically inhibits the protein phosphatases 1 (PPP1) and 2A (PPP2A) that dephosphorylate serine and threonine residues.57,58 When mouse oocytes were treated with OA, MPF activation was inhibited59 and abnormalities involving spindle fibers, multipolar spindles, kinetochores, and chromosome alignment were detected.60–62 Furthermore, elevated frequencies of PCS and aneuploidy were found in mouse oocytes exposed to OA.63 Some of the above observations may be related to the finding that OA treatment resulted in hyperphosphorylation of both microtubuleorganizing centers and microtubule-associated proteins (MAPs)59,60 and that hyperphosphorylated MAPs appear to have a reduced affinity for microtubules.61 The kinase inhibitor 6-dimethylaminopurine (6-DMAP) can disrupt CDC2A kinase and MAPK activities and reversibly block mouse oocytes at various stages during OM.64–65 When mouse dictyate oocytes were exposed to 6-DMAP during in vitro culture, both protein phosphorylation and germinal vesicle breakdown

CHAPTER 54 / INVESTIGATING THE CAUSES AND MECHANISMS OF MAMMALIAN GERM CELL ANEUPLOIDY

(GVBD) were inhibited, and when 6-DMAP was added after GVBD, first polar body formation was prevented.64 Other data showed that 6-DMAP inhibited protein phosphorylation in activated mouse MII oocytes, which resulted in the premature disappearance of phosphorylated proteins and abnormalities involving polar body extrusion and pronuclei formation.65 A relevant point about either in vivo or in vitro oocyte aging is that the pattern of protein dephosphorylation events appears to be correlated with increased frequencies of spontaneous oocyte activation and PCS.15,66–71 SPERMATOGENESIS The tempo and timetable of spermatogenesis differ markedly from that of oogenesis. Germ cells in fetal rodent testes enter into a period of mitotic arrest until shortly after birth. During the same period, the germ cells, positioned in the center of the seminiferous tubules, initiate ameboid activity and move between the Sertoli cells to establish themselves on the basal lamina of the tubules, a niche favorable for stem cell activity.72 During the second week of life, large numbers of differentiated spermatogonia enter meiotic prophase, and by the end of the third week, the meiotic divisions occur and postmeiotic spermatid differentiation, or spermiogenesis, is initiated. Thus, the first wave of spermatogenesis is more or less synchronous, but subsequently, in adult life, these distinct processes of mitotic proliferation, meiosis, and spermiogenesis occur simultaneously in the testis and different generations of germ cells are present simultaneously. Because of the complexity of the adult testis, the first wave of spermatogenesis is frequently studied experimentally to determine temporal relationships and expression patterns, especially for meiotic events. Unlike meiosis in the female germ cell, there is no meiotic arrest during spermatogenesis. After the extended prophase (more than a week long in mice), germ cells proceed through diplonema and the two meiotic divisions rapidly, in a period of 24 h or so. This rapid and uninterrupted timing has important consequences that may be informative about the relative incidence of gametic aneuploidy in males versus females. First, the recombinationbased crossovers, manifest in chiasmata, are not “old” at the time of the meiotic division. Similarly, other “binder” proteins, such as chromosomal cohesins, are relatively “new” in spermatocytes at the time of the meiotic divisions. Together or separately, these could contribute to greater fidelity of metaphase chromosome alignment and subsequent segregation in male gametogenesis. In contrast, in females, both chiasmata and binder proteins are established during prophase, in fetal life, and thus they could be quite “old” at the time of the meiotic divisions, although nothing is known about the potential turnover and replacement of these proteins in oocytes. Experimental analysis of chromosome segregation during male meiosis is hampered by the fact that very little is known about the regulation of the onset of the meiotic division phase in spermatocytes.73,74 Although oocytes spontaneously enter the meiotic division phase upon removal from the follicle, removal of mid-prophase spermatocytes from their surrounding Sertoli cells does not prompt the onset of the division phase. Thus, the signal for the onset of the division phase is likely to be a positive one, either autonomous to the germ cell or emanating from the Sertoli cells. Although genetic evidence is lacking, the G2/MI transition, as the progress from late meiosis I prophase to the first meiotic division is known, is likely to be mediated, as in oocytes, by the universal cell cycle regulator MPF.75 Experimentally, the

529

G2/MI transition can be induced by treating pachytene spermatocytes with OA, but the exposed cells do not complete division, hampering analysis of chromosome segregation. Another experimental approach to the analysis of MI chromosome segregation is the injection of pachytene spermatocytes into oocytes, but in this case, chromosome segregation is not normal,76 thus perhaps providing a model system for analysis of the genesis of gametic aneuploidy.

GENETIC MODELS FOR ANEUPLOIDY The quintessential value of the mouse for the study of aneuploidy is the plethora of genetic models available. These include models of aneuploidy (e.g., mouse models for Down syndrome) for analysis of the consequences of aneuploidy, as well as single gene mutations that can be used to decipher the intricacies of chromosome segregation, contributing to the analysis of causes and mechanisms of aneuploidy. Prior to the development of such mutant gene models (mostly by genetic knockout technologies), studies on aneuploidy had been limited to various chemicals, mainly those that damage microtubules, that induce aneuploidy in mammalian germ cells,77–79 which can be transmitted to progeny.23,80–82 These studies are useful (and are covered in the section on Environmentally Induced Aneuploidy in Germ Cells), but lack the precision and power of genetic models. Gene knockout and knockdown models, RNA interference technology, and mutant genetic strains are now being employed for studying aneuploidy as summarized below. It should be noted that the gene models represent a selective list (Table 54–1) of those proving most useful for analysis of the genesis of aneuploidy, and also, almost by definition, involve only one event in a complex, and yet not completely understood, series of biochemical reactions and cellular organelles that orchestrate chromosome segregation. Most of these models directly relate to aneuploidy, but some involve events that predispose cells to aneuploidy. Importantly, the full potential of these models has not yet been realized; for example, they may provide “sensitized” models for testing of potential environmental disruptors of meiotic chromosome segregation. Note: In the section below and in Table 54–1, standard gene and protein nomenclature, approved by international commissions on nomenclature, is used. GENETIC MODELS FOR ANEUPLOID CONDITIONS The most studied human autosomal aneuploidy is trisomy 21 (Ts21) or Down syndrome. Several mouse models for Ts21 have become available.83–88 The Ts(1716)65Dn, commonly known as Ts65Dn, mouse shows many features characteristic of Down syndrome85,89 and has been particularly informative for teasing apart gene–phenotype relationships for the relevant chromosomal regions in attempts to understand the complexity of Down syndrome manifest in humans. In addition to these models for autosomal aneuploidy, models for sex chromosome aneuploidy have also been exploited to determine the consequences for physiology and meiotic pairing of extra (or deficient) sex chromosomes.90–92 ANEUPLOIDY IN MODELS FOR EVENTS OF CHROMOSOME PAIRING,SYNAPSIS,ANDRECOMBINATION Chromosome pairing is mediated by cohesins, proteins that promote sister-chromatid cohesion, and by proteins of the SC. The synaptonemal complex protein 3, SYCP3 (encoded by the Sycp3 gene in mice and by the SYCP3 gene in humans), is a meiosis-specific structural protein needed for chromosomal structural integrity,

530

SECTION V / MODELS OF BEHAVIOR

Table 54–1 Mammalian genetic models for chromosome missegregation Gene a

Sycp3 Smc1bb Smc1bb Smc1bb Bub1bc Mad2l1d Mad2l1d Mad2l1d Atrxe Top3bf Mei1g Mei1g Mlh1h PL/J mice

Cell type

Cytogenetic event

Mouse oocyte Mouse oocyte Mouse oocyte Mouse spermatocyte Mouse fibroblasts Mouse blastocyst Human/mouse somatic cells Mouse oocyte Mouse oocyte Mouse spermatocytes Mouse spermatocytes Mouse oocytes Mouse oocytes Mouse spermatocytes

Aneuploidy Univalents, single chromatids Abnormal homolog synapsis, premature chromatid separation Meiotic arrest Aneuploidy Chromosome missegregation Premature chromatid separation Aneuploidy Abnormal chromosome alignment Aneuploidy Abnormal homolog synapsis Homolog asynapsis, abnormal chromosome alignment Abnormal homolog synapsis Homolog asynapsis, aneuploidy

Reference 97 101 100 100 114 115 116 19 109 111 117 117 103, 104, 106 119

a

Synaptonemal complex protein. Structural maintenance of chromosomes protein. c BubR1 spindle checkpoint protein. d Mad2 spindle checkpoint protein. e Centromeric binding protein. r Encodes DNA topoisomerase IIIb. t Meiosis defective 1. h DNA mismatch repair protein. b

chiasmata formation, and chromosome synapsis. Expression of SYCP3 protein is restricted to meiotic prophase I and disappears during diakinesis in mouse oocytes.93 In meiotic prophase chromosome preparations, SYCP3 is found in the SC during meiotic prophase,94 where it colocalizes with the RAD21 (also known as SCC1) cohesin subunit.95 In meiosis division-phase spermatocytes, SYCP3 localizes to the centromeres by metaphase I and subsequently disappears by telophase I.95 Spermatocytes in homozygous Sycp3−/− male mice exhibit synaptic failure and meiotic arrest during zygotene, and the male mice are infertile.96 The females, however, are not infertile and oocytes from Sycp3−/− females display incomplete synapsis and alignment of homologs during pachytene and abnormal position and reduced frequency of chiasmata; chromosomes are found as univalents in oocytes and aneuploid zygotes are produced,97 apparently by oocytes that evade checkpoint control.98 This sexually dimorphic phenotype is not clear; it may in some way reflect the fact that SC proteins remain associated with centromeres until meiotic division phase in males, whereas they disappear prior to this in females.93 The “structural maintenance of chromosomes” (SMC) family of proteins includes both cohesins and condensins. SMC1B (also known as SMC1 beta, encoded by the mouse Smc1b gene) is a meiosis-specific isoform of a cohesin subunit.99 Genetic knockout analysis reveals that SMC1B has a role in preserving chiasmata and maintaining chromosome cohesion until anaphase.100,101 Smc1b-deficient mice also display sexual dimorphism; spermatocytes arrest during meiotic prophase, whereas oocytes progress to MII.93,100 The pachytene arrest noted in males was attributed to incomplete synapsis of homologs. In oocytes, reduced recombination and premature loss of sister chromatid cohesion during meiotic prophase I suggest that SMC1B helps stabilize chiasmata until anaphase, and, interestingly, this may be a factor in the agerelated increase in oocyte aneuploidy.101

The MLH1 DNA mismatch repair protein promotes meiotic recombination in yeast.102 Mouse genetic models are available, as the mouse Mlh1 gene has been knocked out.103,104 In spermatocytes of Mlh1-null mice, chromosome pairs fall apart, presenting as univalents at metaphase I,103 the condensed univalents do not assemble onto the spindle apparatus, and spermatocytes die, perhaps in response to a recombination or chiasmata checkpoint.105 This was also found to be the case for female mutants,106 where there is failure to exclude the second polar body and zygotic death.104 These observations suggest that the MLH1 protein is essential for the maintenance of chiasmata. In a robust validation of the utility of such models for basic insights, it was subsequently found that MLH1 localizes to sites of crossing over that become the chiasmata.107 Likewise, deficiency for the related MLH3 mismatch repair protein causes meiotic arrest and aneuploidy phenotypes.108 Other chromosomal proteins have also been found to play a role in chromosome dynamics that lead to correct meiotic chromosome segregation. ATRX (α-thalassemia/mental retardation syndrome X-linked homolog) is a centromeric-binding protein of the SNF2 family of helicase-ATPases. When ATRX activity was inhibited by antibody microinjection or RNA interference in mouse oocytes, chromosome alignment was disrupted during metaphase II.109 TOP3B (DNA topoisomerase III beta) is a type IA DNA topoisomerase.110 Targeted disruption of the Top3b gene in mice causes a homozygous phenotype of infertility with a high incidence of aneuploidy in male germ cells.111 Topoisomerases are required for meiotic chromosome condensation and division in yeast and mammals,112 but much remains to be learned about their precise role. ANEUPLOIDY IN MODELS FOR CHECKPOINTS THAT MONITOR GENETIC INTEGRITY BUB1B (also known as BUB1R) comprises one of several spindle checkpoint proteins.113

CHAPTER 54 / INVESTIGATING THE CAUSES AND MECHANISMS OF MAMMALIAN GERM CELL ANEUPLOIDY

Generation of Bub1b mutant mice resulted from a gene-targeting strategy creating a hypomorphic allele in mice embryonic stem cells.114 Progressive reduction of BUB1B expression in mouse embryonic fibroblasts, spermatocytes, and oocytes resulted in aneuploidy. Also, a correlation between chronological aging of wild-type mice with diminished expression of BUB1B in somatic and germinal cells was noted. This relationship suggested a role for spindle checkpoint protein activity and the maternal age effect of aneuploidy.114 Another spindle checkpoint protein well studied in yeast is MAD2, known in the mouse as MAD2L1. Morpholino-based gene silencing of Mad2l1 transcripts in mouse oocytes during meiosis I resulted in premature proteolysis of both cyclin B and securin, attenuating meiosis I and causing an elevated incidence of aneuploidy. Conversely, overexpression of a human MAD2L1GFP construct in oocytes led to homolog nondisjunction.19 Insufficiency of MAD2 L in mouse,115 as well as partial knockout of Mad2l1 in human and mouse mitotic cells,116 has been shown to result in an accelerated rate of meiosis, PCS, and aneuploidy. Additional data from real-time polymerase chain reaction (PCR) experiments have demonstrated that MAD2L1 transcripts in human oocytes decrease as a function of maternal age17 as well as postovulatory age in mouse oocytes.18 Together, these data indicate an important role for MAD2L1, a spindle checkpoint, in monitoring chromosome segregation. OTHER GENETIC MODELS OF GAMETIC ANEUPLOIDY Meiosis-defective 1 (Mei1−/−) mice were derived from chemically mutagenized embryonic stem cells.117 The Mei1 gene encodes a protein, MEI1, of unknown function.118 Spermatocytes from homozygous Mei1−/− mice exhibited zygotene arrest with abnormal synapsis of homologs; but some oocytes progressed to metaphase I and displayed asynapsis and abnormal chromosome alignment.117 Thus, when revealed, the function of the MEI1 protein may shed light on processes involved in setting up accurate meiotic chromosome segregation. Males of the PL/J mouse strain are characterized by a high frequency of morphologically abnormal sperm, an elevated frequency of aneuploid sperm, as well as spermatocytes exhibiting chromosome asynapsis and other meiotic prophase abnormalities.119 Genetic analysis revealed that these features are complex traits, and thus far the underlying genes have not yet been identified.119 Also, senescence-accelerated mice (SAM) exhibit agerelated defects in chromosome alignment during oocyte meiosis I and II.120 This phenotype is reminiscent of that of the Smc1b-null mice, and it was speculated that cohesion defects in aged mice may be associated with misalignment and PCS.

531

ENVIRONMENTALLY INDUCED ANEUPLOIDY IN GERM CELLS As mentioned previously, many studies have shown that various chemicals, mainly those that damage microtubules, can induce aneuploidy in mammalian germ cells.77–79 Moreover, several studies have demonstrated that aneuploid germ cells can be transmitted to progeny.23,80–82 Animal models for aneuploidy have not been specifically evaluated for their relevance to human aneuploidy. Such comparisons are not possible due to ethical and sample size limitations plus the biological variability inherent among human oocytes. Because of this, animal models are the only experimental approach to obtain information about potential genetic hazards when humans are exposed to certain chemicals and other aneuploidy-inducing agents. As mentioned earlier, it is not only important to identify those agents that can significantly increase the incidence of aneuploidy in germ cells, but also to know if such aneuploid germ cells can be transmitted to offspring. Several relevant agents are described below and in Table 54–2. FEMALE GERM CELLS Several review papers list compounds (aneugens) reported to induce aneuploidy in mammalian female germ cells.23,78,79,121,122 Most of the chemicals reported in the earlier reviews dealt with spindle poisons such as colchicine, vinblastine sulfate, and benomyl, whereas, more recent articles have involved compounds that inhibit unique biochemical63,123 or cellular organelle activities.22,109 Thus, it is well-recognized that various types of compounds are capable of inducing aneuploidy in dividing cells. Based on the numerous unique cellular and biochemical reactions involved with chromosome segregation, the list of aneugens can be expected to increase. For example, it has recently been shown that exposure to bisphenol A can increase meiotic nondisjunction in mouse oocytes.124 Furthermore, environmental agents can be expected to act not only on individual gametes, but also on the conceptus produced by their union. For example, conditions associated with fertilization in vitro may increase mitotic nondisjunction in early cleavage,125 and the mouse is an excellent model for study of this phenomenon. Although identification of aneugens has merit, it is also of importance to describe their mode of action. Agents may act proximally, on mediators of cell division, but may also act distally and upstream, perhaps on endocrine receptors, to create a “susceptible” cellular environment. Thus, for each cell type, the molecular mechanisms of normal chromosome segregation need to be known as well as the effects of a particular perturbation during the chain of events comprising mitosis and meiosis. How such information

Table 54–2 Environmental agents affecting mammalian germ cell chromosome segregation Agent a

MG132 Vanadateb Okadaic acidc Monastrold a

Cell type

Cytogenetic event

Mouse Mouse Mouse Mouse Mouse

Meiotic arrest, aneuploidy, premature chromatid separation Aneuploidy, premature chromatid separation Premature anaphase Aneuploidy, premature chromatid separation Aneuploidy, premature chromatid separation

oocytes somatic cells oocytes oocytes oocytes

Proteasome inhibitor. Tyrosine phosphatase inhibitor. c Protein phosphatase 1 and 2A inhibitor. d Kinesin EG5 inhibitor. b

Reference 167 168 63 124

532

SECTION V / MODELS OF BEHAVIOR

can be utilized for reducing the incidence of human aneuploidy still remains the ultimate task. MALE GERM CELLS Experimental analysis of the effect of environmental agents on genesis of germ cell aneuploidy is facilitated in the male by the relative accessibility of the critical germ cell stages. Two primary germ cell stages are used for analysis of environmental agent effects directly on male germ cells: primary spermatocytes, for assessment of impairment of the SC and/or pairing and recombination, and sperm, for assessment of effects on chromosome segregation. In spite of the availability of these germ cell stages, the effect of environmental agents on transmissible aneuploidy can be detected only at the level of the zygote or offspring. A number of agents can affect the synaptonemal complex structure of mouse spermatocytes, introducing breaks or discontinuities, which are detected after silver staining or with immunofluorescent labeling of surface-spread chromatin. These include colchicine,126,127 cyclophosphamide,128,129 and etoposide. The latter is the most thoroughly studied agent with respect to both the full spectrum of effects and species, and is an excellent example of the power of combined genetic, cellular, and cytogenetic analyses. First found in 1998 to produce peak mutagenicity in primary spermatocytes of the mouse,130 etoposide was subsequently found to reduce crossing over131 and to produce heritable chromosome abnormalities and aneuploidy.132,133 Together, these studies illustrate the spectrum of techniques that can be brought to bear on an analysis of germ cell effects of environmental agents. The first discovery of germ cell stage sensitivity came from analysis of the mutagenicity of etoposide in a genetic-specific locus test,134 relying on detection of specific mutations in offspring. This was followed by careful analysis of SC and chromosome morphology in surface spread chromatin coupled with genetic recombination analysis (of offspring) to show abnormalities in meiotic protein localization and reduced recombination.131 The discovery that etoposide causes nondisjunction and gametic aneuploidy required scoring of offspring and, especially, the use of sperm FISH. This technique, involving hybridization of chromosome-specific probes to denatured sperm DNA in situ,135 is among the most useful for direct determination of gametic aneuploidy. FISH analysis of sperm from etoposide-treated mice revealed increases in aneuploid sperm.133,136,137 Interestingly, FISH data on sperm from treated human males (undergoing chemotherapy) also revealed an increase in the frequency of aneuploid sperm.138 Etoposide and diazepam139 are the only chemicals known for which both mouse and human sperm aneuploidy data are available. Even with the knowledge that sperm are aneuploid, transmissible aneuploidy can be detected only by examination of offspring, which has been a limiting factor in these studies. However, the technique of PAINT/DAPI has facilitated rapid and accurate scoring of chromosomes in zygotic first-cleavage metaphase140; application of this to studies of environmental agents could provide new insights.141 Nonetheless, although these studies highlight the potential utility of rodent models for environmental induction of gametic aneuploidy, we still do not know the exact mechanism(s) by which etoposide or other environmental agents act in promoting nondisjunction and heritable gametic aneuploidy.

ETIOLOGY OF ANEUPLOIDY Although the incidence of aneuploidy has not been routinely studied in mammals other than humans and rodents, the available data show that it occurs more frequently in humans than in rodents,

and more frequently in females than in males. Is there a unique aspect of human female reproduction that renders it more vulnerable to chromosome missegregation? One feature is the absence of a defined estrus period during the menstrual/estrous cycle. Most domestic mammals ovulate during or shortly after the estrus period of their estrous cycle in order to ensure that freshly ovulated oocytes are fertilized.142 Since this situation does not occur in humans, the possibility exists that fertilization of postovulatory aged oocytes might occur. Indeed, it has been proposed that fertilization of postovulatory aged oocytes may contribute to the higher incidence of aneuploidy in humans relative to other species.6,143–147 Furthermore, two epidemiological studies offered support for an association between fertilization of aged human oocytes and early embryonic failure148 and trisomic offspring.143 Postovulatory and in vitro oocyte aging involves a progressive, functional deterioration of the biochemical and cellular organelles essential for accurate chromosome segregation, normal fertilization, and embryonic development.120,148 During this aging process, a narrow window of time exists for the optimal expression of gamete physiology. The fertilizable life span of mammalian oocytes ranges from 12 to 24 h and only 6 to 8 h in mares.142 Although the fertilizable mean life span for both induced and naturally ovulated mouse oocytes is approximately 15 h postovulation, their optimal time for fertilization ranges from 4 to 6 h postovulation.149–151 After this period, time-dependent intraoocyte changes take place among certain cellular organelles and biochemical profiles that can lead to apoptosis.152–155 In addition to postovulatory aging, other hypotheses include reduced and abnormally positioned chiasmata,13,14 biological aging that is associated with reduced oocyte pools and reproductive hormones as maternal age increases,156 compromised gonadal environment,91,157 and premature centromere separation.15,16,20 Recent data suggest that maternal age is positively correlated with progressive deterioration of cohesion between bivalents,21,97 progressive loss of cohesion proteins,95,101 and spindle checkpoint proteins.17 Experimental data have shown that chronological aging of female mice deficient for the SMC1B chiasmata-binding protein led to deterioration of both chiasmata and arm cohesion as well as elevated levels of univalents and single chromatids in oocytes.101 Recognizing that many of the reported aneuploid hypotheses are not necessarily mutually exclusive, it now appears that both preovulatory (maternal aging) and postovulatory-related alterations in the biochemical pathway regulating chromosome segregation can predispose oocytes to aneuploidy. Furthermore, these same cellular features governing chromosome segregation may also contribute to male gametic aneuploidy. Considering the numerous intracellular biochemical and organelle changes that can alter normal chromosome segregation, it seems reasonable to state that various molecular approaches will be needed to better understand the intricate details of aneuploidy.

FUTURE DIRECTIONS AND CONCLUSIONS Since many potential mechanisms may lead to aneuploidy, specific investigations into the molecular mechanisms of aneuploidy that alter one event, or even several events, of the chromosome segregation pathway represent only a small component of the overall format underlying chromosome segregation. It has been estimated that approximately 500 yeast genes directly or indirectly control chromosome segregation.158 Accurate

CHAPTER 54 / INVESTIGATING THE CAUSES AND MECHANISMS OF MAMMALIAN GERM CELL ANEUPLOIDY

chromosome segregation requires the temporally coordinated interaction among unique biochemical and cellular organelles. Some of these events include protein kinases and phosphatases, topoisomerases, microtubule kinetics, kinetochores and their proteins, centrosomes, motor proteins, passenger proteins, spindle checkpoint proteins, anaphase promoting complex, proteasomes, and securin, cohesin, and separin proteins. Abnormalities involving microtubule capture by kinetochores, removal of catenations between sister chromatids, chromosome condensation and biorientation, kinetochore–microtubule tension, and the spindle checkpoint have the potential for predisposing cells to aneuploidy. Technologies such as DNA microarrays159 and RNA-mediated interference of genes and protein kinases involved with cell cycle progression160,161 offer avenues for deciphering cell cycle progression and chromosome segregation pathways. RNAi silencing and gene knockdown technologies have been used to alter the expression of the MAD2L1 protein in mouse oocytes19,162 and BUB1B protein in mouse germ and somatic cells.114 Reduced expression of these SAC proteins was associated with aneuploidy. Additionally, large-scale knockout strategies for genes upregulated during yeast meiosis found that deletion of specific genes required for maintaining centromeric cohesion during anaphase I resulted in chromosome missegregation.163,164 Furthermore, data derived from genomic and proteomic analyses are expected to provide clues about the fundamental processes associated with cell division. Gene expression analyses of cancer cells showed that a subset of genes is universally activated in most cancers,165 and that overexpression of cell division regulatory genes was linked with chromosome aberrations and neoplastic progression.166 Continued research involving the chronological sequence of molecular events comprising chromosome segregation during mitosis and meiosis coupled with cytogenetic analyses is required to further our knowledge about the origin of gametic aneuploidy.167,168

ACKNOWLEDGMENTS We are indebted to Alicia Valenzuela and Drs. Muriel Davisson and Fengyun Sun for helpful comments on the manuscript. M.A.H.’s contributions to this article were supported by a grant from the NIH, HD48998.

REFERENCES 1. Hassold T, Hunt PA. To err (meiotically) is human: The genesis of human aneuploidy. Nat Rev Genet 2001;2:280–291. 2. Ford JH. Nondisjunction. In: Burgio GR, Fraccaro M, Tiepolo L, Wolf U, Eds. Trisomy 21. Berlin: Springer-Verlag,1981:103–143. 3. Hansmann I. Factors and mechanisms involved in nondisjunction and X-chromosome loss. In: Sandberg AA, Ed. Cytogenetics of the Mammalian X Chromosome, Part A: Progress and Topics in Cytogenetics, Vol. 3A. New York: Alan R. Liss, 1983:131–170. 4. Bond DJ, Chandley AC. Aneuploidy. Oxford Monographs on Medical Genetics, No. 11. Oxford, UK: Oxford University Press, 1983. 5. Hook EB. The impact of aneuploidy upon public health: Mortality and morbidity associated with human chromosome abnormalities. In: Dellarco VL, Voytek PE, Hollaender A, Eds. Aneuploidy: Etiology and Mechanisms. New York: Plenum Press, 1985:7–33. 6. Hecht F, Hecht BK. Aneuploidy in humans: Dimensions, demography, and dangers of abnormal numbers of chromosomes. In: Vig BK, Sandberg AA, Eds. Aneuploidy, Part A: Incidence and Etiology. New York: Alan R. Liss, 1987:9–49. 7. Hook EB. Maternal age, paternal age, and human chromosome abnormality: Nature, magnitude, etiology, and mechanisms of

8.

9.

10. 11.

12.

13. 14.

15.

16. 17.

18.

19.

20. 21. 22.

23.

24. 25.

26.

27.

28.

533

effects. In: Dellarco VL, Voytek PE, Hollaender A, Eds. Aneuploidy: Etiology and Mechanisms. New York: Plenum Press, 1985: 117–132. Chandley AC. Aneuploidy: An overview. In: Vig BK, Sandberg AA, Eds. Aneuploidy: Part A: Incidence and Etiology. New York: Alan R. Liss, 1987:1–8. Lamson SH, Hook EB. A simple function for maternal age-specific rates of Down syndrome in the 20-to-48 year age range and its biological implications. Am J Hum Genet 1980;32:743–753. Hassold T, Chiu D, Yamane JA. Parental origin of autosomal trisomies. Ann Hum Genet 1984;48:129–144. Hassold TJ. The origin of aneuploidy in humans. In: Dellarco VL, Voytek PE, Hollaender A, Eds. Aneuploidy: Etiology and Mechanisms. New York: Plenum Press, 1985:103–115. Nicolaidis P, Petersen MB. Origin and mechanisms of nondisjunction in human autosomal trisomies. Hum Reprod 1998;13: 313–319. Henderson SA, Edwards RG. Chiasma frequency and maternal age in mammals. Nature 1968;218:22–28. Lamb NE, Freeman SB, Savage-Austin A, Pettay D, Taft L, Hershey J, Gu YC, Shen J, Saker D, May KM, Avramopoulos D, Petersen MB, Hallberg A, Mikkelsen M, Hassold TJ, Sherman SL. Susceptible chiasmate configurations of chromosome 21 predispose to non-disjunction in both maternal meiosis I and meiosis II. Nat Genet 1996;14:400–405. Angell RR, Xian J, Keith J, Ledger W, Baird DT. First meiotic division abnormalities in human oocytes: Mechanism of trisomy formation. Cytogenet Cell Genet 1994;65:194–202. Angell RR. First-meiotic division nondisjunction in human oocytes. Am J Hum Genet 1997;61:23–32. Steuerwald N, Cohen J, Herrera RJ, Sandalinas M, Brenner CA. Association between spindle assembly checkpoint expression and maternal age in human oocytes. Mol Hum Reprod 2001;7:49–55. Steuerwald N, Steuerwald MD, Mailhes JB. Postovulatory aging of mouse oocytes leads to decreased MAD2 transcripts and increased frequencies of premature centromere separation and anaphase. Mol Hum Reprod 2005;11:623–630. Homer HA, Mcdougall A, Levasseur M, Yallop K, Murdoch AP, Herbert M. Mad2 prevents aneuploidy and premature proteolysis of cyclin B and securin during meiosis I in mouse oocytes. Genes Dev 2005;19:202–207. Angell RR. Predivision in human oocytes at meiosis I: A mechanism for trisomy formation in man. Hum Genet 1991;86:383–387. Wolstenholme J, Angell RR. Maternal age and trisomy–a unifying mechanism of formation. Chromosoma 2000;109:435–438. Sun FY, Schmid TE, Schmid E, Baumgartner A, Adler I-D. Trichlorfon induces spindle disturbances in V79 cells and aneuploidy in male mouse germ cells. Mutagenesis 2000;15:17–24. Eichenlaub-Ritter U. Aneuploidy in aging oocytes and after toxic insult. In: Trounson A, Gosden RG, Eds. Biology and Pathology of the Oocyte; Its Role in Fertility and Reproductive Medicine. Cambridge, UK: Cambridge University Press, 2003:220–257. Dekel N. Regulation of oocyte maturation: The role of cAMP. Ann NY Acad Sci 1988;541:211–216. Schultz RM, Montgomery RR, Belanoff JR. Regulation of mouse oocyte maturation: Implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis. Dev Biol 1983;797:264–273. Schultz RM. Regulatory functions of protein phosphorylation in meiotic maturation of mouse oocytes in vitro. In: Haseltine FP, First NL, Eds. Meiotic Inhibition: Molecular Control of Meiosis. New York: Alan R. Liss, 1988:137–151. Racowsky C. Somatic control of meiotic status in mammalian oocytes. In: Haseltine FP, Heyner S. Eds. Meiosis II, Contemporary Approaches to the Study of Meiosis. Washington, DC: American Association for the Advancement of Science Press, 1993:107– 116. Yamasita M, Mita K, Yoshida N, Kondo T. Molecular mechanisms of the initiation of oocyte maturation: General and species-specific aspects. Prog Cell Cycle Res 2000;4:115–129.

534

SECTION V / MODELS OF BEHAVIOR

29. Boernslaeger EA, Mattei P, Schultz RM. Involvement of cAMPdependent protein kinase and protein phosphorylation in regulation of mouse oocyte maturation. Dev Biol 1986;114:453–462. 30. Downs SM, Daniel SAJ, Bornslaeger EA, Hoppe PC, Eppig JJ. Maintenance of meiotic arrest in mouse oocytes by purines: Modulation of cAMP levels and cAMP phosphodiesterase activity. Gamete Res 1989;23:323–334. 31. Dunphy WG, Kumagai A. The cdc25 protein contains an intrinsic phosphatase activity. Cell 1991;67:189–196. 32. Gautier J, Solomon MJ, Booher RN, Bazan JF, Kirschner ME. cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2. Cell 1991;67:197–211. 33. Strausfeld U, Labbe JC, Fesquet D, Cavadore JC, Picard A, Sadhu K, Russell P, Doree M. Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human CDC25 protein. Nature 1991;351:242–245. 34. Arion D, Meifer L, Brizuela L, Beach D. cdc2 is a component of the M phase-specific histone H1 kinase: Evidence for identity with MPF. Cell 1988;55:371–378. 35. Draetta G, Beach D. Activation of cdc2 protein kinase during mitosis in human cells: Cell cycle dependent phosphorylation and subunit rearrangement. Cell 1988;54:17–26. 36. Choi T, Aoki F, Mori M, Yamashita M, Nagahama Y, Kohmoto K. Activation of p34cdc2 protein kinase activity in meiotic and mitotic cell cycles in mouse oocytes and embryos. Development 1991; 113:789–795. 37. Fulka J Jr, Jung T, Moor RM. The fall of biological maturation promoting factor (MPF) and histone H1 kinase activity during anaphase and telophase in mouse oocytes. Mol Reprod Dev 1992;32:378–382. 38. Collas P, Sullivan EJ, Barnes FL. Histone H1 kinase activity in bovine oocytes following calcium stimulation. Mol Reprod Dev 1993;34:224–231. 39. Kikuchi K, Izaike Y, Noguchi J, Furukawa T, Daen FP, Naito K, Toyoda Y. Decrease of histone H1 kinase activity in relation to parthenogenetic activation of pig follicular oocytes matured and aged in vitro. J Reprod Fertil 1995;105:325–330. 40. Murray AW. MAP kinases in meiosis. Cell 1998;92:157–159. 41. Takenaka K, Moriguchi T, Nishida E. Activation of the protein kinase p38 in the spindle assembly checkpoint and mitotic arrest. Science 1998;280:599–602. 42. Sobajima T, Aoki F, Kohomoto K. Activation of mitogen-activated protein kinase during meiotic maturation in mouse oocytes. J Reprod Fertil 1993;97:389–394. 43. Verlhac MH, Kubiak JZ, Clarke HJ, Maro BH. Microtubule and chromatin behavior follow MAP kinase activity but not MPF activity during meiosis in mouse oocytes. Development 1994;120: 1017–1025. 44. Paules RS, Buccione R, Moschel RC, Vande Woude GF, Eppig JJ. Mouse mos protooncogene product is present and functions during oogenesis. Proc Natl Acad Sci USA 1989;86:5395–5399. 45. Sagata N. What does Mos do in oocytes and somatic cells? BioEssays 1997;19:13–21. 46. Singh B, Arlinghaus RB. Mos and the cell cycle. Prog Cell Cycle Res 1997;3:251–259. 47. Dekel N. Protein phosphorylation-dephosphorylation in the meiotic cell cycle of mammalian oocytes. Rev Reprod 1996;1:82–88. 48. Sagata N. Meiotic metaphase arrest in animal oocytes: Its mechanisms and biological significance. Trends Cell Biol 1996;6: 22–28. 49. Hashimoto N. Role of c-mos proto-oncogene product in the regulation of mouse oocyte maturation. Horm Res 1996;46:11–14. 50. Colledge WH, Carlton MBL, Udy GB, Evans MJ. Disruption of c-mos causes parthenogenetic development of unfertilized mouse eggs. Nature 1994;370:65–68. 51. Hashimoto N, Watanabe N, Furuta Y, Tamemoto H, Sagata N, Yokoyama M, Okazaki K, Nagayoshi M, Takeda N, Ikawa Y, et al. Parthenogenetic activation of oocytes in c-mos deficient mice. Nature 1994;370:68–71.

52. Hyman AA, Mitchison TJ. Two different microtubule-based motor activities with opposite polarities in kinetochores. Nature 1991;351: 206–211. 53. Karsenti E. Mitotic spindle morphogenesis in animal cells. Semin Cell Biol 1991;4:251–260. 54. Dorée M, Le Peuch C, Morin N. Onset of chromosome segregation at the metaphase to anaphase transition of the cell cycle. Prog Cell Cycle Res 1995;1:309–318. 55. Santella L. The role of calcium in the cell cycle: Facts and hypotheses. Biochem Biophys Res Commun 1998;244:317–324. 56. Jesus C, Rime H, Haccard O, Van Lint J, Goris J, Merlevede W, Ozon R. Tyrosine phosphorylation of p34cdc2 and p42 during meiotic maturation of Xenopus oocyte: Antagonistic action of okadaic acid and 6-DMAP. Development 1991;111:813–820. 57. Cohen P, Holmes CF, Tsukitani Y. Okadaic acid: A new probe for the study of cellular regulation. Trends Biochem Sci 1990;15: 98–102. 58. Schönthal S. Okadaic acid—a valuable new tool for the study of signal transduction and cell cycle regulation? New Biol 1992;4:16– 21. 59. Schwartz DA, Schultz RM. Stimulatory effect of okadaic acid, an inhibitor of protein phosphorylation in mouse oocytes and one-cell zygotes. Dev Biol 1991;145:119–127. 60. Vandre DD, Willis VL. Inhibition of mitosis by okadaic acid: Possible involvement of a protein phosphatase 2A in the transition from metaphase to anaphase. J Cell Sci 1992;101:79–91. 61. Zernicka-Goetz M, Kubiak JZ, Antony C, Maro B. Cytoskeletal organization of rat oocytes during metaphase II arrest and following abortive activation: A study by confocal laser scanning microscopy. Mol Reprod Dev 1993;35:165–175. 62. De Pennart H, Helene-Verlhac M, Cibert C, Santa Maria A, Maro B. Okadaic acid induces spindle lengthening and disrupts the interaction of microtubules with the kinetochores in metaphase IIarrested mouse oocytes. Dev Biol 1993;157:170–181. 63. Mailhes JB, Hilliard C, Fuseler JW, London SN. Okadaic acid, an inhibitor of phosphatase 1 and 2A, induces premature separation of sister chromatids during meiosis I and aneuploidy in mouse oocytes in vitro. Chromosome Res 2003;11:619–631. 64. Rime H, Neant I, Guerrier P, Ozon R. 6-Dimethylaminopurine (6DMAP), a reversible inhibitor of the transition to metaphase during the first meiotic cell division of the mouse oocyte. Dev Biol 1989;133:169–179. 65. Szollosi MS, Kubiak JZ, Debey P, de Pennart H, Szollosi D, Maro B. Inhibition of protein kinases by 6-dimethylaminopurine accelerates the transition to interphase in activated mouse oocytes. J Cell Sci 1993;104:861–872. 66. Spielmann H, Krüger C, Stauber M, Vogel R. Abnormal chromosome behavior in human oocytes which remained unfertilized during human in vitro fertilization. J In Vitro Fertil Embryo Transf 1985;2:138–142. 67. Saito H, Koike K, Saito T, Nohara M, Kawagoe S, Hiroi M. Aging changes in the alignment of chromosomes after human chorionic gonadotropin stimulation may be a possible cause of decreased fertility in mice. Horm Res 1993;39:28–31. 68. Sakurada K, Ishikawa H, Endo A. Cytogenetic effects of advanced maternal age and delayed fertilization on first-cleavage mouse embryos. Cytogenet Cell Genet 1996;72:46–49. 69. Dailey T, Dale B, Cohen J, Nunné S. Association between nondisjunction and maternal age in meiosis II oocytes. Am J Hum Genet 1996;59:176–184. 70. Mailhes JB, Young D, London SN. 1,2-Propanediol-induced premature centromere separation in mouse oocytes and aneuploidy in one-cell zygotes. Biol Reprod 1997;57:92–98. 71. Mailhes JB, Young D, London SN. Postovulatory ageing of mouse oocytes in vivo and premature centromere separation and aneuploidy. Biol Reprod 1998;58:1206–1210. 72. Orth JM, Qiu JP, Jester WF, Pilder S. Expression of the c-kit gene is critical for migration of neonatal rat gonocytes in vitro. Biol Reprod 1997;57:676–683.

CHAPTER 54 / INVESTIGATING THE CAUSES AND MECHANISMS OF MAMMALIAN GERM CELL ANEUPLOIDY

73. Handel MA, Cobb J, Eaker S. What are the spermatocyte’s requirements for successful meiotic division? J Exp Zool 1999;285: 243–250. 74. Inselman A, Eaker S, Handel MA. Temporal expression of cell cycle-related proteins during spermatogenesis: Establishing a timeline for the onset of the meiotic divisions. Cytogenet Genome Res 2003;103:277–284. 75. Wiltshire T, Park C, Caldwell KA, Handel MA. Induced premature G2/M-phase transition in pachytene spermatocytes includes events unique to meiosis. Dev Biol 1995;169:557–567. 76. Kimura Y, Tateno H, Handel MA, Yanagimachi R. Factors affecting meiotic and developmental competence of primary spermatocyte nuclei injected into mouse oocytes. Biol Reprod 1998;59: 871–877. 77. Allen JW, Liang JC, Carrano AV, Preston RJ. Review of literature on chemical-induced aneuploidy in mammalian male germ cells. Mutat Res 1986;167:123–137. 78. Mailhes JB, Preston RJ, Lavappa KS. Mammalian in vivo assays for aneuploidy in female germ cells. Mutat Res 1986;167: 139–148. 79. Mailhes JB, Marchetti F. Chemically-induced aneuploidy in mammalian oocytes. Mutat Res 1994;320:87–111. 80. Mailhes JB, Preston RJ, Yuan ZP, Payne HS. Analysis of mouse metaphase II oocytes as an assay for chemically induced aneuploidy. Mutat Res 1988;198:145–152. 81. Mailhes JB, Yuan ZP, Aardema MJ. Cytogenetic analysis of mouse oocytes and one-cell zygotes as a potential assay for heritable germ cell aneuploidy. Mutat Res 1990;242:89–100. 82. Adler I-D, Schmid TE, Baumgartner A. Induction of aneuploidy in male mouse germ cells detected by the sperm-FISH assay: A review of the present data base. Mutat Res 2002;504:173–182. 83. Akeson EC, Lambert JP, Narayanswami S, Gardiner K, Bechtel LJ, Davisson MT. Ts65Dn–localization of the translocation breakpoint and trisomic gene content in a mouse model for Down syndrome. Cytogenet Cell Genet 2001;93:270–276. 84. Davisson MT. Mouse models of Down syndrome. Drug Discov Today: Dis Models 2005;2:103–109. 85. Davisson MT, Costa ACS. Mouse models of Down syndrome. In: Popko B, Ed. Mouse Models in the Study of Genetic Neurological Disorders. Advances in Neurochemistry, Vol. 9. New York: Kluwer Academic/Plenum, 1999. 86. Sago H, Carlson EJ, Smith DJ, Kilbridge J, Rubin EM, Mobley WC, Epstein CJ, Huang TT. Ts1Cje, a partial trisomy 16 mouse model for Down syndrome, exhibits learning and behavioral abnormalities. Proc Natl Acad Sci USA 1998;95:6256–6261. 87. O’Doherty A, Ruf S, Mulligan C, Hildreth V, Errington ML, Sesay A, Modino S, Vanes L, Hernandez D, Linehan JM, Sharpe PT, Brandner S, Bliss TV, Henderson DJ, Nizetic D, et al. An aneuploid mouse strain carrying human chromosome 21 with Down syndrome phenotypes. Science 2005;309(5743):2033–2037. 88. Villar AJ, Belichenko PV, Gillespie AM, Kozy HM, Mobley WC, Epstein CJ. Identification and characterization of a new Down syndrome model, Ts[Rb(12.1716)]2Cje, resulting from a spontaneous Robertsonian fusion between T(171)65Dn and mouse chromosome 12. Mamm Genome 2005;16:79–90. 89. Reeves RH, Irving NG, Moran T, Wohn A, Sisodia SS, Schmidt C, Davisson MT. A mouse model for Down syndrome exhibits learning and behavior deficits. Nat Genet 1995;11:177–183. 90. Hunt PA, Worthman C, Levinson H, Stallings J, LeMaire R, Mroz K, Park C, Handel MA. Germ cell loss in the XXY male mouse: Altered X chromosome dosage affects prenatal development. Mol Reprod Dev 1998;49:101–111. 91. Mroz K, Hassold TJ, Hunt PA. Meiotic aneuploidy in the XXY mouse: Evidence that a compromised testicular environment increases the incidence of meiotic errors. Hum Reprod 1999;14: 1151–1156. 92. Burgoyne PS, Ojarikre OA, Tuner JMA. Evidence that postnatal growth retardation in XO mice is due to haploinsufficiency for a non-PAR X gene. Cytogenet Genome Res 2002;99:252–256.

535

93. Hodges CA, LeMaire-Adkins R, Hunt PA. Coordinating the segregation of sister chromatids during the first meiotic division: Evidence for sexual dimorphism. J Cell Sci 2001;114:2417– 2426. 94. Dobson MJ, Pearlman RE, Karaiskakis A, Spyropoulos B, Moens PB. Synaptonemal complex proteins: Occurrence, epitope mapping and chromosome disjunction. J Cell Sci 1994;107:2749–2760. 95. Parra MT, Viera A, Gomez R, Page J, Benavente R, Santos JL, Rufas JS, Suja JA. Involvement of the cohesion Rad21 and Scp3 in monopolar attachment of sister kinetochores during mouse meiosis I. J Cell Sci 2004;117:1221–1234. 96. Yuan L, Liu JG, Zhao J, Brundell E, Daneholt B, Hoog C. The murine SCP3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility. Mol Cell 2000;5:73–83. 97. Yuan L, Liu JG, Hoja MR, Wilbertz J, Nordqvist K, Hoog C. Female germ cell aneuploidy and embryo death in mice lacking the meiosis-specific protein SCP3. Science 2002;296:1115–1118. 98. Wang H, Hoog C. Structural damage to meiotic chromosomes impairs DNA recombination and checkpoint control in mammalian oocytes. J Cell Biol 2006;173:485–495. 99. Revenkova E, Eijpe M, Heyting C, Gross B, Jessberger R. Novel meiosis-specific isoform of mammalian SMC1. Mol Cell Biol 2001;21:6984–6998. 100. Revenkova E, Eifpe M, Heyting C, Hodges CA, Hunt PA, Liebe B, Scherthan H, Jessberger R. Cohesin SMC1beta is required for meiotic chromosome dynamics, sister chromatid cohesion and DNA recombination. Nat Cell Biol 2004;6:555–562. 101. Hodges CA, Revenkova E, Jessberger R, Hassold T, Hunt PA. SMC1beta-deficient female mice provide evidence that cohesions are a missing link in age-related nondisjunction. Nat Genet 2005;37:1351–1355. 102. Hunter N, Borts RH. Mlh1 is unique among mismatch repair proteins in its ability to promote crossing-over during meiosis. Genes Dev 1997;11:1573–1582. 103. Baker S, Plug A, Prolla T, Bronner C, Harris A, Yao X, Christie DM, Monell C, Arnheim N, Bradley A, et al. Involvement of mouse MLH1 in DNA mismatch repair and meiotic crossing over. Nat Genet 1996;13:336–342. 104. Edelmann W, Cohen PE, Kane M, Lau K, Morrow B, Bennett S, Umar A, Kunkel T, Cattoretti G, Chaganti R, et al. Meiotic pachytene arrest in MLH1-deficient mice. Cell 1996;85:1125–1134. 105. Eaker S, Cobb J, Pyle A, Handel MA. Meiotic prophase abnormalities and metaphase cell death in MLH1-deficient mouse spermatocytes: Insights into regulation of spermatogenic progress. Dev Biol 2002;249:85–95. 106. Woods LM, Hodges CA, Baart E, Baker SM, Liskay M, Hunt PA. Chromosomal influence on meiotic spindle assembly: Abnormal meiosis I in female MLh1 mutant mice. J Cell Biol 1999;145:1395– 1406. 107. Anderson LK, Reeves A, Webb LM, Ashley T. Distribution of crossing over on mouse synaptonemal complexes using immunofluorescent localization of MLH1 protein. Genetics 1999;151:1569– 1579. 108. Lipkin SM, Moens PB, Wang V, Lenzi M, Shanmugarajah D, Gilgeous A, Thomas J, Cheng J, Touchman JW, Green ED, Schwartzberg P, Collins FS, Cohen PE. Meiotic arrest and aneuploidy in MLH3-deficient mice. Nat Genet 2002;31:385–390. 109. De La Fuente R, Viveiros MM, Wigglesworth K, Eppig JJ. ATRX, a member of the SNF2 family of helicase/ATPases, is required for chromosome alignment and meiotic spindle organization in metaphase II stage mouse oocytes. Dev Biol 2004;272:1–14. 110. Champoux JJ. DNA topoisomerases: Structure, function, and mechanism. Annu Rev Biochem 2001;70:369–413. 111. Kwan KY, Moens PB, Wang JC. Infertility and aneuploidy in mice lacking a type IA DNA topoisomerase IIIbeta. Proc Natl Acad Sci USA 2003;100:2526–2531. 112. Cobb J, Reddy RK, Park C, Handel MA. Analysis of expression and function of topoisomerase I and II during meiosis in male mice. Mol Reprod Dev 1997;46:489–498.

536

SECTION V / MODELS OF BEHAVIOR

113. Nasmyth K. How do so few control so many? Cell 2005;120: 739–746. 114. Baker DJ, Jeganathan KB, Cameron JD, Thompson M, Juneja S, Kopecka A, Kumar R, Jenkins RB, de Groen PC, Roche P, van Deursen JM. BubR1 insufficiency causes early onset of aging-associated phenotypes and infertility in mice. Nat Genet 2004; 36:744–749. 115. Dobles M, Liberal V, Scott ML, Benerza R, Sorger PK. Chromosome missegregation and apoptosis in mice lacking the mitotic checkpoint protein Mad2. Cell 2000;101:635–645. 116. Michel LS, Liberal V, Chatterjee A, Kirchwegger R, Pasche B, Gerald W, Dobles M, Lorger PK, Murty VVVS, Benerza R. MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells. Nature 2001;409:355–359. 117. Libby BJ, DeLaFuente R, O’Brien MJ, Wigglesworth K, Cobb J, Inselman A, Eaker S, Handel MA, Eppig JJ, Schimenti JC. The mouse meiotic mutation mei1 disrupts chromosome synapsis with sexually dimorphic consequences for meiotic progression. Dev Biol 2002;242:174–187. 118. Bannister LA, Reinholdt LG, Munroe RJ, Schimenti JC. Positional cloning and characterization of mouse mei8, a disrupted allele of the meiotic cohesin Rec8. Genesis 2004;40:184–194. 119. Pyle A, Handel MA. Meiosis in male PL/J mice: A genetic model for gametic aneuploidy. Mol Reprod Dev 2003;64:471– 481. 120. Liu L, Keefe DL. Aging-associated aberration in meiosis of oocytes from senescence-accelerated mice. Hum Reprod 2002;17:2678– 2685. 121. Pacchierotti F. Chemically induced aneuploidy in germ cells of mouse. In: Vig BK, Sandberg AA, Eds. Aneuploidy, Part B: Induction and Test Systems, Progress and Topics in Cytogenetics, Vol. 7b. New York: Alan R. Liss, 1988:123–139. 122. Mailhes JB, Marchetti F. Mechanisms and chemical induction of aneuploidy in rodent germ cells. Cytogenet Genome Res 2005;111: 384–391. 123. Mailhes JB, Mastromatteo C, Fuseler JW. Transient exposure to the EG5 kinesin inhibitor monastrol leads to syntelic orientation of chromosomes and aneuploidy in mouse oocytes. Mutat Res 2004;559:153–167. 124. Hunt PA, Koehler KE, Susiarjo M, Hodges CA, Ilagan A, Voight RC, Thomas S, Thomas BF, Hassold TJ. Bisphenol a exposure causes meiotic aneuploidy in the female mouse. Curr Biol 2003;13: 546–553. 125. Bean CJ, Hassold TJ, Judis L, Hunt PA. Fertilization in vitro increases non-disjunction during early cleavage divisions in a mouse model system. Hum Reprod 2002;17:2362–2367. 126. Tepperberg JH, Moses MJ, Nath J. Colchicine effects on meiosis in the male mouse .1. Meiotic prophase: Synaptic arrest, univalents, loss of damaged spermatocytes and a possible checkpoint at pachytene. Chromosoma 1997;106:183–192. 127. Tepperberg JH, Moses MJ, Nath J. Colchicine effects on meiosis in the male mouse—II. Inhibition of synapsis and induction of nondisjunction. Mutat Res 1999;429:93–105. 128. Allen JW, Gibson JB, Poorman PA, Backer LC, Moses MJ. Synaptonemal complex damage induced by clastogenic and anti-mitotic chemicals: Implications for non-disjunction and aneuploidy. Mutat Res 1988;201:313–324. 129. Backer LC, Gibson JB, Moses MJ, Allen JW. Synaptonemal complex damage in relation to meiotic chromosome aberrations after exposure of male mice to cyclophosphamide. Mutat Res 1988;203:317–330. 130. Russell LB, Hunsicker PR, Johnson DK, Shelby MD. Unlike other chemicals, etoposide (a topoisomerase-II inhibitor) produces peak mutagenicity in primary spermatocytes of the mouse. Mutat Res 1998;400:279–286. 131. Russell LB, Hunsicker PR, Hack AM, Ashley T. Effect of the topoisomerase-II inhibitor etoposide on meiotic recombination in male mice. Mutat Res 2000;464:201–212. 132. Marchetti F, Bishop JB, Lowe X, Generoso WM, Hozier J, Wyrobek AJ. Etoposide induces heritable chromosomal aberrations and

133.

134.

135.

136.

137.

138.

139.

140.

141.

142. 143.

144.

145.

146. 147.

148. 149. 150.

151.

152.

153. 154. 155.

aneuploidy during male meiosis in the mouse. Proc Natl Acad Sci USA 2001;98:3952–3957. Russell LB, Hunsicker PR, Kerley M, Pyle A, Saxton AM. Etoposide exposure during male mouse pachytene has complex effects on crossing-over and causes nondisjunction. Mutat Res 2004;565: 61–77. Russell LB. Effects of male germ-cell stage on the frequency, nature and spectrum of induced specific-locus mutations in the mouse. Genetica 2004;122:25–36. Davisson MT, Handel MA. Cytogenetics. In: Smith AG, Ed. The Mouse in Biomedical Research. Amsterdam, The Netherlands: Elsevier, 2006:146–164. Attia SM, Schmid TE, Badary OA, Hamada FM, Adler ID. Molecular cytogenetic analysis in mouse sperm of chemically induced aneuploidy: Studies with topoisomerase II inhibitors. Mutat Res 2002;520:1–13. Marchetti F, Pearson FS, Bishop JB, Wyrobek AJ. Etoposide induces chromosomal abnormalities in mouse spermatocytes and stem cell spermatogonia. Hum Reprod 2006;21:888–895. De Mas P, Daudin M, Vincent MC, et al. Increased aneuploidy in spermatozoa from testicular tumour patients after chemotherapy with cisplatin, etoposide and bleomycin. Hum Reprod 2001;16:1204– 1208. Baumgartner A, Schmid TE, Schuetz CG, Adler ID. Detection of aneuploidy in rodent and human sperm by multicolor FISH after chronic exposure to diazepam. Mutat Res 2001;490:11–19. Marchetti F, Lowe X, Moore DI, Bishop J, Wyrobek AJ. Paternally inherited chromosomal structural aberrations detected in mouse first-cleavage zygote metaphases by multicolor fluorescence in situ hybridization painting. Chromosome Res 1996;4:604–613. Marchetti F, Bishop JB, Cosentino L, Moore D, 2nd, Wyrobek AJ. Paternally transmitted chromosomal aberrations in mouse zygotes determine their embryonic fate. Biol Reprod 2004;70: 616–624. Hafez ESE. Reproduction in Farm Animals, 6th ed. Philadelphia, PA: Lea & Febiger, 1993:144–164. Juberg RC. Origin of chromosomal abnormalities: Evidence for delayed fertilization in meiotic nondisjunction. Hum Genet 1983;64:122–127. Blazak WF. Incidence of aneuploidy in farm animals. In: Vig BK, Sandberg AA, Eds. Aneuploidy, Part A: Incidence and Etiology. New York: Alan R. Liss, 1987:103–116. Mailhes JB. Incidence of aneuploidy in rodents. In: Vig BK, Sandberg AA, Eds. Aneuploidy, Part A: Incidence and Etiology. New York: Alan R. Liss,1987:67–101. Pellestor F. Frequency and distribution of aneuploidy in human female gametes. Hum Genet 1991;86:283–288. Zenzes MT, Casper RF. Cytogenetics of human oocytes, zygotes, and embryos after in vitro fertilization. Hum Genet 1992;88: 367–375. Wilcox AJ, Weinberg CR, Baird DD. Post-ovulatory ageing of the human oocyte and embryo failure. Hum Reprod 1998;13:394–397. Lewis WH, Wright ES. On the early development of the mouse egg. Carnegie Inst Contrib Embryol 1935;25:113–143. Edwards RG, Gates AH. Timing of the stages of the maturation divisions, ovulation, fertilization and the first cleavage of eggs of adult mice treated with gonadotrophins. J Endocrinol 1959;18:292– 304. Marston JH, Chang MC. The fertilizable life of ova and their morphology following delayed insemination in mature and immature mice. J Exp Zool 1964;155:237–252. Exley GE, Tang C, McElhinny AS, Warner CM. Expression of caspase and BCL-2 apoptotic family members in mouse preimplantation embryos. Biol Reprod 1999;61:231–239. Morita Y, Tilly JL. Oocyte apoptosis: Like sand through an hourglass. Dev Biol 1999;213:1–17. Perez GI, Tao XJ, Tilly JL. Fragmentation and death (a.k.a. apoptosis) of ovulated oocytes. Mol Human Reprod 1999;5:414–420. Gordo AC, Rodrigues P, Kurokawa M, Jellerette T, Exley GE, Warner C, Fissore R. Intracellular calcium oscillations signal

CHAPTER 54 / INVESTIGATING THE CAUSES AND MECHANISMS OF MAMMALIAN GERM CELL ANEUPLOIDY

156. 157.

158. 159. 160.

161.

162.

apoptosis rather than activation in in vitro aged mouse eggs. Biol Reprod 2002;66:1828–1837. Warburton D. Biological aging and the etiology of aneuploidy. Cytogenet Genome Res 2005;111:266–272. Hodges CA, Ilagan A, Jennings D, Keri R, Nilson J, Hunt PA. Experimental evidence that changes in oocyte growth influence meiotic chromosome segregation. Hum Reprod 2002;17:1171–1180. Yanagida M. Basic mechanisms of eukaryotic chromosome segregation. Phil Trans R Soc B 2005;360:609–621. Schlecht U, Primig M. Mining meiosis and gametogenesis with DNA microarrays. Reproduction 2003;125:447–456. Stein P, Svoboda P, Schultz RM. Transgenic RNAi in mouse oocytes: A simple and fast approach to study gene function. Dev Biol 2003;256:187–193. Bettencourt-Dias M, Giet R, Sinka R, Mazumdar A, Lock WG, Balloux PJ, Safiropoulos PJ, Yamaguchi S, Winter S, Carthew RW, Cooper M, Jones D, Frenz L, Glover DM. Genome-wide survey of protein kinases required for cell cycle progression. Nature 2004;432:980–987. Prawitt D, Brixel L, Spangenberg C. Eshkind L, Heck R, Oesch F, Zabel B, Bockamp E. RNAi knock-down mice: An emerging technology for post-genomic functional genetics. Cytogenet Genome Res 2004;105:412–421.

537

163. Gregan J, Rabitsch PK, Sakem B, Csutak O, Latypov V, Lehmann E, Kohli J, Nasmygh K. Novel genes required for meiotic chromosome segregation are identified by a high-throughput knockout screen in fission yeast. Curr Biol 2005;15:1663–1669. 164. Marston AL, Tham WH, Shah H, Amon A. A genome-wide screen identifies genes required for centromeric cohesion. Science 2004;303:1367–1370. 165. Rhodes DR, Yu J, Shanker K, Deshpande N, Varambally R, Ghosh D, Barrette T, Pandey A, Chinnaiyan AM. Large-scale metaanalysis of cancer microarray data identifies common transcriptional profiles of neoplastic transformation and progression. Proc Natl Acad Sci USA 2004;101:9309–9314. 166. Rajagopalan H, Lengauer C. Aneuploidy and cancer. Nature 2004; 432:338–341. 167. Mailhes JB, Hilliard C, Lowery M, London SN. MG-132, an inhibitor of proteasomes and calpains, induced inhibition of oocyte maturation and aneuploidy in mouse oocytes. Cell Chromosome 2002;1:2. 168. Mailhes JB, Hilliard C, Fuseler JW, London SN. Vanadate, an inhibitor of tyrosine phosphatases, induced premature anaphase in oocytes and aneuploidy and polyploidy in mouse bone marrow cells. Mutat Res 2003;538:101–107.

55

Genetic Models of Alzheimer’s Disease YANN S. MINEUR

ABSTRACT

For a pathological diagnosis of AD, neuritic plaques and NFTs are required. It is now commonly believed that abnormal production and aggregation of Aβ (especially the more fibrillogenic and insoluble Aβ42 isoform) are the primary cause of the plaques found in AD.

This chapter presents and discusses different strategies to dissect genetically the molecular mechanisms of Alzheimer’s disease. First, a short preamble sketches the common phenotypical features of Alzheimer’s disease. Second, a basic introduction defines the characteristics of a good animal model along with the different molecular strategies commonly used to develop transgenic animals. Third, an exclusive census of genetically modified mouse models describes some of the remarkable characteristics observed in these animals and tries to weigh the pros and cons of each of them. Finally, a brief note highlights gene–environment interactions relevant to the field of genetic mouse models and Alzheimer’s disease. All sections are illustrated with examples focusing on behavioral phenotypes with a specific interest on learning and memory. Key Words: Alzheimer’s disease, Transgenic, Mouse models, Neuroanatomy, Behavior, Learning, Memory, Amyloid, Secretases, Tau.

ANIMAL MODELS OF ALZHEIMER’S DISEASE? Investigating the molecular mechanisms of AD in human subjects is particularly difficult because only postmortem tissue can be studied, once the disease has reached its most advanced stages. Neuronal tissue harvested from human brain is generally fragile and very sensitive, affected by aging, medications, and other possible neurological defects. In addition, the complexity is increased by broad individual variability due to genetic and environmental factors. Moreover, the availability of brain tissue is limited. Most of these difficulties can be overcome by using animal models. Given the wealth of data available on models of AD, this chapter will principally focus on the mouse. Mice are the most popular animal models (for AD), primarily because their genome can be manipulated easily. This advantage has led to the generation of a variety of genetic mouse models, although rat models do exist as well (see, for instance, Hu et al.8). Nevertheless, invertebrate models such as Drosophila melanogaster and Caenorhabditis elegans should not be thrust aside too hastily. In spite of their clear limitations—mainly the lack of mammalian brain structures and greater genetic distance from human than mice—these model organisms can aid in the study of AD. These “limitations” can also be an advantage: simpler organisms are generally easier for mapping of neuronal networks and for investigating molecular interactions. From a practical point of view, these animals are also smaller, cheaper to house and breed, and have shorter generation times than any mammal. Thus, many generations can be produced in a short time span. As a result, transgenic fly and worm AD models have been developed for both Aβ and tau expression.9–11

INTRODUCTION Alzheimer’s disease (AD) is expected to affect 16 million individuals in the United States by the year 2050. While the onset of AD-associated dementia can be subtle, the inexorable evolution of this disease ultimately leads to global cognitive deficits including memory, orientation, reasoning, and judgment. Unfortunately, besides limited cholinergic therapies aimed at improving cognitive decline in patients, no effective therapies are currently available. Extracellular neuritic plaques and intraneuronal fibrillary tangles (NFTs) are the two classical hallmarks of AD pathology in the brain.1,2 Neuritic plaques are typically composed of βamyloid oligomers (Aβ) surrounding dystrophic neurites. These plaques will precipitate and accumulate (primarily in the cortex and the limbic system),3–5 leading to neuronal death, which is thought to be responsible for dementia observed in AD patients6 (see Figure 55–1). Tau, a microtubule-associated protein, is the primary component of NFTs.7 In AD, tau is hyperphosphorylated, resulting in disruption of microtubule dynamics, impaired axonal transport, and intraneuronal polymerization of the cytoskeleton. These molecular events will also result in neuronal death.

HOW DO WE DEFINE A “GOOD” MODEL OF ALZHEIMER’S DISEASE? Modeling a disease in an animal can be challenging. A good AD model should, as much as feasible, present the following features: • Reproducibility: a good model should yield consistent phenotypic features across generations and time, and

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

539

540

SECTION V / MODELS OF BEHAVIOR

APP gene

g

a

b Tau gene

Abnormal Aβ biosynthesis

Mutation

Hyperphosphorylation Aggregation

NeuroFibrillary Tangles Plaques

Neurotoxicity Neuronal death Figure 55–1.

Simplified overview of the amyloid cascade hypothesis of Alzheimer’s disease. See text for details.

possibly across investigators and laboratories. However, the latter can be quite difficult to achieve despite the best efforts to do so.12 • Face validity: the phenotypic features should “look” the same as those observed in the human disease. Of course, this can be problematic for phenotypic traits related to higher cognitive function and/or for human characteristics impossible to assess and model in mice. Nevertheless, a plethora of behavioral assays performed in mice permits evaluation of memory processing and cognitive functions, for instance. In addition, physiological and biochemical analyses allow the identification of amyloid plaques and subsequent neuronal damage similar to that observed in AD. • Construct validity: the model either relies on, or reveals, the same basic underlying mechanisms as AD, including the accumulation of Aß peptides, hyperphosphorylation of tau, and subsequent neuronal death. By extension, genetic validity is part of this category when a disease or its risk factors engage genetic components similar to the ones in human subjects. • Predictive validity: the observed effects of particular disease-triggering elements should be similar between the animal model and human subjects. For instance, β amyloidinduced plaques with neuronal losses would constitute

good predictive validity of an animal model of AD. A good predictive validity can also help in defining how useful and/or safe a drug/treatment can be. These three latter categories are not mutually exclusive one with another. Phenotypical factors can belong to one, two, or all of the categories.

GENETIC MOUSE MODELS OF ALZHEIMER’S DISEASE It is crucial to realize that “natural” development of plaques and tangles does not occur in mice (or other species commonly used in scientific research). Thus, engineering a model for AD requires experimental manipulation, either genetic and/or pharmacological, the latter generally consisting of the exogenous administration of different Aβ peptides into the normal rodent brain. Both techniques have advantages and disadvantages, but are essentially complementary. This chapter will focus on genetic models of AD. For a review of mouse models of AD that use exogenous Aβ administration and their comparison to transgenic models, the reader is referred to Stephan and Phillips.13 Genes encoding proteins shown to play a role in the onset of AD pathology would be the prime candidates for manipulations. Consequently, a large number of genetically modified mouse lines

CHAPTER 55 / GENETIC MODELS OF ALZHEIMER’S DISEASE

have been generated using several of the following common techniques: • Gene deletion or knockout (KO): in these models, defined gene(s) expression is absent throughout the life of the animal, from the earliest stage of development. Refinements have been added to this techniques resulting in conditional KO mice, in which the expression of a gene is abolished but only in restricted brain area(s)/neuronal types, and/or at a specific time during development of the animal. The deletion of a gene can also be reversible. • Transgenic overexpression: in this case, a foreign gene, e.g., human APP (hAPP), is introduced into the genome and its expression (level and region) is driven by specific promoters. • Targeted mutation or knockin (KI): a specific region of a gene is mutated, leading to loss or alteration of the proteins encoded by the targeted gene. Combinations of these genetic techniques are possible and are becoming common in mouse models of AD. The following section will describe and compare several AD mouse models generated to date. APP TRANSGENICS The first successful genetically engineered mouse models of AD were transgenic mice, characterized by increased Aβ levels induced by overexpression of its precursor (APP). Commonly, hAPP—and usually a mutant form linked to the early-onset familial form of AD—is introduced into the mouse genome. In turn, this gene transfer leads to the expression of the transgene. To date, numerous hAPP models have been generated including PDAPP, Tg2567, APP23, TgCRNDN8, and J20. Each model uses either different APP mutations, different promoters, or a different genetic background of the host mouse strain (see Table 55–1 for more details and http://www.alzforum.org/res/ com/tra/ for a comprehensive list of transgenic models). Not surprisingly, the phenotypes are therefore dissimilar between these strains, characterized by different qualitative and quantitative levels of neuroanatomical defects. These strains have all been assessed behaviorally, some extensively. PDAPP and Tg2567 have been studied in great detail and high APP levels induced by the transgene were shown to be responsible for cognitive abnormalities in both lines. Both lines are considered to be reliable AD models, because they exhibit clear learning deficits over time and across laboratories. Moreover, the cognitive decline observed in these animals was observed in many cognitive tasks. For instance, Tg2576 mice, developed

541

by Hsiao et al.,14 show a progressive impairment in various forms of the water navigation task,15 deficits in a version of the Barnes maze,16 and poorer performance in a T-maze alternation task and contextual fear conditioning.17 SECRETASE TRANSGENICS The β- and γ-secretases catalyze the processing of APP into the various forms of Aβ, whereas α-secretase is part of the nonamyloidogenic pathway (see Figure 55–1). Thus, genes encoding these secretases are candidate genes of interest for AD. b-Secretase BACE1 is the predominant neuronal βsecretase. Both transgenic mice overexpressing BACE1 and knockout mice lacking BACE1 have been generated and, intriguingly, at least one of their behavioral phenotypes is opposite: BACE1 KO mice exhibit more anxiety-like behaviors than controls, whereas transgenics showed less anxiety-like behaviors. However, cognitive deficits have not been observed. The development of double transgenic mice generated by crossbreeding mice overexpressing hAPP with those either lacking or overexpressing BACE1 has also helped identify the role of BACE1 in AD. As expected, double transgenic mice (hBACE/ Tg2576) have increased amyloid pathology, high concentrations of both total Aβ and Aβ42, and greater numbers of plaques than hAPP mice alone. However, elimination of BACE1 in the presence of hAPP rescues specific cognitive deficits associated with Aβ deposits.18 g-Secretase The secondary cleavage of APP leading to Aß42 involves the activity of γ-secretase. Functional γ-secretase is a complex holoenzyme made of several individual enzymes, including presenilin (PS)1, PS2, Nicastrin, Aph-1, and Pen-2.19,20 The presenilins (PS1, PS2) have been thoroughly investigated since human subjects with mutations in PS1 show early-onset AD. Many genetically engineered mouse models have been developed including KOs, transgenics, and KIs, double and even triple transgenics. While PS1 KO mice are not viable,21 the development of conditional KOs (cKOs), in which the loss of the gene was limited to the postnatal forebrain, circumvented this lethality. PS1 cKO mice show mild cognitive impairments in long-term spatial reference memory and retention.22 Overexpression of a human form of PS1 (hPS1) in mice, however, induced only slight behavioral perturbations. KIs generated by a targeted missense mutation in murine PS1 overproduce Aβ42; however, this does not lead to the aggregation of amyloid plaques. Nonetheless, mice perform poorly in an object recognition task, but are similar to controls in a water navigation test. These results suggest that changes in PS1 activity could influence

Table 55–1 Examples of different APP transgenic mice Common abbreviations

Transgene

Promoter

Genetic background

PDAPP

Human APP with Val-717 substituted by Phe (Indiana mutation) Human APP with mutations Lys-670 substituted to Asn and Met-671 to Leu (Swedish mutation) Human APP751 with Swedish mutation APP695 with Swedish and Indiana mutations APP695 with Swedish and Indiana mutations

Human platelet-derived growth factor β (PDGF- β) chain gene promoter Hamster prion promoter

Swiss × C57BL/6 × DBA2

Murine Thy-1.2 gene Hamster prion promoter Human platelet-derived growth factor β (PDGF- β) chain gene promoter

C57BL/6 C3H/He × C57BL/6J C57BL/6 × DBA2

Tg2567

APP23 TgCRNDN8 J20

C57BL/6 × SJL

542

SECTION V / MODELS OF BEHAVIOR

hippocampus-dependent memory pathways.23,24 Mice lacking PS2 do not exhibit obvious neurological or behavioral abnormalities, and can reproduce successfully. In contrast, PS2 overexpressing transgenic mice and KIs with mutations in PS1 exhibit poor performance in a water navigation task. cPS1/APP double transgenic mice have also been generated (cPS1 carries a neuron-specific conditional KO of the PS1 gene). In this line, accumulation of Aβ is faster and Aβ levels are higher. Behaviorally, cPS1/APP double transgenic mice performed poorly in several cognitive tasks (for details, see Kobayashi and Chen18). As was seen in hAPP/PS double transgenics, the cPS1/APP double transgenic mice do not develop amyloid plaque; behavioral abnormalities in an object recognition task were rescued in the double transgenic mice.25 This reinforces the conclusion that inhibition of secretase could constitute a promising target for AD treatment or prevention. a-Secretase α-secretase cleaves about 90% of APP. However, α-secretase induces production of the P3 peptide, which does not appear to be associated with amyloid neurotoxicity. Although P3 can be detected in amyloid plaques, very few reports suggest deleterious effects on neurons. Hence, the α-secretaseprocessing pathway is referred to as nonamyloidogenic.26,27 Numerous enzymes have α-secretase activity including the ADAM (a disintegrin and metalloproteinase) family. These proteins all have α-secretase cleavage activity, but differential patterns of expression.28–30 In “normal” mice, overexpression of ADAM10 does not appear harmful; however, in transgenic models of AD, neuronal function can be restored by ADAM10 overexpression. For instance, Postina et al.31 demonstrated that increased α-secretase expression prevents plaque deposits in aged animals overexpressing hAPP. In contrast, overexpression of inactivated ADAM10 (mADAM10) in APP mice worsens amyloid aggregation. In a water navigation task, mADAM10/APP double transgenic animals showed deficits in the acquisition phase of place learning and in the probe trial, whereas the ADAM10/APP double transgenics performed similarly to control animals. Long-term potentiation (LTP) was also improved in the double transgenic mice as compared to APP transgenics, suggesting a rescue of synaptic plasticity by increased α-secretase activity. Previous findings from Moechars et al.32 corroborate these results: in transgenic animals carrying a modified hAPP in which the α-secretase cleavage site has been altered (APP/RK), similar results were observed. The APP/RK transgenic animals showed increased APP expression with a shift toward β-site cleavage amyloid peptides. Additionally, these animals had shorter life spans than control mice, were more aggressive and hyperactive, and displayed abnormalities in the amygdala, cortex, and hippocampus.33 TAU In addition to the “amyloid-related” transgenics, complementary mouse models have been developed that overexpress human tau in its normal and/or mutated forms (including a form associated with frontotemporal dementia and Parkinson’s disease, FDPD). Human tau proteins are encoded by a gene on chromosome 17. Six brains isoforms resulting from alternative splicing have been described and can be divided into two classes: proteins with three C-terminal repeat domains (3R) and proteins with four domains (4R). Most transgenic models have been generated with one of the mutated 4R forms, varying from “regular” 4R tau to monomutated forms (P301L, P301S, V337M) and multiple-mutated 4R forms.

Although tau-related pathologies have been detected in the brains of these transgenics,9,10 few studies have characterized these animals behaviorally. In the elevated plus-maze, 11-month-old transgenic mice overexpressing the V337 mutant tau gene displayed less anxiety-like behavior compared to their wild-type littermates. In contrast, no differences were detected in a water navigation test. These animals showed impaired habituation in an open-field exploration task. Additionally, these tau transgenic mice showed an irregular neuronal-shaped hippocampus and altered neuronal activity. Another related line carries the R406W tau mutation (found in human subjects with a tauopathy resembling AD). Observations made in aged animals (ranging from 16 to 23 months of age) revealed a blunted fear response to cues, but not to context, after 2 days of conditioning. The same animals showed lower levels of contextual fear after 15 days of conditioning, suggesting that long-term memory loss may be more dramatic in aged R406W tau transgenic mice. These data suggest that there is a deficit in associative memory in mice overexpressing a mutated tau gene. Neuroanatomically, transgenic mice develop tau inclusions in the hippocampus, amygdala, and neocortex, structures known to be involved in memory formation.34 Subtle differences and specific impairments were also observed in transgenic mice overexpressing the P301L mutation of tau.35 Aggregation and NFTs were observed in many brain areas, including the amygdala and the hippocampus, brain regions that are also severely affected by NFTs in AD patients. Because these mice show accelerated extinction in a conditioned taste aversion test, the authors evaluated their behaviors in tasks requiring an intact hippocampus, including the Morris Water maze and the Y maze. Differences were detected only in spatial working memory, with mice showing greater impairment with age, starting at 6 months. However, the transgenic mice were more active and slightly less prone to novelty-induced anxiety, which suggests that the impairment observed in these mice is not related to affective behavior unrelated to the hippocampus. POLYTRANSGENICS Because Aβ and tau are two primary, and complementary, factors in the development of AD, and because the presenilins are clearly genetic susceptibility loci for the disease,36 the interactions of these proteins were tested by engineering a triple-transgenic mouse model. To generate these mice, two transgenes (Tg2576, an APP mutant, and tau P301L) were transferred onto a PS1-heterozygous knockin mutation background. Triple-transgenic (3xTg) mice developed progressive age-dependent neuropathology, including plaques and tangles. Remarkably, the pattern of evolution of these abnormalities closely mimics some of those described in AD. In these mice, presynaptic dysfunction and LTP deficits precede the accumulation of extracellular Aβ. This suggests a significant role for intracellular Aβ in AD-dependent pathology, even in the absence of structural changes. Plaques first appeared in cortical regions, then in the hippocampus, and later in the amygdala, whereas tangles followed the reversed pattern, beginning in the amygdala. This sequence of events is in line with the amyloid cascade hypothesis of AD (cf. Figure 55–1). 3xTg mice also demonstrate agedependent cognitive deficits.37 The earliest cognitive impairments were recorded at 4 months of age, and were characterized by a deficit in long-term memory correlated with the intraneuronal accumulation of Aβ in the hippocampus and amygdala. No plaques or tangles could be detected at this age, suggesting that

CHAPTER 55 / GENETIC MODELS OF ALZHEIMER’S DISEASE

they contribute to cognitive dysfunction only at older ages. Immunotherapy treatments eliminated intraneuronal Aβ deposits and, consequently, rescued the deficits observed in hippocampaldependent cognitive tasks. The reemergence of Aβ pathology after immunotherapy led to the return of the cognitive deficits observed before treatment. Given these promising results and the very integrative nature of this model, triple transgenic mice have now been studied further and offer promising insights on the sequence of molecular and cellular events leading to AD.38

A NOTE ON EPIGENETIC AND ENVIRONMENTAL FACTORS IN ALZHEIMER’S DISEASE GENETICALLY MODIFIED MOUSE MODELS Because familial cases of AD represent only 15% of AD and because genetic factors are essentially risk factors, an important field of research aims to determine the interaction between epigenetic factors and specific genetic alteration in an effort to identify gene–environment interactions. Two studies by Lazarov et al.39 and Jankowsky et al.40 have investigated the effect of longterm exposure to an enriched environment on double transgenic mice coexpressing APPswe and PS1 polypeptide variants. Although there are some discrepancies in the results (see Lazarov et al.39 for discussion), the results unequivocally demonstrated that environmental factors can influence amyloid deposition in a gene-dependent fashion. Identifying other environmental factors that modulate disease progression could be very valuable in the treatment or prevention of AD.

CONCLUSIONS Genetic mouse models have enhanced our understanding of the pathogenesis of AD and its underlying mechanisms. While inherent differences between mice and humans will always limit our ability to model this disease in animals, recent studies demonstrate that it is possible to generate plaques and tangles in a mouse, similar to human neuropathology. The evolution of both quantitative and qualitative genetic mouse models of AD has provided invaluable tools to better understand this pathology, along with the possibility of developing new therapeutics. When looking at the progress accomplished between the first transgenic models of AD and the current development of neuron-specific, conditional and polytransgenic models, it is clear that genetic models of AD will play an important role in finding a cure for this disease.

ACKNOWLEDGMENTS This chapter was based on a review by Mineur et al.41 I would like to greatly thank Marina Picciotto (Yale University, New Haven, CT) for her helpful comments and suggestions.

REFERENCES 1. Lovestone S, McLoughlin DM. Protein aggregates and dementia: Is there a common toxicity? J Neurol Neurosurg Psychiatry 2002;72: 152–161. 2. Selkoe DJ. Cell biology of protein misfolding: The examples of Alzheimer’s and Parkinson’s diseases. Nat Cell Biol 2004;6: 1054–1061. 3. Glenner GG, Wong CW. Alzheimer’s disease and Down’s syndrome: Sharing of a unique cerebrovascular amyloid fibril protein. Biochem Biophys Res Commun 1984;122:1131–1135.

543

4. Glenner GG, Wong CW. Alzheimer’s disease: Initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 1984;120:885–890. 5. Glenner GG, Wong CW, Quaranta V, Eanes ED. The amyloid deposits in Alzheimer’s disease: Their nature and pathogenesis. Appl Pathol 1984;2:357–369. 6. Wilquet V, De Strooper B. Amyloid-beta precursor protein processing in neurodegeneration. Curr Opin Neurobiol 2004;14: 582–588. 7. Stoothoff WH, Johnson GV. Tau phosphorylation: Physiological and pathological consequences. Biochim Biophys Acta 2005;1739: 280–297. 8. Hu ZH, Wang XC, Li LY, et al. Correlation of behavior changes and BOLD signal in Alzheimer-like rat model. Acta Biochim Biophys Sin (Shanghai) 2004;36:803–810. 9. Brandt R, Hundelt M, Shahani N. Tau alteration and neuronal degeneration in tauopathies: Mechanisms and models. Biochim Biophys Acta 2005;1739:331–354. 10. Lee VM, Kenyon TK, Trojanowski JQ. Transgenic animal models of tauopathies. Biochim Biophys Acta 2005;1739:251–259. 11. Link CD. Invertebrate models of Alzheimer’s disease. Genes Brain Behav 2005;4:147–156. 12. Crabbe JC. Animal model in neurobehavioral genetics: Methods for estimating genetic correlation. In: Jones C, Mormede P, Eds. Neurobehavioral Genetics: Methods and Applications. Boca Raton, FL: CRC Press, 1999. 13. Stephan A, Phillips AG. A case for a non-transgenic animal model of Alzheimer’s disease. Genes Brain Behav 2005;4:157–172. 14. Hsiao K, Chapman P, Nilsen S, et al. Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science 1996;274:99–102. 15. Westerman MA, Cooper-Blacketer D, Mariash A, et al. The relationship between Abeta and memory in the Tg2576 mouse model of Alzheimer’s disease. J Neurosci 2002;22:1858–1867. 16. Pompl PN, Mullan MJ, Bjugstad K, Arendash GW. Adaptation of the circular platform spatial memory task for mice: Use in detecting cognitive impairment in the APP(SW) transgenic mouse model for Alzheimer’s disease. J Neurosci Methods 1999;87:87–95. 17. Corcoran KA, Lu Y, Turner RS, Maren S. Overexpression of hAPPswe impairs rewarded alternation and contextual fear conditioning in a transgenic mouse model of Alzheimer’s disease. Learn Mem 2002;9:243–252. 18. Kobayashi DT, Chen KS. Behavioral phenotypes of amyloid-based genetically modified mouse models of Alzheimer’s disease. Genes Brain Behav 2005;4:173–196. 19. De Strooper B, Woodgett J. Alzheimer’s disease: Mental plaque removal. Nature 2003;423:392–393. 20. Francis R, McGrath G, Zhang J, et al. aph-1 and pen-2 are required for Notch pathway signaling, gamma-secretase cleavage of betaAPP, and presenilin protein accumulation. Dev Cell 2002;3:85–97. 21. Shen J, Bronson RT, Chen DF, Xia W, Selkoe DJ, Tonegawa S. Skeletal and CNS defects in presenilin-1-deficient mice. Cell 1997;89:629–639. 22. Yu H, Saura CA, Choi SY, et al. APP processing and synaptic plasticity in presenilin-1 conditional knockout mice. Neuron 2001;31: 713–726. 23. Huang XG, Yee BK, Nag S, Chan ST, Tang F. Behavioral and neurochemical characterization of transgenic mice carrying the human presenilin-1 gene with or without the leucine-to-proline mutation at codon 235. Exp Neurol 2003;183:673–681. 24. Janus C, D’Amelio S, Amitay O, et al. Spatial learning in transgenic mice expressing human presenilin 1 (PS1) transgenes. Neurobiol Aging 2000;21:541–549. 25. Dewachter I, Reverse D, Caluwaerts N, et al. Neuronal deficiency of presenilin 1 inhibits amyloid plaque formation and corrects hippocampal long-term potentiation but not a cognitive defect of amyloid precursor protein [V717I] transgenic mice. J Neurosci 2002;22:3445–3453. 26. Naslund J, Jensen M, Tjernberg LO, Thyberg J, Terenius L, Nordstedt C. The metabolic pathway generating p3, an A beta-peptide

544

27.

28.

29.

30.

31.

32.

33.

SECTION V / MODELS OF BEHAVIOR

fragment, is probably non-amyloidogenic. Biochem Biophys Res Commun 1994;204:780–787. Wei Q, Holzer M, Brueckner MK, Liu Y, Arendt T. Dephosphorylation of tau protein by calcineurin triturated into neural living cells. Cell Mol Neurobiol 2002;22:13–24. Buxbaum JD, Liu KN, Luo Y, et al. Evidence that tumor necrosis factor alpha converting enzyme is involved in regulated alphasecretase cleavage of the Alzheimer amyloid protein precursor. J Biol Chem 1998;273:27765–27767. Karkkainen I, Rybnikova E, Pelto-Huikko M, Huovila AP. Metalloprotease-disintegrin (ADAM) genes are widely and differentially expressed in the adult CNS. Mol Cell Neurosci 2000;15: 547–560. Lammich S, Kojro E, Postina R, et al. Constitutive and regulated alpha-secretase cleavage of Alzheimer’s amyloid precursor protein by a disintegrin metalloprotease. Proc Natl Acad Sci USA 1999; 96:3922–3927. Postina R, Schroeder A, Dewachter I, et al. A disintegrinmetalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model. J Clin Invest 2004;113:1456–1464. Moechars D, Lorent K, De Strooper B, Dewachter I, Van Leuven F. Expression in brain of amyloid precursor protein mutated in the alpha-secretase site causes disturbed behavior, neuronal degeneration and premature death in transgenic mice. EMBO J 1996;15:1265– 1274. Moechars D, Lorent K, Van Leuven F. Premature death in transgenic mice that overexpress a mutant amyloid precursor protein is preceded

34.

35.

36.

37.

38. 39.

40.

41.

by severe neurodegeneration and apoptosis. Neuroscience 1999;91: 819–830. Tatebayashi Y, Miyasaka T, Chui DH, et al. Tau filament formation and associative memory deficit in aged mice expressing mutant (R406W) human tau. Proc Natl Acad Sci USA 2002;99:13896– 13901. Pennanen L, Wolfer DP, Nitsch RM, Gotz J. Impaired spatial reference memory and increased exploratory behavior in P301L tau transgenic mice. Genes Brain Behav 2006;5:369–379. Oddo S, Caccamo A, Kitazawa M, Tseng BP, LaFerla FM. Amyloid deposition precedes tangle formation in a triple transgenic model of Alzheimer’s disease. Neurobiol Aging 2003;24:1063– 1070. Billings LM, Oddo S, Green KN, McGaugh JL, Laferla FM. Intraneuronal Abeta causes the onset of early Alzheimer’s diseaserelated cognitive deficits in transgenic mice. Neuron 2005;45:675– 688. Laferla FM, Oddo S. Alzheimer’s disease: Abeta, tau and synaptic dysfunction. Trends Mol Med 2005;11:170–176. Lazarov O, Robinson J, Tang YP, et al. Environmental enrichment reduces Abeta levels and amyloid deposition in transgenic mice. Cell 2005;120:701–713. Jankowsky JL, Xu G, Fromholt D, Gonzales V, Borchelt DR. Environmental enrichment exacerbates amyloid plaque formation in a transgenic mouse model of Alzheimer disease. J Neuropathol Exp Neurol 2003;62:1220–1227. Mineur YS, McLoughlin D, Crusio WE, Sluyter F. Genetic mouse models of Alzheimer’s disease. Neural Plast 2005;12:299–310.

56

Rat Models of Experimental Autoimmune Encephalomyelitis ERIK WALLSTRÖM

AND

TOMAS OLSSON

ABSTRACT

currently approved drugs either have only modest effects or can result in rare but fatal adverse events. Since there is a great medical need for better and safer treatments, in turn requiring better knowledge of disease mechanisms, MS continues to be a strong focus for research both in academia and in the pharmaceutical industry; disease-appropriate animal models are therefore indispensable for further progress. This is particularly so in MS, because the CNS is relatively inaccessible for sampling, observations in humans in most cases are descriptive, and experimental manipulation in humans is rarely possible. In principle, a rodent model for MS can be used for studies of disease mechanisms, either by classical hypothesis-driven methods or by unbiased genetic tools, or for studies of new therapeutic strategies. There is no single model of experimental autoimmune encephalomyelitis (EAE) that covers all aspects of human MS, and the choice of a particular EAE variant should be carefully thought through depending on the scientific question. To enable such a selection of a model, in depth knowledge of the natural course of the disease and its basic immunopathology is necessary. We refer the reader to several recent reviews on these topics, and give a summary of the most critical factors in the following. MULTIPLE SCLEROSIS: COURSE AND SUBPHENOTYPES MS rarely starts before the age of 15 or later than 50 years. The average age of onset is 28 years, with a 2/1 female/ male ratio. In about 85% of all cases of MS the disease follows a remitting relapsing course, with relapses of new neurological deficits on average every second year. Recovery can be complete or incomplete. Interestingly, the introduction of magnetic resonance imaging (MRI) into the study of MS has shown that there are up to 10 subclinical inflammatory events in the CNS on each clinical recognizable relapse. With time, most patients enter a secondary chronic progressive course with steadily increasing neurological defects. The mean time from onset to this phase is around 15 years. Current dogma suggests that the mechanisms during this progressive phase differ from those during the relapsing phase. None of the currently approved drugs for MS has had any effect on the progressive phase of the disease. Of MS patients, 10–15% start with a steady progression, categorized as primary progressive MS. Of note, few rodent MS models can mimic these very chronic aspects of MS. The symptoms and signs of MS vary depending on where in the CNS the inflammatory lesions occur, and almost all

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) leading to neurological deficits. The relative inaccessibility of the CNS for sampling and the problems of experimental manipulations in humans make proper experimental models indispensable for progress in the basic understanding of the disease as well as for testing of therapy. There is no single experimental model mimicking all aspects of MS and a model should be chosen based on the scientific question being investigated. We describe our experience with antigeninduced models in the rat species. The clinical course of disease, monophasic versus relapsing/protracted, and the histopathological features depend on the strain of rats used, their MHC and nonMHC genes, as well as the myelin autoantigen used for immunization. Other factors such as age, weight, and gender also influence the outcome. Use of myelin oligodendrocyte glycoprotein in DA rats results in an MS-like relapsing disease with plaques of demyelination. Use of myelin basic protein in LEW rats results in an acute monophasic inflammatory disease with little demyelination. Several disease-determining factors and practical schemes for MS models in rats are presented. Key Words: Multiple sclerosis, Autoimmunity, Myelin, Myelin basic protein, Myelin oligodendrocyte glycoprotein, Proteolipid protein, T cells, B cells, Antibodies, Major histocompatibility complex, Genetics, Demyelination, Inflammation, Axons, Neurons, Oligodendrocytes, Astrocytes, Macrophages, Microglia.

GENERAL CHARACTERISTICS WHY MODEL MULTIPLE SCLEROSIS? Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) with foci of inflammation, demyelination, and damage to axons. It is typically a disease of the Western world with prevalence there varying from 0.5 to 2 per thousand. This distribution most likely depends primarily on a higher genetic predisposition in these countries, but differences in environmental factors may also play a role. Despite its relatively low incidence and prevalence, it has a major impact on the afflicted individual and on society as a whole; its impact is also economic, since the disease starts early in life and persists for decades. In addition, drug companies have realized that MS is a valuable target, since From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

547

548

SECTION V / MODELS OF BEHAVIOR

neurological functions can ultimately be affected. These are focal neurological disturbances. In addition, MS is accompanied by general symptoms and signs. To enhance the understanding of the MS models described below we provide a short summary of common signs. Afferent neurological functions are often affected. Patients may develop regional hypoesthesias/paresthesias and defects in deep sensation. Optic neuritis, with loss of vision in one eye, is common. In most cases vision recovers over weeks. Brainstem lesions may lead to signs such as dysfunction of ocular motility and central vertigo. Cerebellar lesions can lead to dystaxia. Motor tracts are often affected, resulting in monoparesis of an extremity, most often in legs, hemiparesis, or paraparesis. In the chronic progressive phase a slowly increasing paraparesis folowed by tetraparesis is common. General signs include fatigue, which most patients refer to as their worst manifestation of MS. Depression is common. A mild or modest decrease of cognitive function occurs in up to 50% of MS patients. In quite rare instances full blown dementia may develop. Just as there are scoring scales for the EAE models described below, there are rating scales for MS, used in clinical follow-up or during clinical drug trials. The best established is the expanded disability scale (EDSS), with which different neurological functions are assessed and rated in a neuroexamination. These are added together to produce a score from 0 to 10 : 0 is no neurological dysfunction, 6 reflects decreased motor function with the patient able to walk 100 m with unilateral support, and 10 is death due to MS. The multiple sclerosis functional composite (MSFC) is a more recently introduced scale in which performance on a simple cognitive test, motor function in the upper extremity, and walking ability are added together. There are numerous other scales, some based on patient self-report and some measuring quality of life. MULTIPLE SCLEROSIS: HISTOPATHOLOGY MS histopathology is characterized by foci of inflammation, demyelination, and damage to neurons/axons. Initial periventricular lesions are composed of mononuclear cells, mainly macrophages, as well as an abundance of lymphocytes: CD4+, CD8+, T cells, and B cells. Demyelination ensues through macrophage attack. In addition, axons are damaged in this acute inflammatory attack.1 Further damage to axons also occurs more chronically. Classical demyelinated plaques varying in size from millimeters to centimeters ensue; these also contain astroglial scarring. To a variable extent demyelinated axons may become remyelinated by oligodendroglial precursors that are recruited. PRESUMED IMMUNE PATHOGENESIS The formal proof for any autoimmune or alternative viral etiology is lacking. However, there is strong circumstantial evidence suggesting an autoimmune pathogenesis based on the following: (1) Increased numbers of myelin autoreactive T and B cells enriched to the cerebrospinal fluid can be detected in persons with MS. (2) Certain HLA class II alleles, gene products being restriction elements for CD4+ T cells, dispose for MS. (3) Genomic regions disposing for MS overlap regions defined in rodent models known to be autoimmune.2 (4) The organ specificity of the inflammatory attack in MS is most easily reconciled in the context of the immune system, perhaps the only system able to discriminate between such a broad variety of molecular targets. Finally, (5) some of the induced EAE models described in this chapter closely mimic both the clinical course and histopathological features of MS.

Based on immense numbers of studies in EAE and descriptive observations in human MS, a condensed sequential immunopathogenesis of MS can be described as follows. For unknown reasons CNS autoantigen-reactive T cells become activated in the periphery (lymphoid organs/systemic circulation). Upon reaching the endothelium at the blood–brain barrier secretion of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) cause endothelial upregulation of adhesion molecules such as intercellular adhesion molecule (ICAM) and vascular cell adhesion modules (VCAM). The latter seem of special importance as demonstrated in EAE.3 The very late activation (VLA)-4–VCAM interaction allows activated T cells to pass into the CNS parenchyma, and upon rerecognition of a specific CNS autoantigen the T cells become reactivated with secretion of proinflammatory cytokines, inducing a further inflammatory cascade with recruitment of macrophages, chemokine secretion, etc. There is also a potential role for myelin-specific autoantibodies binding to myelin with destructive potential in the form of complement-mediated lysis or opsonization for macrophage attack. Most likely a series of downmodulatory events later takes place involving an endogenous steroid response and cytokines such as tumor growth factor-β (TGF-β) and interleukin 10 (IL-10). One of the few experimental situations we have in human disease is during clinical trials against specific targets. The most effective treatment against MS hitherto demonstrated is blockade of VLA-4 with a humanized monoclonal antibody, reducing relapses by 68% and new MRI lesions by 90%. This is striking evidence in support of the idea that inflammation causes MS pathology, and not the opposite, which occasionally has been suggested. For this reason, experimental models built on induction of a systemic immune response attacking the CNS are relevant for MS pathogenesis. Apart from a purely immunological pathogenesis, there is a growing interest in target-related factors. Thus, the CNS structures in the form of the blood–brain barrier, glial cells, and neuron axons take part in the pathogenesis of the disease, and there may well be genetically regulated differences in the function of these structures upon an inflammatory insult potentially explaining interindividual differences in outcome. For example, certain patients seem to do well despite intense inflammation in the CNS, while others suffer from severe sequelae after less intense inflammatory insults. It is likely that there is an important neurodegenerative aspect of MS in the chronic progressive forms of the disease. THE GENETICS OF MULTIPLE SCLEROSIS Since the genetic background is well known to profoundly affect immune responses in general and also rodent MS models, we here provide a short note on what is known about the genetics of MS. Furthermore, if the particular genes disposing for MS can be found, they will denote primary causative pathogenic pathways, which will pave the way for therapy and prevention. As discussed below, rodent MS models may assist in the finding of such genes. There is solid evidence for genes affecting the risk and course of MS, which is particularly clear from observations in twins, with an approximate concordance rate of 30% in monozygotic twins versus 2–4% in dizygotic twins. The human leukocyte antigen (HLA) complex is the best established and proven genome region regulating MS. It contains around 200 genes, most of which are involved in immune

CHAPTER 56 / RAT MODELS OF EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS

functions. Mapping of influences within the complex is difficult due to a strong linkage disequilibrium between genes in the complex. However, there is strong evidence that the class I and II genes are involved. Different alleles of HLA class II genes may either dispose for or protect against MS.4 Class I genes also affect MS.5 The basic reasons for these effects are not known, but current knowledge strongly suggests that the influence is related to the basic function of the HLA molecules, which is to bind antigenic peptides and present them to T cells. It can be argued that knowledge concerning those genes important for MS has been pivotal in the current focus on new therapies for MS directed toward interfering with T cell function. The major histocompatibility complex (MHC; in humans equal to the HLA) molecules are among the most polymorphic genes existing in mammals, allowing a broad repertoire of peptides at the population level to be presented to the immune system; this in turn helps ensure that infectious agents do not develop strategies to escape host immunity. Thus, preferential or promiscuous binding of myelin autoantigen peptides is an explanation for the MHC regulation of MS. In this context it is worth noting that these phenomena have instrumental impact on the handling of the experimental models discussed below. Non-MHC genes also regulate MS. However, several linkage screens have failed to demonstrate any genome regions apart from the HLA complex with major impact on MS. This in turn suggests that many genes are involved and each has a low or modest impact, which in turn has impeded their positioning. Present speculation suggests odds ratios in the range 1–2. Only a few non-MHC genes, such as the PRKCA and IL-7 receptor and a chemokine gene cluster,6–9 have more definite evidence of support. The outcome of human linkage screens discussed above as well as gene mappings in rodent models suggest that the number of genes with some degree of influence on MS may be around a hundred.10 This non-MHC genetics may also have relevance for experimental modelling of MS, especially when MS genes are better defined. It also relates to the probable genetic heterogeneity of MS, that is, different genes may predispose for the same end phenotype in the form of MS, and thus potentially result in very different mechanisms. This may require different therapeutic strategies. In the future it may be possible to utilize genetic information for tailor-made therapy, in which individuals/families with particular sets of MS-disposing genes accordingly receive adapted therapies. The non-MHC genetic background of different rodent strains is thus important to consider when evaluating the outcome of an experiment or experimental therapy. With time pharmacogenetically defined strains may be at hand (see more below). EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS: HISTORICAL NOTES AND CURRENT DEBATE EAE historically emanated from research devoted to understanding the cause of neuroparalytic accidents of rabies vaccination. Some individuals developed an acute or subacute encephalomyelitis weeks after the rabies vaccination. The virus was grown in brains and the inoculum was contaminated with CNS material. Experiments by Rivers and Schwentker tested the brain material alone, which gave rise to encephalomyelitis in a proportion of monkeys. Later, introduction of Freund’s adjuvant led to a much more reproducible disease induction. This experimental disease reflects acute disseminated encephalomyelitis (ADEM) rather than MS, which

549

has a chronic relapsing course. As described below, there are now a series of chronic relapsing models that better mimic MS. For more than 50 years there has been continuous debate on EAE as a model for MS, relating to different views on the presumed etiology, the predictability of data in the models versus validity for MS, and the “spontaneous” occurrence of MS versus the need for active induction in rodents. John Alvord, Jr., one of the pioneers in research on EAE and myelin basic protein, in 1984 presented a graph charting the ups and downs in his belief in EAE as a model for MS11; this varied from close to 100% when myelin basic protein (MPB) was found to be a discrete encephalitogen to 0% when antimeasles antibodies were found in MS cerebrospinal fluid, suggesting measles as the cause of MS. In 1984 he was 80% convinced that EAE was a good model for MS. Recently at least three studies have addressed these issues. First, a negative study argued that therapies developed in EAE do not work in MS.12 An opposing view was presented by Steinman and Zamvil,13 who argued that at least two therapies in MS, glatiramer acetate and anti-VLA-4 blockade, are completely based on EAE experiments and are of therapeutic use in MS. An intermediate view was presented by Ransohoff.14 It is often argued that EAE is not a good model for MS since it needs to be induced, whereas MS is spontaneous. However, twin data and epidemiological data strongly suggest that MS is also induced, or triggered by so far unknown events (infection is a common speculation). In particular, the 70% discordance rate in monozygotic twins suggests triggering by environmental factors.

PRINCIPAL MODELS OF EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS AND A NOTE ON THE SELECTION OF SPECIES EAE can be induced in practically all species, and most commonly in the mouse or rat. For very select questions nonhuman primate models may be used, for example, when testing reagents that are specific only for primates including humans. The mouse species has a number of advantages: it is small and therefore inexpensive to keep and breed, accessible for gene deletions, which is not yet possible in the rat, and transgenic expression of genes is easier. For genetics, the mouse displays a full genome sequence accessible on the internet, with single nucleotide polymorphism (SNP) and microsatellite maps. However, in our experience the models accessible in the mouse have been less reproducible and there is a paucity of chronic mouse models closely mimicking the course and histopathology of MS. In this chapter we deal only with rat models, with which we have extensive experience. Here, a series of easily reproduced acute and chronic models are at hand. The rat genome sequence is now also available on the internet (ensemble.org), there is a microsatellite map, and SNP maps will appear. There is also access to a wide variety of EAE-susceptible and resistant inbred strains. Well-controlled acute monophasic as well as protracted relapsing EAE forms are at hand. Many of these models are now routinely used by the pharmaceutical industry in evaluating new drug principles. A common problem in such experimentations is that drugs developed to fit human structures, irrespective of whether the drug is a monoclonal antibody, soluble receptor structure, or a low-molecular compound, often either have no or much lower binding affinities

550

SECTION V / MODELS OF BEHAVIOR

to the rodent target. The solution to this is either parallel development of rodent-specific compounds or transgenic expression of the human target in the rodent. The following induction methods can be discerned in principle. Adoptive Transfer Experimental Autoimmune Encephalomyelitis That EAE had a central T cell-mediated pathogenesis was demonstrated in the early 1980s.15,16 Myelin antigen reactive T cells were recovered from actively immunized mice or rats, cultured in vitro in the presence of stimulating antigen, and after several rounds of in vitro stimulation monospecific T cell lines and clones were obtained that upon transfer to naive syngeneic recipients caused EAE, in most cases with an acute monophasic disease course, with widespread inflammation in the CNS, but with little or no demyelination. This methodology has had enormous impact on the understanding of certain aspects of autoimmune disease, especially on questions such as T cell trafficking and the understanding of central features of T cell biology important for neuroinflammation. This form of EAE is therefore useful in studies of similar types of questions, but perhaps not in situations in which a disease course and pathology closely mimicking MS is required. We will not describe this form of EAE further. Cotransfer Experimental Autoimmune Encephalomyelitis For studies of the additive or synergistic role of autoantibodies, research groups invented cotransfer models, in which autoimmune T cells were transferred along with myelin-specific antibodies. For example, in this way it was demonstrated that antibodies alone did not cause disease, but required the elicitation of CNS inflammation by the T cells.17 Consequently this type of methodology can be used to address similar questions on the role of antibodies. We will not discuss this methodology further. Actively Induced Experimental Autoimmune Encephalomyelitis The experimental animal is immunized with a CNS autoantigen, in most cases together with an adjuvant (mineral oil + mycobacteria, and sometimes also injection of pertussis toxin). This recruits an autoimmune T and B cell response causing EAE with either an acute monophasic or chronic relapsing course, depending on the autoantigen and/or the strain of rodent used. Detailed descriptions of this methodology will be provided below. Certain viral infections in rodents, such as Theiler’s murine encephalomyelitis, are accompanied by recruitment of an autoimmune response, where there is strong evidence to support the idea that this autoimmune response may be responsible for the chronic relapses that ensue.18 Transgenic Induction of Experimental Autoimmune Encephalomyelitis With recent still more fine-tuned, elaborate use of transgenic techniques, “spontaneous EAE” may occur. Thus mice are constructed in which a high proportion of the T cells expresses human T cell receptors for myelin autoantigens. The mice can be further manipulated to express human MHC molecules and T cell costimulatory molecules. In addition, they can transgenically express B cells producing myelin-specific antibodies. All these mouse variants, to a variable extent, display spontaneous EAE, which interestingly also varies depending on the laboratory environment in which they are kept. Naturally, these types of mice, although highly artificial, can be used for mechanistic studies.19–21 It is a semantic question as to whether these forms of EAE should be regarded as spontaneous or induced.

We think that the genetic manipulation represents a form of active induction. Potentially, these mice could also be interesting to explore with regard to environmental triggers that may precipitate disease in extremely susceptible rodents. We will not explore these EAE models further. ACUTE MONOPHASIC OR CHRONIC RELAPSING EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS One of the oldest and best characterized EAE models is induced in LEW rats by subcutaneous immunization with MBP (derived from guinea pigs), or MBPgp 63–88 peptide, emulsified in complete Freund´s adjuvant (mineral oil and heat-killed Mycobacterium tuberculosis). The first clinical signs of disease appear approximately day 9–10 postimmunization (pi) in the form of sudden weight loss. By approximately day 10–11 pi tail weakness is evident, progressing to hind leg weakness and eventually complete paralysis of the hind legs at day 12–13 pi A recovery phase follows, with complete (or nearly complete) gain of hind leg and tail function at day 16–17 pi. A small number (0–10%) of animals may have a second relapse around day 22–24 pi, but otherwise they stay healthy, and are resistant to further attempts to induce EAE.22 Similar to MS, inflammatory infiltrates in the CNS are most common around small veins, but in the MBP-induced LEW rat model, lesions are found only in the spinal cord (they develop caudally to rostrally and parallel clinical signs of paresis) and not in the brain. At affected levels in the spinal cord, the whole parenchyma is infiltrated by CD4+ α-β cells and activated macrophages. Some CD8+ α-β cells, NK cells, γ-δ T cells, and B cells may also be present, and there are signs of activation of CNS resident cells (especially microglia).23 Most other EAE models are “variations on the theme” described above, but may include features such as a high frequency of relapsing disease and/or demyelination. In contrast to MBP-induced EAE in the LEW rat, rMOG (aa 1–125)-induced EAE (MOG-EAE) in DA rats displays both a high frequency of relapsing disease and focal inflammation with demyelination in the CNS.24,25 Clinically, relapsing-remitting signs of neurological dysfunction characterize MOG-EAE. However, there are also rats displaying chronic (nonremitting) signs of disease, acute lethal disease, or mild, very late clinical disease [occasionally DA rats have displayed no signs of clinical disease until day 50–60 postimmunization (unpublished observations)]. Therefore, MOGEAE is similar to MS in its diversity in individual clinical courses. Rats may present with only one lesion in the optic nerves and one lesion in the spinal cord, mimicking the MS subtype Devic´s disease. Active demyelination is associated with the deposition of immunoglobulins and complement component 9 (C9), indicating antibody-dependent demyelination. Immunologically, there are signs of activation of both cellular and humoral anti-MOGspecific lymphocytes. This is again similar to MS, where both T and B cell responses to MOG and other myelin antigens are present.26,27 Due to the gradual release of antigen in actively induced disease, it is difficult to determine whether “chronic” EAE can be considered “truly chronic.” If chronicity is defined as a “self-perpetuating” condition in which signs of disease continue indefinitely even after the removal of the initial trigger, then actively induced EAE is not proven to be chronic. Another problem with chronicity in EAE is the occurrence of irreversible neurological deficits. It is not difficult to devise a situation in which an acute inflammation causes irreversible neurological deficits due to

CHAPTER 56 / RAT MODELS OF EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS

destructive lesions in the CNS. Signs of disease may then persist indefinitely after the disappearance of the acute inflammation.

PRACTICAL CONSIDERATIONS CENTRAL NERVE SYSTEM AUTOANTIGEN Autoantigens for EAE-inductions may be purified (myelin) proteins, recombinant proteins, synthesized peptides, or even whole spinal cord and brain homogenates. There are pros and cons with all of these approaches. Purified myelin components may contain contaminations from other myelin proteins and a mixture of antigens is obviously also the case when whole spinal cord homogenates are utilized. Recombinant proteins circumvent the problem of contamination by other myelin proteins, but introduce potential immunologically active contaminations from the production system (often bacteria). Peptide-induced EAE may be complicated by the nonphysiological tertiary structure of peptides28 and nonphysiological epitope selections.29 If a relapsing/remitting disease course is desired, rMOG seems to be the most consistent antigen to use, particularly with DA and LEW.AV1 rats. rMOG here denotes recombinant MOG aa 1–125 (the N-terminal Ig-like extracellular domain), since full-length MOG is difficult to express due to its low solubility. If disease is induced by MBP peptides, monophasic EAE usually ensues.30,31 In adoptive transfer EAE, the antigen specificity of the transferred cells determines the topography of the CNS lesions and the extent of both parenchyma inflammation and macrophage activation.32 Thus, the choice of antigen determines both clinical and histopathological disease expression. DOSE OF ANTIGEN Although rarely discussed in studies on EAE, the dose of antigen may dramatically affect disease expression. Usually, titration experiments with three or four doses of antigen are necessary to set up experimental conditions before the actual experiment is started. In MOG–EAE, a reduction of the amount of antigen in the immunization may result in conversion of an acute lethal disease to a chronic disease, or to no disease.24 Suggested immunization protocols are discussed in the section on Immunization below. ADJUVANTS The minimum adjuvant utilized in most EAE studies is incomplete Freund´s adjuvant (IFA). IFA is a mixture of mineral oils, and it has by itself a complex impact on the immune system, including triggering of oil-induced arthritis in DA rats.33,34 IFA protects the antigen from rapid degradation and thereby prolongs its exposure to the immune system. Macrophage phagocytosis may also be facilitated by a more particulate physical state of the antigen.35 Heat-killed Mycobacterium tuberculosis may be added to the IFA, resulting in complete Freund´s adjuvant (CFA).36 CFA is a classical adjuvant, and has been utilized extensively to boost both cell-mediated and humoral immune responses to antigens with which it has been emulsified. The downside of using CFA as adjuvant is that it adds complexity; mycobacterial antigens interact with the immune system at several levels and responses to mycobacteria are genetically regulated.37 In most EAE induction protocols, CFA is required to induce disease, while IFA induces protective immunity. The ability to induce EAE in DA rats with rMOG/IFA alone is therefore unique and reflects both the strong sensitivity to adjuvants in rats (especially DA rats)38,39 and the potent encephalitogenic properties of MOG. EMULSION The relation between encephalitogenicity and the size of the water/oil emulsion droplets in the inoculum has

551

been studied by Maatta et al.40 Increased encephalitogenicity was associated with the small size of the droplets (achieved by sonication instead of extrusion). A small droplet size corresponded to a large surface area, which preferentially contained antigen. In the extruded emulsion, the antigen was buried in the droplet interior. Even so, most EAE induction protocols do not favor sonication, and a proper emulsion is acquired by mixing the water/oil emulsion in a syringe, or rather two connected syringes. Glass syringes are preferable to plastic, since plastic syringes tend to “clog” from the emulsion. At the beginning of mixing, the emulsion is easily passed through the syringe, but after a while, the texture changes and it takes more force to compress the syringe. The emulsion is then ready to be used. However, before use, the emulsion may be tested by putting a drop in a beaker filled with water. The drop should not immediately dissolve in the water, but rather remain as a drop, or dissolve very slowly. IMMUNIZATION In our hands, subcutaneous EAE induction is less effective than intradermal immunization. However, it is not always easy to achieve a completely intradermal location, especially with a volume of 200 µl, which is a common injection volume. If CFA adjuvant is utilized, rather than IFA, the immunization must always be subcutaneous, to avoid local inflammation and formation of a wound at the site of injection. A needle diameter of 0.6 mm is most often our choice. Thinner needles are difficult to use due to the consistency of the emulsion. It is advisable to make extra emulsion (for 5–10 extra animals) to avoid having to repeat the procedure if spills or other mistakes occur during the emulsification or immunization. It is also important to avoid self-injections, since it may trigger neuroinflammatory disease in certain individuals. We are aware of one such case in which the needle penetrated the skin of the hand with a minimal deposition of inoculum, and the person developed a subacute EAE-like disease. We therefore recommend a minimal surgical excision of the injection site if self-injection indeed occurs. STRESS An old, but seldom systematically studied observation is that nonspecific stress may profoundly suppress EAE. When designing EAE experiments, care should be taken to avoid disturbances such as noise, changes in animal caretaker staff/ routines, and experimental manipulations such as blood samples. Stress effects seem to be most pronounced before the appearance of clinical signs of EAE.41,42 It has been suggested that differences in neuroendocrine function may constitute part of the non-MHC regulation of strain susceptibility/resistance in EAE.43 SEASONAL EFFECTS Seasonal effects are not well recognized in the EAE literature, but there are some reports. In 2004, Teuscher et al. reported increased susceptibility in mice immunized during the summer.44,45 In contrast, our own experience is that EAE susceptibility (and fertility) in rats is reduced during the summer months (unpublished observation). It is unclear if the difference is due to the different types of animals or if the local environment is more important. AGE AND WEIGHT Very young animals with immature immune systems differ in their response to EAE induction.46 We avoid using rats weighing less than 150 g. In adult animals, age may be a factor of importance, in turn intimately linked to weight. Weight is important mainly with “mild” induction protocols, i.e., low doses of rMOG in IFA, while induction protocols with high doses of autoantigen and adjuvant are less sensitive to differences in animal weight. Since age-dependent weight increases differ in different rat strains, it is particularly important to consider weight

552

SECTION V / MODELS OF BEHAVIOR

when comparing different strains. Among the common strains utilized in rat EAE experiments, LEW and PVG rats gain weight faster than DA and ACI rats. Also, male rats gain weight faster than female rats, so all gender comparisons need to consider this factor. GENDER The impact of gender differs depending on the EAE model studied. In monophasic LEW rat EAE, induced by guinea pig spinal cord homogenate, males are usually more susceptible than females.47 In SJL mice injected with PLP 139–151specific T cells, female donor cells mediated more severe EAE than male donor cells.48 In our own experience, monophasic MBP or MBP peptide-induced EAE is more severe and reliable in males, while MOG-EAE is more severe and reliable in females.24,49,50 Mechanisms for gender influences in EAE may include direct hormonal effects on T cells and/or regulation by gender-specific non-MHC loci.51,52 The Y-chromosome itself has been implicated as harboring such loci in mouse EAE.53 In MS, the female-to-male ratio is 2 : 1.54 BREEDER AND SUBSTRAIN Inbred strains are, by definition, genetically homogeneous. However, if identical inbred strains are bred separately for several generations, accumulation of spontaneous mutations (“genetic drift”), residual heterozygosity, and/or accidental contamination by genetic material from other rat strains may well result in the establishment of “substrains.”55,56 Substrains are well known to differ in their susceptibility to EAE (and other autoimmune diseases).57 Among the strains commonly utilized in EAE studies, LEW and BN rat substrains differ in their susceptibility to EAE, while there has been fewer problems regarding substrain differences in DA, PVG, and ACI rats. MAJOR HISTOCOMPATIBILITY COMPLEX AND NONMAJOR HISTOCOMPATIBILITY COMPLEX GENES Classically a series of different rat strains, differing in both their MHC and non-MHC genes has been divided into resistant or susceptible strains, mainly based on susceptibility to MBP or whole spinal cord-induced disease. First, resistance or susceptibility should be used as relative terms since use of stronger immunization protocols can overcome genetic barriers. Second, both features may reside in both the MHC complex and among non-MHC genes. As an example, the classical MBP EAE-resistant strain, the BN rat, is relatively resistant to EAE due to both non-MHC genes and an MBP nonresponder MHC haplotype.58,59 However, the BN rat MHC (RT1N) is strikingly responsive to MOG, as demonstrated with RT1N on the EAE-susceptible Lewis non-MHC background.24 In the following section we discuss these matters in some detail.

WHY IS THE RAT STRAIN CRUCIAL IN SETTING UP AN EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS MODEL? MAJOR HISTOCOMPATIBILITY COMPLEX GENE IMPACT ON RAT EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS The influence of the MHC gene on EAE was recognized in the early 1970s,60 as well as the influence of the HLA gene on MS.61–63 The full genetic composition and distribution of rat genes are known in detail.64 There are about 200 genes in the rat species, with the region divided into classical class I

(Ia), class II, class III, and nonclassical class I (Ib) genes, situated in that order on rat chromosome 20. The nomenclature for the rat MHC is RT1, in which RT1A corresponds to classical class Ia genes, which corresponds to HLA A in humans. RT1B denotes class II genes corresponding to human HLA-DQ and mouse H2A. The rat RT1D corresponds to HLA-DR and mouse H2-E. Rat strains can be selected according to their MHC, based on their response to particular autoantigens (Table 56–1). Thus, there are different MHC restriction patterns depending on the particular autoantigen used for immunization. It is possible to select inbred rat strains with varying MHC on different genetic backgrounds, either with a relative non-MHC resistance or susceptibility. In addition, there are strains with recombinations within the MHC providing options for a rough positioning of MHC regulation within the MHC complex. Use of such rats has positioned the major EAE regulation to the class II genes, with additional influences from other parts of the MHC.24,5865 Mechanistically, the influence of class II is perhaps most easy to understand, especially with regard to peptide-induced EAE. Starting with the most classical rat EAE, induced with MBP in the Lewis rat (RT1L), immunodominant stretches of the molecule have been defined. These comprise the MBP 63–88 region and the 89–101 region. Classically heterologous MBP of guinea pig origin has been most effective in inducing EAE in the Lewis rat. Interestingly, on the RT1U haplotype the rat sequence is as effective.66 There is only a one amino acid difference in the core region of the peptide-binding site at position 79, by a single exchange of threonine (T) to serine (S). Despite the heterologous character of this guinea pig peptide, the fact that disease ensues must mean that recruited T cells recognize endogenous rat MBP. With these peptides, the use of intra-MHC recombinant strains between responder and nonresponder MHC has resulted in the mapping of major influences to the class II genes.59 Use of peptidebinding assays has demonstrated that RT1B molecules restrict the response to the 63–88 peptide, while RT1D restricts the MBP 89–101 response.67 Similar studies with MOG peptides on a series of rat MHC haplotypes have shown that MOG 91–108 is dominantly encephalitogenic.68 Since the basic function of class II genes is to present peptides to CD4+ T cells, the regulation of class II MHC is likely to depend on differences in the peptide-binding abilities of the different class II variants. Peptide-binding studies with purified class II molecules from different MHC haplotypes have shown that dominantly immunogenic peptides in the rat bind with high or moderate affinity.67 Peptides that bind poorly tend to be nonimmunogenic. This may be a difference compared to one example in the mouse in which the encephalitogenic MBP 1–10 is a poor class II binder. In addition, only a fraction of immunogenic peptides in the rat is encephalitogenic, suggesting that there are additional factors, perhaps in the quality of a class II-regulated immune response, that determine pathogenicity of the ensuing immune response, a topic that still is unresolved in autoimmunology. Use of intra-MHC recombinant rats has also evidenced a function of the class I-CD8+ T cell pathway in EAE, in which certain alleles may recruit a protective disease downregulatory immune response.65 Less well studied are influences from the class III region on EAE. However, there are indications that genes in this region may influence histopathological features of MOG EAE, such as the degree of axon loss and demyelination.69

CHAPTER 56 / RAT MODELS OF EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS

553

Table 56–1 Selected EAE models: strains, MHC haplotypes, and autoantigensa,b Strain

MHC Haplotype

Autoantigen

Type of EAE

Comment

DA

AV1

rMOG(1–125) MBPrat69–87 MBP 87–101 PLPbovine Spinal cord homogenate MBPgp63–88 MBPrat63–88 MBP 89–101 rMOG(1–125) PLPbovine rMOG(1–125) rMOG(1–125) MBPgp63–88 MBP 89–101 MBP 87–110 rMOG(1–125) MBPgp63–88 MBPrat63–88 rMOG(1–125) rMOG(1–125) rMOG(1–125)

Relapsing Protracted in some rats Protracted in some rats Severe EAE Prolonged, relapses

MS-like model

LEW

L

LEW.AV1 LEW.N

AV1 N

LEW.W

U

PVG PVG.AV1 ACI

C AV1 AV1

Monophasic Monophasic Monophasic Resistant 20 million years, since before the first humans evolved. It remains unclear how hantavirus exist within a rodent reservoir, particularly how they establish a persistent infection. In experiments with laboratory rats and mice, several groups have shown that an experimentally infected newborn animal readily develops a persistent infection, whereas an adult animal develops only a transient infection and recovers completely. On the other hand, epizootiological investigations have demonstrated that virus is transmitted between adult animals through wounds, and the adults develop a persistent infection. EBOLA VIRUS Ebola virus is a nonsegmented RNA virus, which, together with Marburg virus, makes up the filovirus family. This now notorious group of viruses was discovered in 1967 when Marburg virus was identified as the etiological agent of a hemorrhagic fever outbreak in research facilities in Europe, which handled tissues from African green monkeys imported from Uganda. Subsequently, Ebola viruses were shown to be the cause of simultaneously occurring hemorrhagic fever outbreaks in 1976 in the Democratic Republic of Congo (DRC, formerly Zaire) and Sudan. These outbreaks were shown to be caused by two different subtypes of Ebola virus, which became known as the Zaire and Sudan subtypes. Mortality rates of up to 80% were recorded in these and more recent outbreaks in DRC and Gabon in 1995– 1996. Epidemiological data from recent outbreaks indicate that close contact is necessary for efficient transmission of Ebola virus from one individual to another, and little evidence can be found for aerosol transmission of the virus. Despite considerable efforts to identify the natural reservoir for Ebola and Marburg viruses, the host species remains an enigma. Although nonhuman primates have been implicated as the source of the introduction of the virus into humans during several of the identified outbreaks, they are not considered likely to represent reservoir species because of their susceptibility to high-mortality hemorrhagic disease similar to that seen in humans. NIPAH VIRUS Nipah virus is a newly discovered member of the paramyxovirus family of nonsegmented RNA viruses. This virus was responsible for a viral encephalitis outbreak in Malaysia that was first recognized in October 1998 and ended in midsummer

561

1999. This outbreak resulted in almost 300 confirmed infections, and the mortality rate for hospitalized cases was approximately 35%. Initially, Malaysian authorities thought the outbreak was caused by Japanese encephalitis (JE virus, a mosquito-borne RNA virus). However, JE vaccination and mosquito control efforts failed to halt the epidemic. The virus appeared to be first introduced into pigs, where close contact caused by intensive farming practices led to efficient pig-to-pig transmission, and subsequently pig-to-human transmission. Most human cases were in close proximity to the infected pigs. Genetic analysis showed Nipah virus to be closely related to Hendra virus, which recently was discovered in Australia as a cause of disease in horses and humans and also is maintained in Pteropus species fruit bats. These examples highlight the subtle balance of environmental and genetic factors that can mold the diverse evolutionary patterns observed for RNA viruses and illustrate the complexity of these systems, which makes it difficult to predict future viral disease emergences. Much more research is needed and animal models will continue to be needed to ultimately understand and control these diseases.

RELEVANT ANIMAL MODELS Use of animals as models for human disease has been indispensable in understanding the cause, biology, and prevention of disease. The Animal Model Division at the Institute of Human Virology has developed several relevant animal models, particularly for the study of AIDS and AIDS-associated cancers. Animal models are required for the study of human diseases. However, the relevance of small animal models such as mice to natural human in vivo physiological and metabolic kinetics remains unclear. Large animal species may provide far more appropriate preclinical models that will more closely reflect human physiological characteristics and behavior. Among large animals, nonhuman primates may provide the best models because of their close phylogenetic relationship to humans It is essential to develop animal models for human diseases to reveal its mechanisms and to develop new therapeutic interventions. Led by the author of this review at the Institute of Human Virology, the Animal Models Division is a unique feature of the Institute, enabling scientists to work with relatively inexpensive models to study AIDS and new drugs or therapies without risk to humans. Developing animal subjects for use in viral research is a science unto itself, and it is essential in taking a discovery from laboratory to clinic. The use of animals as models for human disease has been indispensable in understanding the causes, biology, and prevention of disease. “To my knowledge, almost all major scientific successes on unraveling and conquering human diseases have been with the use of animal models. AIDS and AIDS-associated diseases, will be no exception” (J.L. Bryant, personal communication).

VIRAL DISEASE IN SEARCH OF AN ANIMAL MODEL Hepatitis viruses belong to different families and have in common a striking hepatotropism and restrictions of propagation in cell culture. Viral hepatitis represents a global public health problem. Over the past 20+ years, the chimpanzee model has served as the backbone for advancements in the hepatitis C virus (HCV) research field. Despite this remarkable progress, the chimpanzee model has some important disadvantages. Perhaps most

562

SECTION V / MODELS OF BEHAVIOR

importantly, chimpanzees are rare, expensive, and difficult to handle. Limitations to the chimpanzee model have stimulated progress toward developing alternative animal modes for HCV research. Transgenic technology, coupled with the relative ease and low cost by which mice can be reared and maintained, along with the availability of inbred mouse strains, have made the laboratory mouse an attractive animal model for HCV research. For some human viruses [e.g., hepatitis B (HBV) and HCV], no satisfactory cell culture or animal model exists, and in these cases, inhibition of an essential viral function or activity against related viruses can be used to indicate potential activity. When no satisfactory cell culture or animal model exists for the target human virus, it is particularly important to know whether a drug’s active moiety enters cells, if it has a proposed intracellular site of action, and if the intracellular concentration is consistent with biochemical studies to identify an inhibitory concentration. Cell-based assays and host cell lines for studying viruses such as HBV and HCV replication may advance and improve, but at the present time are limited. For analysis of HCV replication, a replicon system has been developed that permits studies of viral replication and can be used to assess antiviral activity of some anti-HCV drugs. More than 200 million people worldwide are now believed to be infected with HCV, including 4 million individuals within the United States. HCV accounts for the deaths of at least 8000 to 10,000 Americans each year and is now one of the most common indicators for adult liver transplants in developed countries. Before the introduction of anti-HCV screening in mid-1990, HCV accounted for 80–90% of new cases of posttransfusion hepatitis in the United States. Currently, injection drug use is probably the most common risk factor for HCV infection, with approximately 80% of this population seropositive for HCV. A high rate of HCV infection is also seen in individuals with bleeding disorders or chronic renal failure, groups that have had frequent exposure to blood and blood products. HCV is a global public health problem, with approximately 3% of the world population now infected. The clinical course of HCV often involves chronic infection, which can lead to liver dysfunction and hepatocellular carcinoma. Because HCV cannot be efficiently propagated in cell culture, research has relied heavily on animal models to study the physical characteristics of HCV and the course of events associated with HCV infection. The chimpanzee is the only nonhuman primate actually proven to be susceptible to HCV infection and has commonly been used to study viral hepatitis induced by HCV. Molecular cloning of the HCV genome has now allowed HCV transmission studies in chimpanzees to progress from the early work of characterizing infectious serum to a current focus of characterizing infectious HCV molecular clones. Moreover, the cloned HCV genome has paved the way for the development of alternative animal models for HCV, most notably transgenic mouse models for the study of HCV pathogenesis.33 The expression of specific viral protein products in these animal models will provide important insights into the structure–function relation that specific HCV genome sequences impart on virus replication and pathogenesis. Despite this remarkable progress, the chimpanzee model has some important disadvantages. Perhaps most importantly, chimpanzees are rare, expensive, and difficult to handle, and must be housed and cared for in appropriate nonhuman primate research facilities. Such research facilities must possess proper surgical support and specialized veterinary care. Moreover, the chimpan-

zee has been listed as an endangered species since 1988, and appropriate safeguards must be considered whenever selecting such a model for research. These and other limitations to the chimpanzee model have stimulated progress toward developing alternative animal models for HCV research. Transgenic technology, coupled with the relative ease and low cost by which mice can be reared and maintained, along with the availability of inbred mouse strains, have made the laboratory mouse an attractive animal model for HCV research.34 An HCV infection cannot be propagated in mouse tissues, which obviously limits the research application of mouse models. However, expression of the HCV genome or subgenomic fragments of HCV within inbred strains of mice has demonstrated the utility of mouse models for research geared toward understanding mechanisms of HCV pathogenesis. Transgenic mice expressing HCV proteins are only beginning to provide insights into the pathobiology of HCV infection. From the studies presented, it is clear that expression of HCV proteins per se is not directly cytopathic to hepatocytes, even when expression levels are greater than those observed in HCV-infected individuals. This evidence supports the notion that the host immune response to HCV may play a significant role in HCV pathogenesis. The use of conditional transgenic expression systems, such as the Cre/LoxP system, offers exciting prospects to study the immune-mediated mechanisms of HCV-related liver injury in transgenic mice. However, the recent development of mice immunocompetent for the HCV proteins using this system awaits full characterization. It is clear that in some transgenic mouse strains, expression of the HCV genome or the core protein alone results in the development of steatosis and HCC. How does the core protein induce these pathologies, and how may the other HCV proteins contribute to this phenotype? It is unclear whether this phenotype is a result of HCV protein expression directly or whether it occurs in combination with environmental or hostderived factors leading to chronic liver cell injury. Importantly, steatosis and HCC are relevant clinical manifestations seen in HCV-infected individuals. These transgenic mice should therefore play a pivotal role in elucidating the molecular mechanisms of HCV-related liver pathology.

CONCLUSIONS There should be no doubt that the knowledge gained form the study of viral diseases in humans in different animal species has enhanced our knowledge and understanding of the pathogenesis of the diseases in humans. One important component of the value of using animals in viral research is that the pooling of knowledge of diseases in different animal species has led to more rapid progress in understanding the pathogenesis of diseases. It was not the intent here to discuss comprehensively the contribution of animals in viral research but to historically acknowledge that they have played and will continue to play a major role in solving many health problems, including viral diseases in humans. A review of the literature clearly shows that depending on the training and background of the scientists concerning the use of animals in viral research, there are numerous differences of animal models in viral research. It is clear that the more knowledge the scientist has of diseases in animals the better the understanding of how a model can be used. For example, the direct application of knowledge of a disease in an animal, scrapie in sheep, has led to a better understanding of a human disease,

CHAPTER 57 / ANIMAL MODELS IN VIROLOGY

kuru. It was a veterinarian and pathologist, Dr. W. J. Hadlow, who through his experience with scrapie in sheep and goats noted the similarities of the histological lesions in the brains of kuru patients and those of sheep and goats. He suggested the inoculation of brain material from kuru patients into animals. Dr. Carlton Gajdusek, using chimpanzees, produced a fatal disease in these animals after a long inoculation period. This established a viral etiology for kuru and led to the prevention of the diseases. This is one of numerous examples of how applying the knowledge of veterinary medicine and human medicine can lead to the prevention and treatment of diseases caused by viruses. Our understanding of emerging and reemerging viruses from an evolutionary perspective offers clear directions to follow. If we seriously plan to develop efficient and reasonable control strategies, an international concerted effort involving a thorough survey of the genetic diversity of viruses in time and space is a must. This will also help in understanding several processes that take place at the molecular level in a comparative context. More emphasis on multidisciplinary sciences is needed. Molecular biology and systematics, bioinformatics, data mining, and analysis are all becoming considerably sophisticated lately. Hepatitis C and HIV-1 are glaring proof that disease knows no national borders. The use of and development of animal models for studying viral diseases of humans will continue to be a necessary part of biomedical research in understanding the current viral diseases that affect humans and the new emerging viruses.

REFERENCES 1. Ghiara JB, Stura EA, Stanfield RL, Profy AT, Wilson IA. Crystal structure of the principal neutralization site of HIV-1. Science 1994;264(5155):82–85. 2. Gottfredsson M, Bohjanen PR. Human immunodeficiency virus type I as a target for gene therapy. Front Biosci 1997;2:619–634. 3. Drotman DP. Emerging Infectious Diseases. Vol. 12, No. 11, November 2006. www.cdc.gov/eid. 4. McFadden G. Smallpox: An ancient disease enters the modern era of virogenomics Proc Natl Acad Sci USA 2004;101(42):14994–14995. 5. Campo MS. Animal models of papillomavirus pathogenesis Virus Res 2002;89(2):249–261. 6. March RF. Animal model of human disease: Kuru, Creutzfeldt-Jakob disease (slow virus infections). Animal model: Transmissible mink encephalopathy, scrapie-like disease of mink. Am J Pathol 1972; 69(1):209–212. 7. 19th annual UCLA symposium: Animal models of human viral diseases: Relevance to developmental therapeutics. J Cell Biochem Suppl 1990;14D:41–75. 8. Levy JA. The value of primate models for studying human immunodeficiency virus pathogenesis. J Med Primatol 1996;25(3): 163–174. 9. Langley RJ, Prince GA, Ginsberg HS. HIV type-1 infection of the cotton rat (Sigmodon fulviventer and S. hispidus). Proc Natl Acad Sci USA 1998;95(24):14355–14360. 10. Miller CJ, Alexander NJ, Sutjipto S, Lacker AA, Gettie A, Hendrikx AG, Lowenstine LJ, Jennings M, Marx PA. Genital mucosal transmission of simian immunodeficiency virus: Animal model for heterosexual transmission of human immunodeficiency virus. J Virol 1989;63(10):4277–4284. 11. Lackner AA, Smith MO, Munn RJ, Martfeld DJ, Gardener MB, Marx PA, Dandekar S. Localization of simian immunodeficiency virus. Am J Pathol 1991;145:428–439.

563

12. Letvin NL, Daniel MD, Seghal PK, Desrosiers RC, Hunt RD, Waldron LM, McKey JJ, Schmidt DK, Chalifoux LV, King NW. Induction of AIDS-like disease in macaque monkeys with T-cell tropic retrovirus STLV-III. Science 1985;230:71–73. 13. Cohen J. Building a small-animal model for AIDS, block by block. Science 2001;293(5532):1034–1036. 14. Nesbit CE, Schwartz SA. In vitro and animal models of human immunodeficiency virus infection of the central nervous system. Clin Diagn Lab Immunol 2002;9:515–524. 15. Avgeropoulos N, Kelley B, Middaugh L, Arrigo S, Persidsky Y, Gendelman HE, Tyor WR. SCID mice with HIV encephalitis develop behavioral abnormalities. J Acquir Immune Defic Syndr Hum Retrovirol 1998;18:13–20. 16. Gallo R. History of The Institute of Human Virology. http://www. ihv.org/aboutihv/index.html (personal communication), 2006. 17. Bennett M, Hart CA. Feline immunodeficiency virus infection-model for HIV and AIDS? J Med Microbiol 1995;42:233–236. 18. McCune JM. Animal models of HIV-1 disease. Science 1997; 278(5346):2141–2142. 19. Reid W, Sadowska M, Denaro F, Rao S, Foulk J Jr, Hayes N, Jones O, Doodnauth D, Davis H, Sill A, O’Driscoll P, Huso D, Fouts T, Lewis G, Hill M, Kamin-Lewis R, Wei C, Ray P, Gallo R, Reitz M, Bryant J. An HIV-1 transgenic rat that develops HIV-related pathology and immunologic dysfunction Proc Natl Acad Sci USA 2001;98(16):9271–9276. 20. Martina BE, Haagmans BL, Kuiken T, Fouchier RA, Rimmelzwaan GF, Van Amerongen G, Peiris JS, Lim W, Osterhaus AD. Virology: SARS virus infection of cats and ferrets Nature 2003;425(6961): 915. 21. Navas-Martin S, Weiss SR. SARS: Lessons learned from other coronaviruses. Viral Immunol 2003;16(4):461–474. 22. Prince AM, Brotman B. Perspectives on hepatitis B studies with chimpanzees. ILAR J 2001;42(2):85–88. 23. de Harven E. Viral etiology of human cancer: A historical perspective. Haematologica 1999;84(5):385–389. 24. Chen Z, Zhou P, Ho DD, Landau NR, Marx PA. Genetically divergent strains of simian immunodeficiency virus use CCR5 as a coreceptor for entry. J Virol 1997;71:2705–2714. 25. Klotman PE, Notkins AL. Transgenic models of human immunodeficiency virus type-1. Curr Top Microbiol Immunol 1996;206: 198–222. 26. Murakami M, Gurski KJ, Steller MA. Human papillomavirus vaccines for cervical cancer. J Immunother 1999;22:212–218. 27. Govorkova EA, Rehg JE, Krauss S, et al. Lethality to ferrets of H5N1 influenza viruses isolated from humans and poultry in 2004. J Virol 2005;79:2191–2198. 28. Chotpitayasunondh T, Ungchusak K, Hanshaoworakul W, et al. Human disease from influenza A (H5N1), Thailand, 2004. Emerg Infect Dis 2005;11:201–209. 29. Zitzow LA, Rowe T, Morken T, Shieh W-J, Zaki S, Katz JM. Pathogenesis of avian influenza A (H5N1) viruses in ferrets. J Virol 2002;76(9):4420–4429. 30. Martin G. Ottolini JC, Blanco G, Eichelberger MC, Porter DD, Pletneva L, Richardson JY, Prince GA. The cotton rat provides a useful small-animal model for the study of influenza virus pathogenesis. J Gen Virol 2005;86:2823–2830. 31. CDC—Influenza http://www.cdc.gov/flue/about/fluviruses.htm. 32. Hart CA, Bennett M. Hantavirus infections: Epidemiology and pathogenesis. Microbes Infect 1999;1(14):1229–1237. 33. Mercer DR, Schiller DE, Elliott JF, Douglas DN, Hao C, Rinfret A, Addison WR, Fischer KP, Churchill TA, Lakey JRT, Tyrrell DLJ, Kneteman NM. Hepatitis C virus replication in mice with chimeric human livers. Nat Med 2001;7:927–933. 34. Barth H, Liang TJ, Baumert TF. Hepatitis C virus entry: Molecular biology and clinical implications. Hepatology 2006;44(3):527–535.

58

Nonhuman Primate Models for AIDS NANCY L. HAIGWOOD, RAYMOND R. O’NEILL, AND VANESSA M. HIRSCH

ABSTRACT

private and government-sponsored research funding commitments in the United States, Canada, the United Kingdom, and Europe, in part due to misplaced optimism about the ease of developing HIV vaccines. Much of this funding was directed toward the discovery and development of appropriate animal models to study antiviral immunity and to evaluate vaccine candidates and strategies. Although natural lentiviral infections in nonlaboratory animal species are known, none develops into an AIDS-like illness. Attempts to create transgenic mice or rats that develop AIDS have been unsuccessful to date. Importantly, early research in academic laboratories and in industry did lead to anti-HIV, or antiretroviral (ARV) drug development in the absence of animal models. These early drugs targeted viral enzymes such as reverse transcriptase (RT) and protease (PR) with no human counterparts, and drugs could be screened for activity against the virus using in vitro enzymatic inhibition assays. Because this type of drug research did not require an animal model to demonstrate drug potency, nearly all of the ARVs in use at present were approved without animal model efficacy testing. With the development of PR inhibitors and their use in a cocktail of anti-RT inhibitors, patients could expect to experience significant, sustained control of virus replication and in many cases delay or prevention of opportunistic infections. These combination drug regimens were termed highly active antiretroviral treatment, or HAART.1 Various improved formulations and combinations of HAART are the current standard of care in the developed world, and there are continuing efforts to find economic mechanisms to make these drugs available worldwide. Despite success in developing HAART, the epidemic continues to spread worldwide through sexual transmission, primarily heterosexual, via mother-to-child transmission, and drug use via blood-contaminated needles. As with other epidemics, an important public health goal is to develop vaccines that can prevent the acquisition or limit the spread of a lethal or seriously debilitating infection. These vaccines, once discovered, can then be utilized in concert with campaigns to limit transmission via behavioral changes. AIDS vaccine research has been far more challenging than drug research due to a number of factors. First, HIV-1 is a lethal virus that persists in the host due to its integration into the genome. Therefore the use of human challenge models is prohibited, even after the development of relatively effective ARV drugs. Second, the virus targets many of the very immune cells that are needed to eliminate or control it, leading to death by

Since the discovery of acquired immune deficiency syndrome (AIDS) and the human immunodeficiency viruses HIV-1 and HIV-2 in patients 25 years ago, efforts have been directed toward identifying or developing appropriate models for this disease. Several species of nonhuman primates can be productively infected with primate lentiviruses HIV-1, HIV-2, SIV, and genetic chimeras of HIV and SIV (SHIV), and the ensuing pathology has many elements in common with HIV infection of humans. This chapter summarizes the key features of the nonhuman primate models that have been developed for AIDS research as of 2006. It focuses on (1) the major contributions and limitations of these models to our understanding of HIV infection and pathogenesis and (2) uses of the models to test prophylactic and therapeutic approaches to prevent infection and/or limit disease. It is intended as a reference for investigators in the field as well as for those considering utilizing the models to address specific questions in AIDS research. In an overview of this type, comprehensive referencing of studies is not possible, and key examples are included in an effort to cite some of the many important studies performed to date. Many excellent comprehensive and focused reviews on the subject of nonhuman primate models for AIDS have been written in the past several years, and readers are encouraged to consult these reviews for details, examples, and additional references. Key Words: HIV-1, HIV-2, SIV, SHIV, AIDS, Baboons, Chimpanzees, Macaques.

GENERAL CHARACTERISTICS HISTORY OF AIDS MODELS Since the discovery of the etiological agent of AIDS, human immunodeficiency virus (HIV), efforts to combat the HIV/AIDS epidemic have been focused on the development of vaccines and drug treatment, as well as on behavioral interventions. Because the disease is known to be a caused by a blood-borne virus, some of the earliest efforts were directed to developing tests for screening the human blood supply. Recombinant viral antigens that were developed for diagnostic tests were also tested for their suitability as early vaccine candidates. The early vaccine work was bolstered with significant

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

565

566

SECTION V / MODELS OF BEHAVIOR

infection with opportunistic agents. Thus, vaccines will need to be provided prior to or early in infection to stimulate maximal immunity before the loss of key immune cells. Finally, animal models described to date are imperfect and do not recapitulate all aspects of the infection. This is a particular irony, as the viruses HIV-1 and HIV-2 certainly were endemic to nonhuman primates before bridging the gap to productive infection of humans.2 Nonhuman primates were imported to the United States and other European countries from Africa and Asia during the 1960s continuing through the 1980s to provide animals for testing in cancer and other biomedical research. In the early 1980s, investigators observed that some of their Asian macaques that had been exposed to African sooty mangabey monkeys harboring their own lentivirus (SIVsm) were succumbing to a disease that had remarkable similarities to AIDS in humans. This disease was termed “simian AIDS.” The etiological agent was a blood-borne virus that showed morphological similarities to HIV-1 in electron micrographs and was designated SIVmac to indicate its origin in macaque monkeys. This fortuitous observation was the genesis of today’s models that are in wide use for vaccine,3–5 microbicide, transmission,6 pathogenesis and treatment,7,8 and behavioral research.9,10 As noted in the abstract, many fine reviews have been published to describe the models and their uses, and the reader is encouraged to pursue these for additional information. COMPARISON OF HUMAN AND SIMIAN LENTIVIRUSES In addition to animal passage of infectious blood and tissues, molecular cloning was a key technology in the identification of virus isolates for use in nonhuman primate studies.11,12 Fulfilling Koch’s postulate, it was demonstrated directly that blood from simian immunodeficiency virus (SIV)-infected macaques13 as well as cloned SIV was the causative agent of this nonhuman primate version of AIDS in Asian macaques.12 Like HIV-1, this SIV is a member of the lentivirus group of retroviruses, characterized by the presence of regulatory genes in addition to the standard retroviral gag, pol, and env genes. These viruses cause a persistent infection and a relatively slow disease course. It was subsequently demonstrated through molecular cloning of viruses from various African species including chimpanzees, sooty mangabeys, and African green monkeys that the SIVs are endemic to many African primates14,15 and are by definition well adapted, failing to cause disease in their natural host. While there are a variety of different SIVs that are classified based on their African primate of origin, this chapter focuses on the SIVmac/SIVsm strains that are most widely used in vaccine and pathogenesis research. This research to determine the etiology of the simian disease was performed in parallel with early work to establish an animal host for HIV-1 and HIV-2. Originally imported for research in other diseases, the macaque models arose by accidental exposure to other endemic viruses, as noted above. Some of these monkeys (sooty mangabeys) harbor an endemic nonpathogenic virus, SIVsm, which was transmitted to the Asian macaques.16 More comprehensive searches have shown that this group of viruses is widespread in many species of African primates. Painstaking field work to obtain samples and detailed phylogenetic analyses of viruses found in these samples17 have led to our current understanding of the sources of HIV-1 and HIV-2 in humans. HIV-1 arose by exposure of humans to a virus endemic in chimpanzees, and HIV-2 from similar exposure to macaques infected with SIV, most likely SIVsm.18 Susceptibility of specific cell types to viral entry by the nonhuman primate lentiviruses is enabled by the envelope glycoprotein, which binds to the cellular

receptor (CD4, found on monocytes, macrophages, and subpopulations of T cells) and one or more of the 7-transmembrane chemokine coreceptors (typically either CCR5 or CXCR4).19 HIV-1 laboratory isolates that are adapted for growth in tissue culture utilize CD4 and the chemokine receptor CXCR4, while primary HIV-1 isolates utilize CCR5.20 SIV is known to utilize CCR5 and potentially other coreceptors as yet uncharacterized.21 Mutations in the gene encoding CCR5 can confer partial or full resistance to HIV-1 infection in humans who are heterozygous and homozygous, respectively, for a 32-base pair deletion in gene that prevents expression of functional CCR5.22 Despite conservation of receptor and some coreceptor utilization, the primate lentivirus envelope proteins, while structurally similar, differ as much as 20% in sequence, limiting cross-protective immunity. Recent studies have identified two important restriction factors unique to different primate species that operate at a cellular level to inhibit HIV or SIV viral replication. These are APOBEC3G, which exerts a late block to retroviral replication,23 and TRIM5α, which exerts a dominant block to infection immediately after entry.24 Further studies of how these restriction factors limit the acquisition of the ancestors of our current human epidemic will no doubt cast light on changes that accrued during cross-species transmission and important adaptations of the current viruses to increase transmission to humans.25 SPECIES UTILIZED FOR AIDS RESEARCH Early work to develop an infectivity model focused on infection of chimpanzees (Pan troglodytes) with HIV-1,26 since they are the closest genetic relative of Homo sapiens. Although this species is readily infected with HIV-1 and there is a robust antiviral immune response, there was a puzzling lack of evidence for disease progression. Evidence for disease was found only recently in captive chimpanzees infected with one or more HIV-1 isolates for 10 years or more.27 In retrospect, this finding makes sense, as the viruses being used to infect the chimpanzees had been selected and adapted for growth and pathogenesis in humans. There was a significant effort in the 1980s to expand the number of captive chimpanzees available for AIDS research via breeding programs, since the U.S. laboratory population was severely limited in number, and additional importation from Africa was prohibited. However, even with increased breeding, the cost of performing experiments with even a small number of chimpanzees was prohibitive for all but a few groups. These studies were limited to testing approaches that could block infection, such as an early study that showed an envelope-specific monoclonal antibody directed to the third hypervariable region could block intravenous infection with HIVIIIB.28 Although baboons (Papio species) were not productively infected with HIV-1, they could be infected with HIV-2, and some efforts were directed at developing a pathogenic HIV-2 via passage in vivo.29 Macaque (Macaca nemestrina)-passaged HIV-2 led to a model characterized by high peripheral and tissue virus and rapid, irreversible loss of CD4+ T cells.30 Species that are susceptible to infection with SIV include Macaca fascicularis (cynomolgus), Macaca mulatta (rhesus), and M. nemestrina (pigtailed). An excellent discussion of the merits and limitations of each of these species can be found in the review by Hu.5 PATHOGENESIS In humans, HIV infection with a single or small number of variants quickly expands with viral replication to a group of genetically31 and biologically distinguishable viruses.32 Different virus–host combinations lead to differing degrees of pathology, with some of the equation dependent upon the virus and some dependent upon the host. Typically, macaques

CHAPTER 58 / NONHUMAN PRIMATE MODELS FOR AIDS

567

Table 58–1 Nonhuman primate models for human AIDS Virus

Animal

Comments

HIV-1

Chimpanzee

HIV-2

Pigtailed macaque Baboon Pigtailed macaque

Replicates but usually no disease, or disease in >10 years Chimps are limited in number, expensive, and require long-term care Very poor replication, not a useful model for disease Moderate replication, not a useful model for disease One pathogenic strain replicates to a high level and causes an acute CD4+ T cell loss (HIV-2–287) Various strains that induce AIDS in macaques (SIVmac251, SIVmac239mc, SIVsmB670, SIVsmE660, SIVsmE543mc, SIVsmm9, SIVsmPBj) Rhesus macaques of Indian origin the most frequently used model The majority of strains use the CXCR4 coreceptor expressed on naive T cells and cause an acute CD4+ T cell loss (SHIV-SF33, SHIV-KU1, SHIV89.6P, SHIV-DH12) One pathogenic strain uses the CCR5 coreceptor on memory T cells and causes more of an AIDS-like disease (SHIV-SF162)

SIVsm/mac Rhesus macaques Pigtailed macaques Cynomolgus macaques SHIV

Rhesus macaques

Pigtailed macaques

experience high viral loads and loss of CD4+ T cells within 6 months to 2 years of infection, a course that is significantly foreshortened compared with HIV-1 or HIV-2 in humans. In comparative infection studies with SIVsmE660, for example, the time to loss of CD4+ T cells and disease from opportunistic infection is significantly shortened in M. nemestrina compared with M. mulatta. Experiments with a shorter duration using host and virus pairs with high pathogenicity are less expensive and can provide greater throughput at primate centers. Therefore these models are frequently favored. A number of AIDS-inducing SIV isolates and molecular clones have been derived as detailed in Table 58–1. These include the SIVmac239 and SIVmac251 viruses originally described by investigators at Harvard, SIVmne from the National Cancer Institute and University of Washington research groups, and related strains SIVsmm9, SIVsmB670, SIVsmE543, and SIVsmE660 isolated by investigators at the Yerkes Primate Center and Tulane University.7,33 The pathogenesis of disease is similar between these viruses and is characterized by (1) an acute phase of high viremia in which rapid loss of mucosal CD4+ memory T cells occurs, (2) the development of cellular and humoral immune responses and a long, relatively asymptomatic period in which a gradual decline in immune function and loss of CD4+ T cells occurs, and (3) the development terminally of opportunistic infections. Most of these viruses also cause SIV encephalitis, which has provided a model for AIDS dementia.34 Loss of gut-associated lymphoid tissue (GALT) in SIV-infected macaques is a hallmark of pathogenesis35,36 and was shown prior to such studies in humans.37,38 Two important findings were derived from nonhuman primate studies: (1) the pathogenic nature of live attenuated vaccines in newborn rhesus macaques, despite lack of pathogenesis in juveniles;39,40 and (2) the reemergence of virus from vaccinated “protected” macaques after several years, underscoring the difficulty of detecting some infections41 as well as the permanence of lentivirus infection and the potential for lethal outcomes after years of control despite years of viral control.42–44

TRANSMISSION One of the most useful applications of the nonhuman primate models has been to explore the routes of infection by various isolates and clones. Early research involved intravenous inoculation of chimpanzees, baboons, or macaques with blood or infected tissues, and later infectious molecular clones. Each of the known routes of transmission in humans has been demonstrated in the monkey for both SIV45 and for SHIV46: oral,47 vaginal,48 rectal,49 and perinatal.6,50 The importance of hormonal changes in susceptibility of transmission could be explored in a controlled manner, demonstrating higher susceptibility during different stages of the menstrual cycle.51 Using these models, it has been possible to quantify the difference in infectiousness of various stocks by different routes, thereby showing that SIV and SHIV are approximately 1000-fold more infectious by the intravenous route than by any mucosal route.48 Mucosal transmission of HIV-1 in chimpanzees was shown to be relatively inefficient,52,53 leading to loss of CD4 cells.54 Viral divergence was seen with dual infection in chimpanzees,55 foreshadowing findings in the clinic56 that have shown intersubtype and even intrasubtype recombination to be an important outcome of infection. Importantly, the nonhuman primate models allow the comprehensive investigation of cells that are infected in the genital tract57 as well as investigations as to how the virus spreads following infection.58

HUSBANDRY/AVAILABLE STOCKS OF NONHUMAN PRIMATES AND LENTIVIRUSES LOCATIONS OF MAJOR RESEARCH CENTERS USING NONHUMAN PRIMATES FOR AIDS RESEARCH Scientists working in nonhuman primate models for AIDS perform this research in academic, nonprofit, industrial, and governmentsponsored laboratories and centers. Numerous private concerns provide purchase, care, and infrastructure for nonhuman primate work for investigators throughout the world. In the United States, there are eight National Primate Research Centers (NPRCs) for

568

SECTION V / MODELS OF BEHAVIOR

Table 58–2 National Primate Research Centers in the United States, 2006 Primate center name and website

Academic affiliate

City and state

California National Primate Research Center http://www.cnprc.ucdavis.edu New England National Primate Research Center http://www.hms.harvard.edu/nerprc/ Oregon National Primate Research Center http://onprc.ohsu.edu/ Southwest National Primate Research Center http://www.sfbr.org/pages/snprc_index.php Tulane National Primate Research Center http://www.tpc.tulane.edu/ Washington National Primate Research Center http://www.wanprc.org/WaNPRC/ Wisconsin National Primate Research Center http://www.primate.wisc.edu/ Yerkes National Primate Research Center http://www.yerkes.emory.edu

University of California at Davis

Davis, CA

Harvard University

Southborough, MA

Oregon Health and Sciences University

Portland, OR

Southwest Foundation for Biomedical Research

San Antonio, TX

Tulane University

Covington, LA

University of Washington

Seattle, WA

University of Wisconsin

Madison, WI

Emory University

Atlanta, GA

which long-term core support is provided by center grants from the National Center for Research Resources (NCRR) of the United States National Institutes of Health. Their locations, academic affiliations, and websites are listed in Table 58–2. The mission of these centers is to provide nonhuman primate resources, relevant scientific expertise, and biological samples to the research community, and each of the centers has active research in AIDS pathogenesis, vaccines, and treatments. Because of their historical involvement in AIDS research, these centers remain important resources to the research community worldwide. A current list of contact information for the NPRC, and an overview of how outside investigators may access these resources, is maintained at www.ncrr.nih.gov/ncrrprog/cmpdir/PRIMATES.asp. For specific experiments, specific pathogen-free (SPF) rhesus and pigtailed monkeys initially free of SIV, simian retrovirus D, STLV-1, and monkey herpes B virus are available from the eight NPRCs and from the Caribbean primate center in Puerto Rico (http://rcmi. rcm.upr.edu/docs/primate.html). In addition to these centers, many academic institutions, pharmaceutical and biotechnology companies, and the U.S. National Institutes of Health maintain primate colonies for in-house research. In Europe, the German Primate Center and the Biomedical Primate Research Center in the Netherlands house primates and have historically been major centers for AIDS research. This is by no means an exhaustive list; rather its objective is to acquaint the investigator seeking possible research collaborations with the national centers in the United States as a starting point. VIRAL STOCKS The HIV-1 stocks that have been tested in chimpanzees and subsequently in macaques were derived from the early, laboratory-passaged viruses such as HIV-IIIB or HIVBRU, HIV-LAI, HIV-MN, or HIV-SF2. These viruses replicated to high titers in vivo and elicited robust antiviral antibodies, including neutralizing antibodies. Although attempts were made by several investigators to infect macaques with HIV, sustained replication and pathogenesis were not observed. Today, it is strongly suspected that restriction by TRIM5α in cells from most macaque species was responsible for prevention of cross-species

transmission. HIV-2 isolates were serially passaged in baboons with some efficient replication, but little or no pathogenesis.59 After the observation that SIV infection caused pathogenesis in the Asian macaques, a number of virus isolates were derived from the blood and tissues of serially infected macaques. These three major types of SIV are based on SIVsm (naturally found in sooty mangabeys), SIVmac (from an experimental infection of M. mulatta), and SIVmne (from an experimental infection of M. nemestrina). Members of these isolates were further characterized by obtaining full-length infectious molecular clones that could recapitulate the pathogenic capacity of the isolate. Subsequently other members of the SIV family have been identified from naturally infected African green monkeys (SIVagm), mandrills (SIVmnd), colobus monkeys (SIVcol), and almost all of the various African monkeys, but not baboons. For the most part, the SIV/macaque model has utilized SIVsm or SIVmac in M. mulatta or M. fascicularis; other groups have favored the use of SIVmne in M. fascicularis or M. nemestrina. Hayami and colleagues made a seminal contribution to the field when they constructed the first viral chimera between HIV-1 and SIV.60–62 This virus consisted of the SIV “backbone” with the env gene replaced by that from HIV-1. Since this first SHIV construct, several additional SHIVs have been described.63 However, the development of chimeric viruses has not been a trivial pursuit, and only one virus to date, SHIV-SF162P3, bears an HIV-1 envelope that utilizes the CCR5 coreceptor.64 This virus was shown to cause depletion of CD4+ T cells in the GALT, unlike CXCR4utilizing SHIVs.65 These chimeras have been very useful for vaccine efforts that target the development of neutralizing antibodies, since the env genes of SIV and HIV-1 are poorly crossneutralized, if at all. SHIVs may also be useful in the pursuit of live attenuated vaccines.66 Other important constructs have included replacement of other SIV genes, such as the RT gene, with that from HIV-1, in order to test RT inhibitors in vivo.67 Many investigators have developed stocks of SIV and SHIV that are made widely available to researchers who wish to perform challenge studies. The expense of preparing and titering these

CHAPTER 58 / NONHUMAN PRIMATE MODELS FOR AIDS

large scale (300 vials or greater) stocks in vivo in the relevant nonhuman primate host is quite high, certainly well above $100,000 (U.S.). Typically a full in vivo titration requires purchasing, infecting, and housing at least 6 and up to 10 animals for up to 1 year in BSL-2 or BSL-3 containment, in order to support statistically robust outcomes.68 In part to defray this cost, several primate centers have prepared and titered stocks that are widely distributed, and the NIH Division of AIDS also has made several stocks available, including a recent stock of SHIV-SF162P3 grown in M. mulatta. Clearly the use of identical challenge stocks is a variable that can be controlled by this judicious sharing of resources. The U.S. NIH has developed a shared resource for qualified investigators that is entitled the AIDS Research Reference Reagent Repository (http://www.aidsreagent.org/ ecommerce/). This resource provides a very comprehensive and well-referenced set of viral stocks, cell lines for growing virus, monoclonal antibodies, recombinant antigens, etc. to investigators who are registrants and can show that their laboratories have met any required biological and biosafety containment criteria.

ADVANTAGES/DISADVANTAGES There are many advantages to the utilization of nonhuman primate models for AIDS research. Some examples include increased understanding of viral and host factors that affect pathogenic outcomes, infectiousness via diverse routes of entry, and the roles of specific factors in limiting infection. The use of these models allows investigators to compare outcomes of infection in multiple individuals using the identical, characterized—in many cases cloned—virus stock. By this type of study, it is apparent that there are host factors contributing to pathogenic outcomes. Conversely, viruses derived at different times of infection can themselves have differential pathogenic potential.69 Direct inoculation of virus at various mucosal sites has allowed a clearer understanding of the differential infectivity of stocks given by different routes. Similarly, the ability to perform timed necropsies of primates infected with well-characterized and genetically identical stocks has facilitated the identification of specific cell types involved in the earliest stages of infection by SIV and SHIV, as well as the rates of mutation that accrue after infection. Information about neurovirulence has been much more approachable in the SIV model than in infected humans.70 The contribution of regions of the genome to the pathogenic potential of a given virus has been demonstrated with viral chimeras, and more recently the study of changes in the viral envelope over time has yielded interesting information about the inherent ability of the HIV-1 envelope to tolerate mutations in vivo.71 One of the key questions in vaccine research is how neutralizing antibodies are generated, particularly those with the ability to neutralize multiple viruses. Neutralizing antibodies with broad capacity can be generated by infection with the primate lentiviruses, but they are rare, as in humans.72 These models may offer insights into the mechanisms of development of key immune responses, including innate, cellular, and humoral immunity. Laboratory nonhuman primate models for AIDS allow comprehensive studies of the events occurring in the days immediately following exposure and infection, and experiments with various combinations of competing microbes, which cannot be accomplished for exposed humans. Another area that can be addressed much more easily in the nonhuman primate than in humans is the evaluation of vaccines and therapies to limit mother-to-child transmission. Some new

569

models were developed in recent years to test active and passive approaches for vaccination in very young primates. These include breast milk transmission,73 perinatal transmission,74 and the oral inoculation of newborns with SIV75 or SHIV.76 Risky approaches that would be prohibited in humans, such as live attenuated vaccines39 and passive antibody transfer,77–79 have been important tools to understanding host immunity and protection. Even with less than fully effective vaccines, it is possible to test how well newborns and infants respond to vaccination. Newborn rhesus macaques have been vaccinated with live attenuated vaccines or recombinant viral vectors expressing SIV antigens in the first few weeks of life and they have been able to mount significant immune responses and control pathogenic viral challenge,80 which is encouraging news for the future once more effective vaccines are developed. Important information about passive immunity81 such as dosing and timing82 and active vaccination of newborns80 can be derived from these models without risking human infants. These advantages are balanced by a number of disadvantages. Obviously, the absence of a model that uses HIV-1 as the infectious agent is the most glaring problem for the field. Although certain aspects of this problem can be overcome by the use of SHIV clones and isolates, there is as yet no substitute for clinical work to understand human immune responses to the virus and pathogenic sequelae of the resulting immune depletion. Most nonSPF adult macaques in captivity harbor a virus that is lethal to humans, herpes B. This problem has led to the development of safety regulations to prevent exposure, but there have still been rare infections of animal handlers. One approach is to develop SPF breeding colonies, and many of the centers are making great strides in this area, eliminating (as noted above) SIV, simian retrovirus (SRV), simian T-lymphotropic virus-1 (STLV-1), and herpes B. The lack of complete genotyping, specifically with a focus on MHC Class I, has made the use of nonhuman primates more challenging for both pathogenesis and vaccine studies. Certain alleles in macaques that allow responses to key epitopes in SIV Gag, for example, were very useful in understanding escape from cytotoxic T cell (CTL) control of SIV.83 This problem is complicated by the use of multiple species of macaques by different investigators, at a time when genetic characterization of the different species is not well developed. To some degree, the use of certain species to the exclusion of others (e.g., M. mulatta of Indian origin) has created a shortage that can be alleviated only by expanded breeding or acceptance of other species and subspecies. Differential virus replication in rhesus macaques of Indian and Chinese origin calls into question the interchangeability of the two populations for research.84 Certainly, another major disadvantage is the expense of purchase and maintenance of these valuable primates, which has limited the size of studies. Thus, analyses of subtle differences in immune parameters in immunogenicity studies, for examples, have been much easier to evaluate in clinical studies in humans. Some of the recent efforts from the HIV Vaccine Enterprise include infusing HIV vaccine research with funding that can support much larger nonhuman primate studies that may have sufficient statistical power to inform the choice of immunogens for clinical development. However, most of the primate facilities are not designed to handle multiple large (>100 animal) experiments at one time, which will require careful planning and shepherding of resources. Finally, research in nonhuman primates requires an appreciation of the model at many levels, and for the first time investigator there are many complex

570

SECTION V / MODELS OF BEHAVIOR

issues to consider. This last problem is one of the easiest to address, as seasoned investigators can be found to collaborate, and the NPRCs have as their mandate to serve the research community. Diverse samples of blood, tissues, and organs are available to all research investigators. Pilot studies are available by competitive application, and these can serve to match an investigator with a primate modeler, facilitating the development of preliminary data for a larger study or grant. Although the NCRR provides core support for the eight NPRCs, scores of additional grants from other Institutes of NIH that are awarded to numerous academic and research institutions provide the majority of the funding for the NPRC work. An additional resource to consider is attendance of the yearly Nonhuman Primate Models for AIDS Symposium, presently having completed its 24th Annual conference (http://www.hivatlas.org/24th_Annual_Symposium_on_ Nonhuman_Primate_Models_for_AIDS) where current results and future collaborative efforts and opportunities are discussed. PREDICTIVE VALUE OF THE MODELS As noted above, the uses of the nonhuman primate models are diverse, thus making the careful choice of the appropriate model for a particular study absolutely key to understanding the outcome(s). In addition, frequently due to availability of species and particular virus stocks, the same experimental design has been tested in different laboratories with divergent outcomes. Thus, one of the major issues confounding AIDS research has been the interpretation of results obtained utilizing different virus–primate pairs. These differences have also been compounded by the unfortunate limitations in nonhuman primate funding during the first 10 years of the epidemic, which led to use of sizes of experimental arms that were very small (three or four animals per group), insufficient to provide statistical power unless there were all-or-none outcomes. Recent studies have utilized much larger groups (6–10), allowing the interrogation of more subtle questions and comparisons between groups receiving different vaccines, for example. The current primate models for AIDS, as well as future models, would be considerably less expensive, allow greater throughput at the primate centers, and reduce the number of monkeys used yearly, if surrogate markers of vaccine success could be assessed before, or instead of, live virus challenge. Surrogate markers currently include both T cell and antibody responses in blood and at mucosal sites. The most compelling question for the nonhuman primate models is their predictive value for human studies. In cases in which direct comparison can be made such as drug development or transmission studies, it appears that the predictive value is strong.85 A great deal of our understanding about the biology of infection and dissemination via mucosal routes has been obtained from nonhuman primate studies, where both qualitative and quantitative data are forthcoming. Three recent reviews have touched on this point of predictive value for vaccine studies: Staprans and Feinberg,3 Haigwood,85 and Hu5. Once there is an HIV vaccine in humans with some efficacy, it will be possible to validate one or more of the nonhuman primate animal–virus pairs and to proceed with some confidence to use this model for future development. Until that time, these models will continue to serve as important adjuncts to clinical studies to understand the issues in transmission, persistence, and pathogenesis,86 to elucidate innate and adaptive parameters of viral control,87 and to serve as testing grounds for vaccine strategies.

REFERENCES 1. Ho DD. Time to hit HIV, early and hard. N Engl J Med 1995;333(7):450–451. 2. Gao F, Bailes E, Robertson DL, et al. Origin of HIV-1 in the chimpanzee Pan troglodytes troglodytes. Nature 1999;397(6718): 436–441. 3. Staprans SI, Feinberg MB. The roles of nonhuman primates in the preclinical evaluation of candidate AIDS vaccines. Expert Rev Vaccines 2004;3(4Suppl.):S5–32. 4. Schmidt B, Ashlock BM, Foster H, Fujimura SH, Levy JA. HIVinfected cells are major inducers of plasmacytoid dendritic cell interferon production, maturation, and migration. Virology 2005;343(2): 256–266. 5. Hu SL. Non-human primate models for AIDS vaccine research. Curr Drug Targets Infect Disord 2005;5(2):193–201. 6. Jayaraman P, Haigwood NL. Animal models for perinatal transmission of HIV-1. Front Biosci 2006;11:2828–2844. 7. Johnson PR, Hirsch VM. Genetic variation of simian immunodeficiency viruses in nonhuman primates. AIDS Res Hum Retroviruses 1992;8(3):367–372. 8. Hirsch VM, Lifson JD. Simian immunodeficiency virus infection of monkeys as a model system for the study of AIDS pathogenesis, treatment, and prevention. Adv Pharmacol 2000;49:437–477. 9. Weed MR, Steward DJ. Neuropsychopathology in the SIV/macaque model of AIDS. Front Biosci 2005;10:710–727. 10. Zink MC, Laast VA, Helke KL, et al. From mice to macaques–animal models of HIV nervous system disease. Curr HIV Res 2006; 4(3):293–305. 11. Daniel MD, Letvin NL, King NW, et al. Isolation of T-cell tropic HTLV-III-like retrovirus from macaques. Science 1985;228(4704): 1201–1204. 12. Letvin NL, Daniel MD, Sehgal PK, et al. Induction of AIDS-like disease in macaque monkeys with T-cell tropic retrovirus STLV-III. Science 1985;230(4721):71–73. 13. Gravell M, London WT, Houff SA, et al. Transmission of simian acquired immunodeficiency syndrome (SAIDS) with blood or filtered plasma. Science 1984;223(4631):74–76. 14. Sharp PM, Shaw GM, Hahn BH. Simian immunodeficiency virus infection of chimpanzees. J Virol 2005;79(7):3891–3902. 15. Keele BF, Van Heuverswyn F, Li Y, et al. Chimpanzee reservoirs of pandemic and nonpandemic HIV-1. Science 2006;313(5786):523– 526. 16. Hirsch VM, Olmsted RA, Murphey-Corb M, Purcell RH, Johnson PR. An African primate lentivirus (SIVsm) closely related to HIV-2. Nature 1989;339(6223):389–392. 17. Gojobori T, Moriyama EN, Ina Y, et al. Evolutionary origin of human and simian immunodeficiency viruses. Proc Natl Acad Sci USA 1990;87(11):4108–4111. 18. Chen Z, Luckay A, Sodora DL, et al. Human immunodeficiency virus type 2 (HIV-2) seroprevalence and characterization of a distinct HIV2 genetic subtype from the natural range of simian immunodeficiency virus-infected sooty mangabeys. J Virol 1997;71(5):3953–3960. 19. Choe H, Farzan M, Konkel M, et al. The orphan seventransmembrane receptor apj supports the entry of primary T-cell-linetropic and dualtropic human immunodeficiency virus type 1. J Virol 1998;72(7):6113–6118. 20. Long EM, Rainwater SM, Lavreys L, Mandaliya K, Overbaugh J. HIV type 1 variants transmitted to women in Kenya require the CCR5 coreceptor for entry, regardless of the genetic complexity of the infecting virus. AIDS Res Hum Retroviruses 2002;18(8):567–576. 21. Chen Z, Gettie A, Ho DD, Marx PA. Primary SIVsm isolates use the CCR5 coreceptor from sooty mangabeys naturally infected in west Africa: A comparison of coreceptor usage of primary SIVsm, HIV-2, and SIVmac. Virology 1998;246(1):113–124. 22. Bakshi SS, Zhang L, Ho D, Than S, Pahwa SG. Distribution of CCR5delta32 in human immunodeficiency virus-infected children and its relationship to disease course. Clin Diagn Lab Immunol 1998;5(1):38–40.

CHAPTER 58 / NONHUMAN PRIMATE MODELS FOR AIDS

23. Sheehy AM, Gaddis NC, Choi JD, Malim MH. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 2002;418(6898):646–650. 24. Stremlau M, Owens CM, Perron MJ, Kiessling M, Autissier P, Sodroski J. The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 2004;427(6977): 848–853. 25. Heeney JL, Dalgleish AG, Weiss RA. Origins of HIV and the evolution of resistance to AIDS. Science 2006;313(5786):462–466. 26. Alter HJ, Eichberg JW, Masur H, et al. Transmission of HTLV-III infection from human plasma to chimpanzees: An animal model for AIDS. Science 1984;226(4674):549–552. 27. Novembre FJ, Saucier M, Anderson DC, et al. Development of AIDS in a chimpanzee infected with human immunodeficiency virus type 1. J Virol 1997;71(5):4086–4091. 28. Emini EA, Schleif WA, Nunberg JH, et al. Prevention of HIV-1 infection in chimpanzees by gp120 V3 domain-specific monoclonal antibody. Nature 1992;355(6362):728–730. 29. Castro BA, Nepomuceno M, Lerche NW, Eichberg JW, Levy JA. Persistent infection of baboons and rhesus monkeys with different strains of HIV-2. Virology 1991;184(1):219–226. 30. Watson A, McClure J, Ranchalis J, et al. Early postinfection antiviral treatment reduces viral load and prevents CD4+ cell decline in HIV type 2-infected macaques. AIDS Res Hum Retroviruses 1997;13(16): 1375–1381. 31. Saag MS, Hahn BH, Gibbons J, et al. Extensive variation of human immunodeficiency virus type-1 in vivo. Nature 1988;334(6181): 440–444. 32. Fisher AG, Ensoli B, Looney D, et al. Biologically diverse molecular variants within a single HIV-1 isolate. Nature 1988;334(6181): 444–447. 33. Hirsch VM, Johnson PR. Pathogenic diversity of simian immunodeficiency viruses. Virus Res 1994;32(2):183–203. 34. Lackner AA, Smith MO, Munn RJ, et al. Localization of simian immunodeficiency virus in the central nervous system of rhesus monkeys. Am J Pathol 1991;139(3):609–621. 35. Heise C, Vogel P, Miller CJ, Halsted CH, Dandekar S. Simian immunodeficiency virus infection of the gastrointestinal tract of rhesus macaques. Functional, pathological, and morphological changes. Am J Pathol 1993;142(6):1759–1771. 36. Heise C, Miller CJ, Lackner A, Dandekar S. Primary acute simian immunodeficiency virus infection of intestinal lymphoid tissue is associated with gastrointestinal dysfunction. J Infect Dis 1994; 169(5):1116–1120. 37. Guadalupe M, Reay E, Sankaran S, et al. Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy. J Virol 2003;77(21):11708– 11717. 38. Chase A, Zhou Y, Siliciano RF. HIV-1-induced depletion of CD4+ T cells in the gut: Mechanism and therapeutic implications. Trends Pharmacol Sci 2006;27(1):4–7. 39. Daniel MD, Kirchhoff F, Czajak SC, Sehgal PK, Desrosiers RC. Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene. Science 1992;258(5090):1938–1941. 40. Ruprecht RM. Live attenuated AIDS viruses as vaccines: Promise or peril? Immunol Rev 1999;170:135–149. 41. McChesney MB, Collins JR, Lu D, et al. Occult systemic infection and persistent simian immunodeficiency virus (SIV)-specific CD4(+)T-cell proliferative responses in rhesus macaques that were transiently viremic after intravaginal inoculation of SIV. J Virol 1998; 72(12):10029–10035. 42. Finzi D, Hermankova M, Pierson T, et al. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 1997;278(5341):1295–1300. 43. Ho DD. Toward HIV eradication or remission: The tasks ahead. Science 1998;280(5371):1866–1867. 44. Finzi D, Blankson J, Siliciano JD, et al. Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even

45.

46.

47. 48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

571

in patients on effective combination therapy. Nat Med 1999;5(5): 512–517. Trichel AM, Roberts ED, Wilson LA, Martin LN, Ruprecht RM, Murphey-Corb M. SIV/DeltaB670 transmission across oral, colonic, and vaginal mucosae in the macaque. J Med Primatol 1997; 26(1–2):3–10. Lu Y, Brosio P, Lafaile M, et al. Vaginal transmission of chimeric simian/human immunodeficiency viruses in rhesus macaques. J Virol 1996;70(5):3045–3050. Ruprecht RM, Baba TW, Liska V, et al. Oral transmission of primate lentiviruses. J Infect Dis 1999;179(Suppl. 3):S408–412. Miller CJ, Alexander NJ, Sutjipto S, et al. Genital mucosal transmission of simian immunodeficiency virus: Animal model for heterosexual transmission of human immunodeficiency virus. J Virol 1989;63(10):4277–4284. Doria-Rose NA, Ohlen C, Polacino P, et al. Multigene DNA primingboosting vaccines protect macaques from acute CD4+-T-cell depletion after simian-human immunodeficiency virus SHIV89.6P mucosal challenge. J Virol 2003;77(21):11563–11577. Ruprecht RM, Fratazzi C, Sharma PL, Greene MF, Penninck D, Wyand M. Animal models for perinatal transmission of pathogenic viruses. Ann NY Acad Sci 1993;693:213–628. Sodora DL, Gettie A, Miller CJ, Marx PA. Vaginal transmission of SIV: Assessing infectivity and hormonal influences in macaques inoculated with cell-free and cell-associated viral stocks. AIDS Res Hum Retroviruses 1998;14(Suppl. 1):S119–123. Fultz PN, Wei Q, Yue L. Rectal transmission of human immunodeficiency virus type 1 to chimpanzees. J Infect Dis 1999;179(Suppl. 3):S418–421. Girard M, Mahoney J, Wei Q, et al. Genital infection of female chimpanzees with human immunodeficiency virus type 1. AIDS Res Hum Retroviruses 1998;14(15):1357–1367. Davis IC, Girard M, Fultz PN. Loss of CD4+ T cells in human immunodeficiency virus type 1-infected chimpanzees is associated with increased lymphocyte apoptosis. J Virol 1998;72(6):4623– 4632. Wei Q, Fultz PN. Extensive diversification of human immunodeficiency virus type 1 subtype B strains during dual infection of a chimpanzee that progressed to AIDS. J Virol 1998;72(4):3005– 3017. Zhu T, Wang N, Carr A, Wolinsky S, Ho DD. Evidence for coinfection by multiple strains of human immunodeficiency virus type 1 subtype B in an acute seroconvertor. J Virol 1995;69(2):1324–1327. Hu J, Gardner MB, Miller CJ. Simian immunodeficiency virus rapidly penetrates the cervicovaginal mucosa after intravaginal inoculation and infects intraepithelial dendritic cells. J Virol 2000; 74(13):6087–6095. Miller CJ, Li Q, Abel K, et al. Propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus. J Virol 2005;79(14):9217–9227. Barnett SW, Murthy KK, Herndier BG, Levy JA. An AIDS-like condition induced in baboons by HIV-2. Science 1994;266(5185): 642–646. Kuwata T, Igarashi T, Ido E, et al. Construction of human immunodeficiency virus 1/simian immunodeficiency virus strain mac chimeric viruses having vpr and/or nef of different parental origins and their in vitro and in vivo replication. J Gen Virol 1995;76(Pt. 9):2181–2191. Kuwata T, Shioda T, Igarashi T, et al. Chimeric viruses between SIVmac and various HIV-1 isolates have biological properties that are similar to those of the parental HIV-1. AIDS 1996;10(12): 1331–1337. Hayami M, Igarashi T. SIV/HIV-1 chimeric viruses having HIV-1 env gene: A new animal model and a candidate for attenuated live vaccine. Leukemia 1997;11(Suppl.)3:95–97. Karlsson GB, Halloran M, Li J, et al. Characterization of molecularly cloned simian-human immunodeficiency viruses causing rapid CD4+ lymphocyte depletion in rhesus monkeys. J Virol 1997;71(6): 4218–4225.

572

SECTION V / MODELS OF BEHAVIOR

64. Harouse JM, Gettie A, Eshetu T, et al. Mucosal transmission and induction of simian AIDS by CCR5-specific simian/human immunodeficiency virus SHIV(SF162P3). J Virol 2001;75(4):1990–1995. 65. Harouse JM, Gettie A, Tan RC, Blanchard J, Cheng-Mayer C. Distinct pathogenic sequela in rhesus macaques infected with CCR5 or CXCR4 utilizing SHIVs. Science 1999;284(5415):816–819. 66. Kuwata T, Miura T, Hayami M. Using SHIVs to develop an antiHIV-1 live-attenuated vaccine. Trends Microbiol 2001;9(10): 475–480. 67. Isaka Y, Sato A, Kawauchi S, et al. Construction of the chimeric reverse transcriptase of simian immunodeficiency virus sensitive to nonnucleoside reverse transcriptase inhibitor. Microbiol Immunol 1998;42(3):195–202. 68. Spouge JL, Layne SP, Dembo M. Analytic results for quantifying HIV infectivity. Bull Math Biol 1989;51(6):715–730. 69. Kimata JT, Kuller L, Anderson DB, Dailey P, Overbaugh J. Emerging cytopathic and antigenic simian immunodeficiency virus variants influence AIDS progression. Nat Med 1999;5(5):535–541. 70. Mankowski JL, Flaherty MT, Spelman JP, et al. Pathogenesis of simian immunodeficiency virus encephalitis: Viral determinants of neurovirulence. J Virol 1997;71(8):6055–6060. 71. Blay WM, Gnanakaran S, Foley B, Doria-Rose NA, Korber BT, Haigwood NL. Consistent patterns of change during the divergence of human immunodeficiency virus type 1 envelope from that of the inoculated virus in simian/human immunodeficiency virus-infected macaques. J Virol 2006;80(2):999–1014. 72. Yuste E, Sanford HB, Carmody J, et al. Simian immunodeficiency virus engrafted with human immunodeficiency virus Type 1 (HIV1)-specific epitopes: Replication, neutralization, and survey of HIV1-positive plasma. J Virol 2006;80(6):3030–3041. 73. Amedee AM, Lacour N, Ratterree M. Mother-to-infant transmission of SIV via breast-feeding in rhesus macaques. J Med Primatol 2003;32(4–5):187–193. 74. Jayaraman P, Mohan D, Polacino P, et al. Perinatal transmission of SHIV-SF162P3 in Macaca nemestrina. J Med Primatol 2004;33(5–6):243–250. 75. Baba TW, Koch J, Mittler ES, et al. Mucosal infection of neonatal rhesus monkeys with cell-free SIV. AIDS Res Hum Retroviruses 1994;10(4):351–357. 76. Baba TW, Trichel AM, An L, et al. Infection and AIDS in adult macaques after nontraumatic oral exposure to cell-free SIV. Science 1996;272(5267):1486–1489.

77. Shibata R, Igarashi T, Haigwood N, et al. Neutralizing antibody directed against the HIV-1 envelope glycoprotein can completely block HIV-1/SIV chimeric virus infections of macaque monkeys. Nat Med 1999;5(2):204–210. 78. Mascola JR, Stiegler G, VanCott TC, et al. Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat Med 2000;6:207–210. 79. Haigwood NL, Montefiori DC, Sutton WF, et al. Passive immunotherapy in simian immunodeficiency virus-infected macaques accelerates the development of neutralizing antibodies. J Virol 2004; 78(11):5983–5995. 80. Van Rompay KK, Greenier JL, Cole KS, et al. Immunization of newborn rhesus macaques with simian immunodeficiency virus (SIV) vaccines prolongs survival after oral challenge with virulent SIVmac251. J Virol 2003;77(1):179–190. 81. Baba TW, Liska V, Hofmann-Lehmann R, et al. Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection. Nat Med 2000; 6(2):200–206. 82. Nishimura Y, Igarashi T, Haigwood NL, et al. Transfer of neutralizing IgG to macaques 6 h but not 24 h after SHIV infection confers sterilizing protection: Implications for HIV-1 vaccine development. Proc Natl Acad Sci USA 2003;100(25):15131–15136. 83. Allen TM, O’Connor DH, Jing P, et al. Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia. Nature 2000;407(6802):386–390. 84. Marthas ML, Lu D, Penedo MC, Hendrickx AG, Miller CJ. Titration of an SIVmac251 stock by vaginal inoculation of Indian and Chinese origin rhesus macaques: Transmission efficiency, viral loads, and antibody responses. AIDS Res Hum Retroviruses 2001;17(15): 1455–1466. 85. Haigwood NL. Predictive value of primate models for AIDS. AIDS Rev 2004;6(4):187–198. 86. Johnson WE, Desrosiers RC. Viral persistence: HIV’s strategies of immune system evasion. Annu Rev Med 2002;53:499–518. 87. Evans DT, Desrosiers RC. Immune evasion strategies of the primate lentiviruses. Immunol Rev 2001;183:141–158.

MODELS OF OTHER VI HUMAN DISEASES

59

Use of Congenic Mouse Strains for Candidate Disease Gene Identification in Complex Traits UTE CHRISTINE ROGNER AND PHILIP AVNER

ABSTRACT

The breeding systems underlying the development of key genetic resources are shown in Figure 59–1. Each recombinant inbred (RI) strain contains a unique admixture of genetic contributions—in approximately equal proportions—from its two original progenitor inbred strains. Traditionally, such RI strains are established by crossing animals of two inbred strains, followed by 20 or more consecutive generations of brother/sister matings.3 Recombinant congenic (RC) strains are also established by an initial crossing between two inbred strains, but this is followed by a few, usually two, backcrosses of the resulting F1 hybrids to one of the parental strains, called the “recipient” strain, with subsequent brother/sister intercrossing as for RI lines.4 In both RI and RC strains the result is a mosaic genetic structure with blocks of genetic material from one parent interspersed with blocks of genetic material from the other parent. RI and RC strains differ, however, in the relative contribution of the two parents to the final strains. The Complex Trait Consortium5 represents the largest community effort to date to generate and maintain collections of RI lines having sufficient analytical power to allow for systematic quantitative trait locus (QTL) analysis. The current project aims to generate some 1000 RIs from eight different parental strains to sort out the genes behind different complex disorders. Consomics and congenics are special types of inbred strains in which part of the genome of one mouse strain is transferred to another by backcrossing the mouse strain acting as the donor to the recipient strain followed by intercrossing in later generations to ensure fixation and homozygosity. Selection is systematically practiced to ensure retention of the desired genetic material from the donor strain. The method was first introduced by Snell who produced histoincompatible congenic strains, which he originally called “congenic resistant” strains.6 In the case of a consomic, a whole chromosome is transferred.7,8 In the case of a congenic strain it is a defined chromosomal segment, also termed the differential segment that is transferred9,10 (Figure 59–1). Such congenic strains will normally carry differential regions of 10–20 Mb in size11 unless specific efforts are made to reduce the size of the differential segment. Several hundred congenic strains currently exist. Many of these can be retrieved directly from the Jackson Laboratories. Congenic strains need to be distinguished from coisogenic strains that differ at only a single locus from their parental strain.12 Coisogenics can be derived by both gene targeting involving homologous recombination or mutagenesis

Genetic analysis aims to establish the relationship between a gene and a specific trait through the parallel study of phenotype and underlying genetic variation. Current tools for genetic analysis in the mouse extend from standard matings such as intercrosses and backcrosses to the use of more specialized resources such as recombinant inbred (RI) strains and recombinant congenic (RC) strains. Both RI and RC strains are characterized by the fixation of a genome-wide admixture of novel genetic combinations subsequent to meiotic recombination and segregation. Other resources of particular interest for the analysis of complex traits include consomic and congenic strains. In consomic or chromosome substitution strains, the genetic admixture is restricted to a single chromosome, and in congenic strains it is restricted to a single chromosomal region. Consomic and congenic mouse stocks have played a pivotal role in the genetic analysis of complex diseases that are often under the control of both environmental and multiple genetic factors and include type 1 diabetes, multiple sclerosis, obesity, and cancer. This chapter will provide an overview of the different tools available in the mouse for the genetic analysis of complex traits, with particular emphasis on different aspects of the use of congenic mouse strains. Key Words: Consomic, Congenic, Inbred mice, Complex disease, Quantitative trait locus (QTL), Gene.

TOOLS AND SYSTEMS OF GENETIC ANALYSIS IN THE MOUSE The mouse is the organism of choice for modeling human disease with not only many thousands of mutations already isolated and projects to inactivate all mouse genes well underway,1 but also with 450 described inbred strains,2 a wealth of genetic and phenotypic diversity. It is this collection of inbred strains, rather than the collection of mutations, that has provided the raw material for studying phenotypes under complex genetic control. This has, in recent times, been facilitated by the Mouse Phenome project (see database links), which aims at garnering and curating, through a coordinated international effort, baseline phenotypic data on most of the common inbred strains. From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

575

576

SECTION VI / MODELS OF OTHER HUMAN DISEASES

Figure 59–1. Genetic admixture in recombinant inbred strains, consomics, and congenics. Recombinant inbred strains are generated by intercrosses of F1 mice and subsequent brother–sister interbreeding. Consomic strains are generated by repeated backcrossing of F1 animals to the parental receiver strain, but only one chromosome is derived from the donor strain. In congenic strains only the differential chromosome segment is derived from the donor strain.

approaches. RI and RC consomic and congenic strains all have in common the advantage that they allow repeat phenotyping of large numbers of genetically homogeneous animals under defined environmental conditions.

APPROACHES TO THE IDENTIFICATION OF A GENETIC INTERVAL CONTROLLING A COMPLEX TRAIT The starting point for the identification of a genetic locus controlling a complex trait, or QTL, is most often the generation of an F1 intercross from two parental inbred strains differing in phenotype for the trait under study and the subsequent intercrossing of F1 progeny to produce an F2 generation that can be subject to genetic analysis. Alternative analytical schemes may involve the backcrossing of the F1 progeny to either one or the other parental strains, to generate first-stage backcross generation animals (BC1) and/or eventually backcross 2 animals (BC2). The number of animals needed for the F2, BC1, or BC2 analysis depends on the strength of the phenotypic effect conferred by each QTL under study and also on the size of the genetic interval(s) to be identified. Generally, experimental cohort sizes are in the range of several hundred animals. Most of the currently identified QTL genes are slightly atypical in presenting rather extreme phenotypes that confer above average contributions to the overall phenotype. Contributions of the average single QTL to the overall phenotype has been estimated to be 5% or less.13 While increasing the number of F2 or BC animals under study and using a higher marker density may increase statistical power and allow the candidate region to be better localized, the additional effort would sometimes be better directed elsewhere, for example, to fine mapping and use of congenic strains (see the discussion below). Alternatives to the generation of de novo crosses involve the use of sets of RI lines and/or RC strains. The analytical power of an RI line set depends on the number of available sublines and the degree of genetic/phenotypic variation in the parental strains. While RI sets can deliver higher mapping resolution than F2 mice,13 current RI sets will often have insufficient power to iden-

tify genes encoding small-effect QTLs. In contrast to RI sets, sets of RC strains have the property of limiting the amount of the genome that has to be searched for multiple genes involved in quantitative traits as long as they have been selected for the phenotype of interest. The genome of a standard RC strain comprises, on average, only 12.5% from the donor strain.14 Irrespective of the approach used in the initial QTL localization, moving on to subsequent candidate gene identification and characterization will often prove difficult with both genetic complexity and genetic background effects hampering progress. Consulting the Mouse Genome Informatics (MGI) database (see below) reveals, for example, that while an initial localization has been described for some 2000 quantitative trait loci, in less than 1% of the cases has the gene underlying the QTLs been identified. Recent advances have on occasion been linked to the use of innovative in silico “Hapmap” type strategies, but mostly to the exploitation of consomic and especially congenic strains, which are increasingly playing a pivotal role in the dissection of QTLs.

HAPLOTYPE MAPPING AS AN IN SILICO ALTERNATIVE AND COMPLEMENTATION In silico mapping is a powerful computational-based method for predicting chromosomal regions regulating phenotypic traits.15 The discovery based on the distribution of single nucleotide polymorphisms (SNPs) in different inbred mouse strains of an alternating mosaic pattern of relatively large, typically 1–2 Mb, genomic regions (blocks) of low or high polymorphic variation (SNPs)16,17 is the foundation for this approach. Regions that are poor in polymorphic markers have been designated as regions of common ancestry. Haplotype blocks are conceptually defined as genomic segments harboring sets of coupled polymorphisms that reflect a common ancestral origin.18,19 Genome-wide association studies involving the correlation of a phenotype, for instance, disease prone and disease resistant, over a wide selection of different inbred mouse strains to patterns of genetic variation in these same strains have provided powerful indications of potential candidate regions.15 QTL genes are likely, although not necessarily, to be found in regions of different ancestry among pairs of differentially affected strains, while a given phenotype observed in common in different strains will likely be controlled by a region that is held in common between these strains.20 The approach requires that well-standardized parental phenotype data are available for many inbred strains. The PHENOME project was established in part with this type of application in mind. Applied to congenic strain analysis, knowledge of the haplotype block structure within a congenic interval may focus interest on a particular subregion within the congenic candidate region (Figure 59–2). There are several examples in the literature that have demonstrated the value of this type of combined approach.21–23 In whatever way in silico mapping is applied, the success of the strategy depends on the size of the underlying haplotype blocks and the panel of relevant mouse strains. The approach also depends on rates of mutation within regions of shared haplotype being sufficiently low as to not obscure the underlying patterns of haplotype variation. Recent work indicates that the definition of haplotype blocks is not that robust and that methods for QTL mapping may fail if they assume a simple block-like structure.19

CHAPTER 59 / USE OF CONGENIC MOUSE STRAINS FOR CANDIDATE DISEASE GENE IDENTIFICATION IN COMPLEX TRAITS

577

Figure 59–2. Haplotype mapping. Comparison of the SNP distribution within the candidate interval between disease-sensitive and -resistant strains may allow a candidate region to be further reduced.

USING CONSOMICS AND CONGENICS FOR DISSECTING QUANTITATIVE TRAIT LOCI INTO GENES Consomic and especially congenic strains are proving to be key resources for the dissection of QTLs whose genetic control is very distant from the simplicity of characters showing singlegene dominant or single-gene recessive Mendelian inheritance. Understanding the role of an individual QTL is often hampered by the complexity of its genetic and phenotypic interactions with other participating QTLs. As an example, consider susceptibility to type 1 diabetes and the well-known disease model for type 1 diabetes, the nondiabetes (NOD) mouse.24,25 It turns out that diabetes-sensitive strains such as NOD carry not only diabetes sensitivity loci but also QTL loci conferring diabetes resistance. Conversely, diabetes-resistant strains such as C57BL/6 carry loci conferring diabetes susceptibility as well as diabetes resistance genes. It is the overall balance and interactions that determine the final phenotype.26 Some idea of the overall complexity is given by the 30 or more murine insulin-dependent diabetes loci (Idd) that have been genetically identified. In this complex situation, congenic strains often carrying segments derived from the diabetes-resistant C57BL/6 mouse strain have been of critical importance for breaking down the overall genetic complexity, for ensuring reproducibility of the detailed phenotyping studies, and for the comparative transcriptional profiling approaches used to narrow down the candidate region. Among non-major histocompatibility complex (MHC) Idd genes that have been cloned using a congenic strategy are Idd3, where Il2 has been implicated,22 Idd5.1, where Ctla4 and Icos were suggested,27 and Idd5.2, where Nramp is a likely candidate.28 The second reason to generate congenic strains is to study the effects of a gene mutation, knockout, knockin, or transgene on one or several different genetic backgrounds. This approach is of particular interest for studies on genetic modifiers.29,30 The use of congenics, although often onerous, remains less time consuming than introducing the same modification in parallel onto different genetic backgrounds by gene targeting or transgenesis. Indeed, in many cases the latter approach may be impossible as the appropriate ES cell lines may not be available. When congenic construction is used, it should, however, be borne in mind, as already pointed out, that it is not a single gene that is being transferred but a gene and its surrounding genetic region. There have been examples showing that such genomic fragments can contribute to

the phenotype or in a worst-case scenario even confer a new phenotype. Different breeding strategies to minimize such problems have been discussed.31

CLASSICAL AND SPEED BREEDING STRATEGIES FOR CONGENIC STRAINS Congenic strains are derived by repeated backcrossing of the desired donor strain to the recipient strain with selection for the differential segment. This breeding is then followed by sister/ brother interbreeding of the backcrossed progeny. In practice, female F1 animals are mated with recipient strain males to establish the BC1 generation. Males heterozygous for the selected chromosome region are then repeatedly backcrossed to recipient females during congenic strain derivation. Congenic strains are then rendered homozygous for the genetic intervals under study by intercrossing heterozygous males and females of the same genotype and subsequently maintained by brother and sister mating. When repeated backcrossing is used to establish a congenic strain a minimum of nine generations of backcrossing is normally recommended to remove 99.9% of the unlinked and unwanted donor material,32 though the exact number of required backcross generations appears somewhat arbitrary.33 A genome scan, minimally, for other known loci relevant to the disease under study should be carried out before fixing the congenic interval so that if further backcrossing is necessary to remove a contaminating genomic fragment this can be carried out before the congenic strain is rendered homozygous. Alternative congenic breeding schemes have been established that involve both positive selection for the desired differential segment and selection against the rest of the donor genome during early backcross generations. In such breeding schemes, called “speed congenics,” the genetically “best” animals, i.e., those carrying the differential segment and minimal detectable donor strain material elsewhere in the genome, are selected. Theoretically the process can lead to the creation of a congenic strain with less than 0.5% contaminating donor genome unlinked to the differential segment within a total of five generations or four backcrosses.34 Simulations suggest that screening between 16 and 20 male progeny per generation with markers spaced every 25 cM most efficiently reduces unlinked contaminating donor genome and represents an effective organizational strategy. Use of larger progeny cohorts and higher marker density seems of little advantage in reducing the contaminating donor genome until later back-

578

SECTION VI / MODELS OF OTHER HUMAN DISEASES

cross generations. High-density genotyping of the differential segment in later generations is, however, necessary to reduce the size of the target region below the 20–30 cM otherwise obtained at the N4 generation.10 Experience suggests that both “best” and “second best” males should routinely be kept for breeding, in particular when intersubspecies congenic strains are being constructed and poor breeding performance may occur. Recent simulation studies have suggested that marker-assisted breeding strategies can lead to increased background heterogeneity, or “gaps,” in the recipient genetic background as compared to standard breeding procedures. This suggests that additional backcrossing may still be required in order to reduce the number and length of such gaps.35 On the assumption of putative remaining gaps, it may be of interest to derive a given set of congenic strains from a single breeding pair and to generate at least one congenic strain carrying no differential fragment as an internal control for phenotyping. When choosing recombinants to fix genetic intervals we recommend ensuring the highest possible density of markers within the differential fragment (1–2 cM) to avoid partial heterozygosity. While the genetic interval conferring a particular phenotype in a given congenic strain can often be reduced and refined by identification of new recombinants during further backcrossing, it becomes increasingly difficult to obtain the necessary recombinants as the genetic distance under study is reduced and much larger breeding populations are needed. Several studies have shown the existence of sex-specific differences in recombination frequency36–38 and it can, therefore, on occasion, be of use to change the direction of the cross and use heterozygous females instead of males or vice versa. It should be noted that recombination does not occur with random efficiency throughout the genome and is often higher at so-called hotspots. Certain theories predict that recombination will occur more often in regions in which gene density is higher and less often in “gene deserts,” an expectation borne out for the human genome, where recombination rates are found to be higher in regions of the genome with higher gene density.39 The informative polymorphic markers necessary to characterize the recombinants can be normally identified from the MGI database. Current densities of available markers ensure that only rarely will additional comparative sequencing efforts be required to identify additional SNPs. When analyzing congenic strains, it has been observed that the phenotypic effects often get smaller as the genetic interval is reduced and subcongenics are generated. This most often occurs when the original effect was due to the combination of several genes and may reflect the relatively frequent occurrence of QTLs as haplotype blocks. In other cases, genetic interactions may lead to the suppression of phenotypes when intervals are combined and in such cases the generation of subcongenics is accompanied by an increase in the penetrance of the trait.40 In theses cases, complexity within the genetic interval can normally still be successfully addressed, although it may require larger numbers of animals to be phenotyped and studied. The benefit that can be obtained from a panel of congenic strains is critically dependent on the quality of the phenotyping available, which, in turn, is obviously dependent on the disease under study. The availability of subphenotypes for characterization is often critical to the fine dissection of the trait. Analysis often starts with the most robust and basic phenotype before pro-

ceeding to more subtle analysis of subphenotypes. Phenotyping methods often employed range from histology, behavioral studies, and the evaluation of physiological parameters to metabolomics and transcriptional profiling. At this stage of the project, the efforts employed in the construction of the congenic strains are usually rewarded.

CANDIDATE GENE IDENTIFICATION AND VALIDATION Once a candidate region has been defined and characterized, the crucial difficulty of identifying and validating the causative gene(s) arises. It is important to realize that there is rarely one single approach, but rather a spectrum of complementary approaches that can be used to identify without ambiguity the gene(s) underlying a QTL. The identification of changes in the primary nucleotide sequence, which is powerfully diagnostic in the case of mutations in monogenic disorders, is of much less certain value in the case of QTL characterization. First, because nonsense or stop codons that completely abolish gene function are much less likely to underlie QTL variation than in mutations affecting monogenic traits. Second, because the investigator cannot, a priori, know whether to expect changes in coding sequences or in noncoding regulatory sequences. And third, because extensive nucleotide variation may occur outside of the exon sequences of some genes, which renders the identification of causal polymorphisms in such regions potentially perilous. This indeterminacy may moreover be compounded if the causal gene lies within a region that shows a high degree of polymorphism between the parental strains. In such cases the polymorphism within the genomic sequence of the causal gene will likely be no more marked than that of the surrounding genes. Such regions of elevated polymorphism are to be found throughout the mouse genome and reflect the breeding history of mouse inbred strains (see above). Such caveats suggest that resequencing of entire regions in the donor and recipient strains is an approach that can help to exclude a certain proportion of candidate genes rather than lead to the unambiguous identification of the responsible gene. The use of expression profiling for candidate gene identification is based on the idea that in many cases the QTL will reflect quantitative changes in the expression of the underlying gene(s). This approach has proven to be particularly fruitful in cases in which the phenotype of the disease under study has provided clues as to the class(es) of genes or to the tissues in which the candidate gene is likely to be expressed. Annotated lists of genes for the region under study obtained from Ensembl, MGI, or NCBI databases, including in silico expression profiling approaches based on exploiting data from sources such as Serial Analysis of Gene Expression (SAGE) libraries, microarray analysis-based datasets, and cumulative data on expressed sequence tags (ESTs) (see Table 59–1), allow the tissue expression profiles of the genes to be established. Where necessary these data are then validated for the most promising candidates by comparative expression profiling of the discriminatory congenic strains using techniques such as quantitative real-time polymerase chain reaction (PCR). A complementary variant of this approach uses genome-wide microarray-based expression profiling to identify possible genetic pathways.41 The efficiency of both strategies depends on the completeness of the gene annotations and the exhaustiveness of

CHAPTER 59 / USE OF CONGENIC MOUSE STRAINS FOR CANDIDATE DISEASE GENE IDENTIFICATION IN COMPLEX TRAITS

579

Table 59–1 Some useful web links Mouse Genome Informatics (MGI) database Complex Trait Consortium Mouse Phenome Database Center of Rodent Genetics Mouse Genome Resequencing and SNP Discovery Project Online books on mouse genetics and human molecular genetics Single nucleotide polymorphisms (SNPs) Mouse sequence databases

Gene expression data

BAC mapping The RNAi WEB Transgenic animal web (links) Knockout mouse project Available mouse models Standard genotyping protocol Online Mendelian Inheritance in Man, OMIM Human/mouse relationships

the gene representation that is being exploited and is influenced by both the cellular complexity of the target tissue and relative transcript expression levels. In the case of complex tissues sensitivity may be increased by analyzing cellular subpopulations of the tissue in question.42,43 Neither the identification of sequence variation nor of altered expression profiles is of itself sufficient to establish causality. For this, techniques of gene inactivation, gene overexpression, and the gold standard, replacement of the allele of one strain by that of the other strain—referred to here as “allele shuffling”—are required. Ongoing programs for inactivation or mutating all mouse genes will increasingly provide “off the shelf” ES cells carrying a knockout for the candidate genes under study. Database links that provide information about existing knockouts and mutants are listed in Table 59–1. Exploiting such resources for QTL validation may in many cases be complicated by the need to cross the knockout onto the relevant genetic background. Interestingly, some of the constructs used in these global knockout programs may facilitate application of a knockin strategy, which would enable the integration of alternative functional allelic forms of the gene at the disabled locus to be undertaken.44 An alternative and potentially very efficient strategy for gene disabling is the use of small interfering RNA (siRNA) to inhibit/ knock down gene expression. RNA interference (RNAi) is a highly evolutionary conserved process of posttranscriptional gene silencing (PTGS) in which double-stranded RNA (dsRNA), when introduced into a cell, causes sequence-specific degradation of homologous mRNA sequences.45 Double-stranded RNAs of around 21 nucleotides in length inhibit the expression of specific genes and can work effectively both ex vivo and in vivo.46–48 The RNAi WEB

http://www.informatics.jax.org/ http://www.complextrait.org/ http://www.jax.org/phenome http://www.niehs.nih.gov/crg/ http://www.niehs.nih.gov/crg/cprc.htm http://www.informatics.jax.org/silver/index.shtml http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=hmg http://www.broad.mit.edu/snp/mouse/ http://mousesnp.roche.com/ http://www.ncbi.nlm.nih.gov/ http://www.ensembl.org/ http://mrcseq.har.mrc.ac.uk/ http://www.genome.ucsc.edu/ http://www.informatics.jax.org/menus/expression_menu.shtml http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Mm http://bodymap.ims.u-tokyo.ac.jp/ http://www.bcgsc.ca/lab/mapping/mouse http://www.rnaiweb.com/ http://www.med.umich.edu/tamc/links.html http://www.nih.gov/science/models/mouse/knockout/index.html http://jaxmice.jax.org/index.html http://www.jax.org/mmr/Standard_mapping_protocol.html http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM http://www.ncbi.nlm.nih.gov/Omim/Homology/

database provides a good summary of such studies and excellent practical advice. The long-term and stable inhibition of gene function normally necessary for assessing the effect of a QTL implies that delivery systems capable of supporting stable and persistent expression in vivo are necessary. Cloning of stable interfering short hairpin (sh) RNA molecules and use of several viral vectors and transposon-based nonviral vectors have been reported that fulfill this requirement.49,50 Interestingly, such RNAi approaches can be used with embryos circumventing the strain restriction bottleneck associated with ES cell use. Although there are problems associated with off target non-specificity of RNAi that require stringent control, RNAi can be engineered to be specific for particular allelic forms of a given gene. Providing technical problems can be overcome, experiments aimed at targeting RNAi to alleles of a candidate gene in F1 animals from two discriminatory congenic strains should prove particularly informative. Overexpression studies provide an alternative if less stringent way to either further reduce the size of the candidate region or to validate formally candidate genes, when the trait under consideration is dominantly or codominantly expressed.51,52 Such approaches are often carried out using BAC clones, which may be one to several hundred kilobases in size and therefore allow the gene to be tested along with many of the cis-acting sequences necessary for its regulated expression. Such studies are being facilitated by the construction of BAC libraries for many mouse strains other than C57BL/6 and 129/Sv.53 The fingerprinting of these libraries and the ready availability of both mouse genomic draft and finished sequences54 and of strain resequencing programs allow the DNA hybridization probes necessary for BAC isolation to be easily designed. Although BAC transgenesis is an

580

SECTION VI / MODELS OF OTHER HUMAN DISEASES

efficient process, it should be noted that both copy number variation and variation in the site of integration in the genome leading to position effects may lead to modifications in gene expression profiles that might hamper or obscure the identification of the gene when subtle phenotypes are concerned.

COMPARATIVE GENOMICS BETWEEN MOUSE AND HUMAN Humans and mouse have both evolved from an ancestral small mammal that split into two species toward the end of the dinosaur era. Despite 75 million years of separate evolution, only about 300 genes—1% of the 25,000–30,000 genes in the mouse genome—were found to be without an obvious counterpart in the human genome and vice versa,55,56 leading to the idea that in the majority of cases the underlying biology and physiology of processes occurring in humans and mouse will be similar or identical. Most studies of mouse models of human disease are predicated on this. Diseases under monogenic control most often, although not necessarily always, provide support for the assumption.57 Where the assumption fails to hold, because mutations in a given gene fail to produce the same phenotype in both species, the differences are mainly imputable to differences in physiology, to subtle differences in the gene regulation, and or/to differences in the specific mutation itself rather than to the absence of the gene. The predictive value of QTLs identified in one species for the other is generally considerably weaker. This is almost certainly due to the genetic heterogeneity underlying most complex traits, to differences in penetrance even when the same gene may be concerned, and to differences in the spectrum of naturally occurring variation at a given locus in humans and mouse. This, however, does not imply that the underlying genetic networks are highly divergent and if we take the case of type 1 diabetes this is clearly not the case. Indeed, the identification of causative genes for the autoimmune disease type 1 diabetes (T1D) in humans and candidate genes in the NOD mouse has shown that susceptibility or resistance to type 1 diabetes, involving genes and pathways contributing to autoimmune pathogenesis, are primarily held in common by the two species. To cite but one example, gene variants for the interacting molecules IL2 and CD25, members of a pathway that is essential for immune homeostasis, are present in mice and humans.58 In this context, identifying the mouse genes involved in type1 diabetes by consolidating our knowledge of the pathway underlying the pathogenesis will both indirectly and directly identify novel targets, which can be studied for their possible role in the human pathogenic process. Congenic strains have and will continue to play a central role in the panoply of genetic tools available to the research scientist. Indeed, their importance and pivotal role are likely to rise as the tools necessary for high-throughput systematic functional testing of candidate genes lying within the candidate regions defined by using congenic strains become more widely available.

ACKNOWLEDGMENTS We thank Richard Harvey and Christophe Rachez for critical reading of the manuscript. This work was supported by grants from the Juvenile Diabetes Research Foundation International (1–2000–600), the Association pour la recherche sur le Diabète, and recurrent funding from the CNRS, INSERM, and the Pasteur Institute.

REFERENCES 1. Grimm D. Mouse genetics. A mouse for every gene. Science 2006;312(5782):1862–1866. 2. Beck JA, Lloyd S, Hafezparast M, et al. Genealogies of mouse inbred strains. Nat Genet 2000;24(1):23–25. 3. Bailey DW. Recombinant-inbred strains. An aid to finding identity, linkage, and function of histocompatibility and other genes. Transplantation 1971;11(3):325–327. 4. Demant P, Hart AA. Recombinant congenic strains—a new tool for analyzing genetic traits determined by more than one gene. Immunogenetics 1986;24(6):416–422. 5. Churchill GA, Airey DC, Allayee H, et al. The Collaborative Cross, a community resource for the genetic analysis of complex traits. Nat Genet 2004;36(11):1133–1137. 6. Snell GD. Congenic resistant strains of mice. In: Morse HC, Ed. Origins of Inbred Mice. New York: Academic Press, 1978:1–31. 7. Nadeau JH, Singer JB, Matin A, Lander ES. Analysing complex genetic traits with chromosome substitution strains. Nat Genet 2000;24(3):221–225. 8. Santos J, Montagutelli X, Acevedo A, et al. A new locus for resistance to gamma-radiation-induced thymic lymphoma identified using inter-specific consomic and inter-specific recombinant congenic strains of mice. Oncogene 2002;21(43):6680–6683. 9. Boyse EA, Bentley DR. The increasing value of congenic mice in biomedical research. Lab Anim Sci 1977;27(5Pt. 2):771–781. 10. Wakeland E, Morel L, Achey K, Yui M, Longmate J. Speed congenics: A classic technique in the fast lane (relatively speaking). Immunol Today 1997;18(10):472–477. 11. Peirce J. Looking at old tools in new ways: Using knockouts as congenics to study QTLs. Genome Res 2001;11(9):1469–1471. 12. Roths JB, Murphy ED, Eicher EM. A new mutation, gld, that produces lymphoproliferation and autoimmunity in C3H/HeJ mice. J Exp Med 1984;159(1):1–20. 13. Flint J, Valdar W, Shifman S, Mott R. Strategies for mapping and cloning quantitative trait genes in rodents. Nat Rev Genet 2005;6(4):271–286. 14. Stassen AP, Groot PC, Eppig JT, Demant P. Genetic composition of the recombinant congenic strains. Mamm Genome 1996;7(1): 55–58. 15. Grupe A, Germer S, Usuka J, et al. In silico mapping of complex disease-related traits in mice. Science 2001;292(5523):1915–1918. 16. Lindblad-Toh K, Winchester E, Daly MJ, et al. Large-scale discovery and genotyping of single-nucleotide polymorphisms in the mouse. Nat Genet 2000;24(4):381–386. 17. Wade CM, Kulbokas EJ, Kirby AW, et al. The mosaic structure of variation in the laboratory mouse genome. Nature 2002; 420(6915):574–578. 18. Frazer KA, Wade CM, Hinds DA, Patil N, Cox DR, Daly MJ. Segmental phylogenetic relationships of inbred mouse strains revealed by fine-scale analysis of sequence variation across 4.6 mb of mouse genome. Genome Res 2004;14(8):1493–1500. 19. Yalcin B, Fullerton J, Miller S, et al. Unexpected complexity in the haplotypes of commonly used inbred strains of laboratory mice. Proc Natl Acad Sci USA 2004;101(26):9734–9739. 20. Guo Y, Weller P, Farrell E, et al. In silico pharmacogenetics of warfarin metabolism. Nat Biotechnol 2006;24(5):531–536. 21. Hillebrandt S, Wasmuth HE, Weiskirchen R, et al. Complement factor 5 is a quantitative trait gene that modifies liver fibrogenesis in mice and humans. Nat Genet 2005;37(8):835–843. 22. Lyons PA, Armitage N, Argentina F, et al. Congenic mapping of the type 1 diabetes locus, Idd3, to a 780-kb region of mouse chromosome 3: Identification of a candidate segment of ancestral DNA by haplotype mapping. Genome Res 2000;10(4):446–453. 23. Ikegami H, Makino S, Yamato E, et al. Identification of a new susceptibility locus for insulin-dependent diabetes mellitus by ancestral haplotype congenic mapping. J Clin Invest 1995;96(4):1936– 1942. 24. Makino S, Kunimoto K, Muraoka Y, Mizushima Y, Katagiri K, Tochino Y. Breeding of a non-obese, diabetic strain of mice. Jikken Dobutsu 1980;29(1):1–13.

CHAPTER 59 / USE OF CONGENIC MOUSE STRAINS FOR CANDIDATE DISEASE GENE IDENTIFICATION IN COMPLEX TRAITS

25. Hattori M, Buse JB, Jackson RA, et al. The NOD mouse: Recessive diabetogenic gene in the major histocompatibility complex. Science 1986;231(4739):733–735. 26. Yang Y, Santamaria P. Lessons on autoimmune diabetes from animal models. Clin Sci (Lond) 2006;110(6):627–639. 27. Greve B, Vijayakrishnan L, Kubal A, et al. The diabetes susceptibility locus Idd5.1 on mouse chromosome 1 regulates ICOS expression and modulates murine experimental autoimmune encephalomyelitis. J Immunol 2004;173(1):157–163. 28. Kissler S, Stern P, Takahashi K, Hunter K, Peterson LB, Wicker LS. In vivo RNA interference demonstrates a role for Nramp1 in modifying susceptibility to type 1 diabetes. Nat Genet 2006;38(4):479–483. 29. Montagutelli X. Effect of the genetic background on the phenotype of mouse mutations. J Am Soc Nephrol 2000;11(Suppl. 1)6: S101–105. 30. Nadeau JH. Modifier genes in mice and humans. Nat Rev Genet 2001;2(3):165–174. 31. Wolfer DP, Crusio WE, Lipp HP. Knockout mice: Simple solutions to the problems of genetic background and flanking genes. Trends Neurosci 2002;25(7):336–340. 32. Silver LM. Mouse Genetics Concepts and Applications. Oxford, UK: Oxford University Press, 1995. 33. Festing MFW. Inbred Strains in Biomedical Research. London: The Macmillian Press LTD, 1979. 34. Markel P, Shu P, Ebeling C, et al. Theoretical and empirical issues for marker-assisted breeding of congenic mouse strains. Nat Genet 1997;17(3):280–284. 35. Armstrong NJ, Brodnicki TC, Speed TP. Mind the gap: Analysis of marker-assisted breeding strategies for inbred mouse strains. Mamm Genome 2006;17(4):273–287. 36. Lynn A, Schrump S, Cherry J, Hassold T, Hunt P. Sex, not genotype, determines recombination levels in mice. Am J Hum Genet 2005;77(4):670–675. 37. Morelli MA, Cohen PE. Not all germ cells are created equal: Aspects of sexual dimorphism in mammalian meiosis. Reproduction 2005;130(6):761–781. 38. Shiroishi T, Sagai T, Hanzawa N, Gotoh H, Moriwaki K. Genetic control of sex-dependent meiotic recombination in the major histocompatibility complex of the mouse. EMBO J 1991;10(3):681– 686. 39. Fullerton SM, Bernardo Carvalho A, Clark AG. Local rates of recombination are positively correlated with GC content in the human genome. Mol Biol Evol 2001;18(6):1139–1142. 40. Rogner UC, Boitard C, Morin J, Melanitou E, Avner P. Three loci on mouse chromosome 6 influence onset and final incidence of type I diabetes in NOD.C3H congenic strains. Genomics 2001;74(2):163–171. 41. Eaves IA, Wicker LS, Ghandour G, et al. Combining mouse congenic strains and microarray gene expression analyses to study a complex trait: The NOD model of type 1 diabetes. Genome Res 2002;12(2): 232–243.

581

42. Lock C, Hermans G, Pedotti R, et al. Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis. Nat Med 2002;8(5):500–508. 43. Scearce LM, Brestelli JE, McWeeney SK, et al. Functional genomics of the endocrine pancreas: The pancreas clone set and PancChip, new resources for diabetes research. Diabetes 2002;51(7):1997– 2004. 44. Garcia-Otin AL, Guillou F. Mammalian genome targeting using site-specific recombinases. Front Biosci 2006;11:1108–1136. 45. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998;391(6669):806–811. 46. Hasuwa H, Kaseda K, Einarsdottir T, Okabe M. Small interfering RNA and gene silencing in transgenic mice and rats. FEBS Lett 2002;532(1–2):227–230. 47. Xia H, Mao Q, Paulson HL, Davidson BL. siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 2002;20(10): 1006–1010. 48. Qin XF, An DS, Chen IS, Baltimore D. Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl Acad Sci USA 2003;100(1): 183–188. 49. Naldini L. Lentiviruses as gene transfer agents for delivery to nondividing cells. Curr Opin Biotechnol 1998;9(5):457–463. 50. Yant SR, Ehrhardt A, Mikkelsen JG, Meuse L, Pham T, Kay MA. Transposition from a gutless adeno-transposon vector stabilizes transgene expression in vivo. Nat Biotechnol 2002;20(10): 999–1005. 51. Symula DJ, Frazer KA, Ueda Y, et al. Functional screening of an asthma QTL in YAC transgenic mice. Nat Genet 1999;23(2): 241–244. 52. Giraldo P, Montoliu L. Size matters: Use of YACs, BACs and PACs in transgenic animals. Transgenic Res 2001;10(2):83–103. 53. Osoegawa K, Tateno M, Woon PY, et al. Bacterial artificial chromosome libraries for mouse sequencing and functional analysis. Genome Res 2000;10(1):116–128. 54. Waterston RH, Lindblad-Toh K, Birney E, et al. Initial sequencing and comparative analysis of the mouse genome. Nature 2002; 420(6915):520–562. 55. Okazaki Y, Furuno M, Kasukawa T, et al. Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs. Nature 2002;420(6915):563–573. 56. Marshall E. Genome sequencing. Public group completes draft of the mouse. Science 2002;296(5570):1005. 57. Villasenor J, Benoist C, Mathis D. AIRE and APECED: Molecular insights into an autoimmune disease. Immunol Rev 2005;204: 156–164. 58. Wicker LS, Clark J, Fraser HI, et al. Type 1 diabetes genes and pathways shared by humans and NOD mice. J Autoimmun 2005; 25(Suppl.):29–33.

60

Animal Models of Sudden Infant Death Syndrome JANE BLOOD-SIEGFRIED

ABSTRACT

model literature, in particular, pathology related to respiration. Changes, both central and systemic, that can affect oxygenation, rebreathing of carbon dioxide, and recurrent apnea are a prime focus of animal research.8–11 Other animal studies have focused on damage to the fetal neurological system. Autopsy evaluation of many SIDS cases reveals histological and chemical changes that indicate abnormal development in the brain.12,13 These abnormalities are located primarily in the brainstem but are found throughout the brain as well. They reflect possible damage from anoxia, but are not limited to that particular insult. For example, prenatal nicotine exposure without hypoxia may cause structural and neurochemical changes in the brain.14–18 An animal model offers a way to examine the developing nervous system and damage from possible insults. A more recent area of animal research focuses on mechanisms that challenge cardiovascular responsiveness through overheating, hypoglycemia, infection, and toxins. Risk factors such as the increased incidence of SIDS in winter months parallel closely those associated with these cardiovascular challenges.19 Prone sleeping position is thought to play a significant role in risk because of changes in gas exchange; however, elevated body temperature may also be a factor. It is believed that the fatal event involves the inability to compensate for a vital physiological challenge to a compromised infant during sleep. Compensatory mechanisms of gasping ability, arousal, and efforts to overcome lowered blood pressure and heart rate are currently being examined.13,20 Some autopsy findings observed in SIDS infants have been difficult to explain. Many SIDS infants have characteristic intrathoracic petechiae and liquid blood in the heart.21,22 These could be associated with efforts to overcome hypoxia, respiratory distress, suffocation, or resuscitation; however, they could also be due to coagulation problems associated with infection and inflammation.23,24 Autopsy findings are varied, but there are often signs of inflammation and response to infection that are out of proportion to preexisting symptoms. Thymic involution, a sign that the infant has been responding to excessive stress, is observed in more than half of SIDS cases.25 A good animal model must reflect the characteristics of an uneventful history without a clear cause for death. Careful consideration must be given to associations between the human condition and the findings in the model. Insults should be plausible and compatible with daily living, and they cannot be so severe that they are obviously the cause of the death.

Sudden infant death syndrome (SIDS) is the most common cause of postneonatal infant mortality in the developed world. It is a diagnosis of exclusion, defined as the sudden and unexplained death of an infant between 1 month and 1 year of life. Examination of specific pathology may be possible only in an animal model. The three animal models most commonly used in SIDS research to examine risk factors are the piglet, neonatal rabbit, and neonatal rat. We also describe a developmental rat model of sudden unexplained death in response to viral and bacterial infections. This model offers a unique way to evaluate the role of immune development and its relationship to potential risk factors believed to be important in the etiology of SIDS. Key Words: Animal model, Cytokine, Endotoxin, Inflammation, Influenza virus, Neonatal rabbit, Neonatal rat, Piglet, SIDS.

INTRODUCTION Sudden infant death syndrome (SIDS) is the most common cause of postneonatal infant mortality in the developed world.1,2 It is a diagnosis of exclusion, often defined as the sudden death of an infant not explained by a thorough autopsy, review of the clinical history, and examination of the death scene.2,3 SIDS occurs from 1 month to 1 year with the peak incidence between 2 and 6 months of age. Although infants dying of SIDS appear normal and healthy, there is often a history of a mild viral illness that is not thought to be the cause of death.1,2 Epidemiological research has helped establish risk factors for SIDS including ethnicity, maternal smoking and drug use during pregnancy, environmental tobacco smoke exposure, concurrent viral illness, overheating, and sleep position; in addition, the incidence is highest in the winter months.4 Before 1990, prone sleeping was considered the greatest risk factor for SIDS. With the decrease in SIDS due to the campaign to put infants to sleep on their backs, the most significant risk factor has become maternal smoking. Although passive smoke exposure can cause respiratory problems in infants, maternal smoking during gestation is the most important risk for SIDS.5–7 The etiology of SIDS is thought to be multifactorial, and it is quite likely that many different means can cause the unexplained death. Several theories of causation are prevalent in the animal From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

583

584

SECTION VI / MODELS OF OTHER HUMAN DISEASES

ANIMAL MODELS OF SUDDEN INFANT DEATH SYNDROME Many animals have been used to study sudden unexplained death in cats,26,27 chick embryo,28–31 dogs,8 ferrets,32,33 guinea pigs,34 mice,35–37 and several primates.38,39 Although primates are more like humans than the other species, they present difficulties for researchers. Primates have fewer offspring and much longer gestations than other laboratory animals. In addition, many species of primates are endangered, adding moral and legal reasons not to subject them to science that involves the death of offspring. Therefore the majority of research on sudden unexplained death has been done using the piglet, neonatal rabbit, and neonatal rat (Table 60–1). This chapter therefore focuses on models developed in these three species. PIGLET The piglet is commonly used in neonatal research, in part because it is a large animal, which allows easy manipulation of equipment and procedures. The piglet also has a tracheal morphology that is very similar to a newborn infant.40 Many processes have been well characterized in perinatal swine models. Piglets have been used primarily for respiratory research in SIDS, but they have also been used to examine the effects of heat and heat stress, reflux, and cardiac development. A large study published in 1994 evaluated the natural history of a group of 1921 piglets, examining all animals that died for cause of death. Eight animals died suddenly or unexpectedly; however, only one animal represented a “SIDS-like” death. It was concluded that although there was a high incidence of mortality, the incidence of sudden unexplained death in the piglet was very low, and thus it would be pointless to study the piglet in the natural state.41 Nevertheless, studies in piglet models have been very productive in focusing on specific systems. Galland and colleagues,42,43 for example, in an early study, looked at the effects of hyperther-

mia and oxygen consumption and tested the hypothesis that a cold face and hot body might increase hyperthermia and drop O2 consumption in a cold climate. They found that face cooling could also increase rapid eye movement (REM) sleep, a time of risk for SIDS.42 Additionally, this study supported the idea that head covering does not increase CO2 or decrease O2 but has its primary effect by increasing body temperature.44 In another study, heated piglets developed tachycardia, hypotension, and metabolic acidosis. They also became tachypneic, with hypocapneic alkalosis and neutrophil leukocytosis. Interestingly, these animals had excessive hemorrhage in the lungs and alveolar edema, similar to that seen in SIDS cases.45 In addition, piglets have been shown to have a delayed response to airway obstruction while they are recovering from fever.46 Xia and colleagues used decerebrated, vagotomized piglets to evaluate the combined effects of hyperthermia and apnea in sudden death. They found that respiratory inhibition associated with chemoreflex was prolonged when body temperature was elevated,47 and these changes were probably mediated by the central nervous system (CNS).48 It has been proposed that reflux during sleep has a negative effect on the ability of the human infant to autoresuscitate.49 The piglet model allows for manipulation of the gastrointestinal (GI) tract and artificial instillation of a mildly acidic compound to stimulate airway protective responses in naturally sleeping and sedated animals.49,50 Richardson and Adams examined lung changes postmortem following laryngeal chemoreflux and found that these piglets had petechiae in their lungs.51 Togari and colleagues examined the compensatory role of cerebral circulation in respiratory control in a high CO2 environment and found that reduction of cerebral blood flow caused respiratory suppression in hypercarbic piglets.52 The piglet model has also been used to examine hypoxic responses to infection and smoking. Pretreatment with nicotine

Table 60–1 Animal models examining risk factors for sudden infant death syndrome in the piglet, neonatal rabbit, and neonatal rat Risk factors

Animal

Conclusion

Reference

Hyperthermia

Piglet

Head covering does not decrease O2 or increase CO2 but has its primary effect by increasing body temperature Piglets have a delayed response to airway obstruction while recovering from fever Respiratory inhibition in response to chemoreflex was prolonged with elevated body temperature; these changes are probably mediated by the central nervous system (CNS) The reduction of cerebral blood flow caused respiratory suppression in hypercarbic piglets Maternal cocaine exposure alters the maturation of centers in the brain important for autonomic and respiratory function Imbalance in autonomic tone favoring parasympathetic tone could cause a problem in the developing piglet Nicotine and infection interfere with autoresuscitation from chemoreflex or hypoxia Prenatal nicotine exposure decreases autoresuscitation in response to apnea; this appears to be related to the affect on autonomic nervous system development Intravenous administration of different bacterial toxins increased catecholamine levels, and induced bradycardia, hypotension, and apnea in neonatal rabbits Combinations of bacterial isolates were lethal to rat pups There is a transitory period of imbalance in brainstem respiratory control in the 12day-old rat Lethal response to influenza A and endotoxin challenge in the 12-day-old rat pup

42–44

Piglet Piglet

CNS

Piglet Rabbit

Autonomic tone

Piglet

Nicotine

Piglet Rat

Toxins and infection

Rabbit

Development

Rat Rat Rat

46 47, 48

52 58–60 55–57 53, 54 72–79 62, 65 66, 83, 84 86–88 83, 84

CHAPTER 60 / ANIMAL MODELS OF SUDDEN INFANT DEATH SYNDROME

and endotoxin has been shown to interfere with autoresuscitation and produce prolonged apnea in experimental piglets exposed to a subglottic acidified saline solution.53 Nicotine and interleukin (IL)-1β had synergistic effects, decreasing the animal’s ability to respond to apnea.53 This response was also stimulated by intermittent hypoxia down to 6% O2, then reoxygenation. Nicotine and inflammatory mediators appear to act as cofactors in hypoxic– ischemic neurological injury.54 Froen and colleagues did not examine the synergy between endotoxin and nicotine; however, their work suggests a relationship between either nicotine or an inflammatory response to infection and lethal apnea.53 SIDS infants may succumb to a fatal cardiac arrhythmia that is not detectable on autopsy, and researchers have used the piglet to examine the development of cardiac sympathetic innervation and evaluate arrhythmia.55 These studies have established that an imbalance in the maturation of autonomic function favoring a parasympathetic response could pose a problem to the developing piglet.56 They have also shown that the piglet can be used to evaluate prolonged QT syndrome in swine.55,57 This syndrome can cause lethal arrhythmias in all age groups and may possibly be a mechanism of SIDS. RABBIT The rabbit allows researchers to do larger scale manipulations than are possible in rodents. In SIDS research, rabbits have been used primarily to examine the effects of respiratory insults, infectious agents, and gastric reflux. In an early study, a series of experiments examined the effect of maternal cocaine use in pregnancy on respiratory response in neonatal rabbits.58,59 The researchers exposed rabbit pups to different levels of FiO2 and found that prenatal cocaine changed the maturational response of respiratory control.60 This supported the basic hypothesis that cocaine use alters the maturation of centers in the brain important for autonomic and respiratory function. Gastrointestinal pathogens are known to be a leading cause of morbidity and mortality in children under the age of 5 years.61 Siarakas and colleagues examined the effects of intravenous administration of six common bacterial toxins on the cardiorespiratory system in 1–3 kg rabbits. They observed bradycardia, hypotension, and apnea with sudden death and concluded that under the right conditions, bacteria could produce toxins that cause inflammatory responses similar to those associated with endotoxin-induced shock.62 The rabbit has also been used to evaluate the response to acid reflux by instilling an acid solution in the larynx of neonatal rabbits, inducing obstructive, central, and mixed apnea.63 Similarities between the gastrointestinal tract of the young rabbit and the human infant provide a good model for evaluating potential effects of toxigenic bacteria.64 A study using this model measured catecholamine responses to intravenous and intraluminary exposure in the gastrointestinal tract to bacterial toxins. With increasing intravenous doses, the study found a dose-related increase in catecholamine levels and sudden death. However, intraluminal doses did not increase sudden death. It appears that a healthy gut provides a good protective barrier.65 RAT The rat is a wonderful research animal because it is large enough to allow manipulation using standard equipment and it has the added advantage of large litters and fast turnover of progeny. A drawback to rat research is that there are fewer antibodies available for immune studies, and knockout strains are much less developed than with mice. In spite of this, the rat has often been used in SIDS research. Rat models have been devel-

585

oped to examine both infectious and respiratory insults, and current work is focusing on neurological changes in the brain following prenatal nicotine exposure. Building on a body of work showing that infectious insults could cause unexplained death, Lee and colleagues showed that following subcutaneous injection of nasopharyngeal bacterial isolates cultured from human SIDS infants, 21-day-old weanling rats died rapidly without terminal signs of illness and with minimal inflammatory changes in lungs, liver, and heart.66 When pathogen samples were paired (Escherichia coli and Staphylococcus aureus) mortality occurred much more abruptly, demonstrating the synergy between pathogens. The identification of Helicobacter pylori DNA in postmortem tissues of SIDS infants led to the use of a rat model to examine fatal apnea as a response to gastroesophageal reflux in infants.67,68 There is little evidence that H. pylori is the primary cause of SIDS; however, this model is consistent with the infectious challenge theories of SIDS.69,70 Maternal smoking is an independent risk factor for SIDS.71 Passive exposure to environmental tobacco smoke increases risk,5,6 but the risk of SIDS doubles with maternal smoking and is three to four times higher if the mother smokes more than 10 cigarettes per day.7 Nicotine is currently considered a neuroteratogen and thus it is possible that maternal smoking during pregnancy is more harmful than postnatal exposure to environmental tobacco smoke.5,72 Prenatal exposure to nicotine has been shown to cause a decreased response to apnea and respiratory insult; in addition, it is believed that nicotine affects the development of autonomic responsiveness and delays maturity in the newborn, increasing the risk of SIDS.73 The rat offers an excellent model in which to evaluate the effects of nicotine on development of the respiratory and central nervous systems. Perinatal nicotine has been shown to impair respiratory function and response to hypoxia in neonatal rats during the first week of life.73–75 More recently, investigators have used the rat model to evaluate changes in autonomic function as a result of perinatal nicotine exposure, as well as its effects on the heart,76 brain,77 and respiration.78,79 The changes appear to last well into adulthood.15,80

A DEVELOPMENTAL MODEL OF SUDDEN UNEXPLAINED DEATH The studies reviewed above did not always address the consistent observation that the peak incidence of SIDS occurs between 2 and 4 months in all countries for which accurate data are available.1 The factors underlying the increased susceptibility of this particular age range need to be examined. Developmental responses to infectious challenges may be crucial in this regard. Animal models have been important for developing and testing ideas about the physiological events leading to SIDS; however, it is difficult to show a direct connection to human immune development. We think that rat immune development may provide a useful model for human infants. The rapid development of the immune and neurological systems in the rat makes each day a significant time point for comparison to human infants. There may be changes in the development of immune responses that turn a minor illness into a lethal event.81 We have addressed this issue by assessing an age-dependent model of susceptibility in rats to a nonlethal strain of influenza A virus and a sublethal dose of endotoxin.

586

SECTION VI / MODELS OF OTHER HUMAN DISEASES

Initial experiments were done on postnatal day (PND) 10 to ascertain mortality, timing between doses, and dose response. Pups were treated with 25 µl of a rat-adapted influenza virus (RAIV) given intranasally.82 One to 5 days post-RAIV treatment, five pups per day, per dose, were given an intraperitoneal dose of endotoxin (2 mg/kg, 0.5 mg/kg, 0.2 mg/kg, or 0.05 mg/kg). A second control group of RAIV-naive pups were given the same dose of endotoxin at each time point. RAIV controls received virus only on PND 10. Pups were examined every 4 h postendotoxin for morbidity and mortality.83 The animals died quietly without significant symptoms 8–10 h postendotoxin. This model produced 20% mortality on PND 10, 70–80% mortality on PND 12, but no mortality after PND 14.83,84 This narrow window of susceptibility on PND 12 is similar to the narrow window of susceptibility to SIDS in human infants between 2 and 4 months of age. Additional studies were conducted to establish the critical age at which the dual challenge resulted in unexplained death. Pups were treated intranasally with RAIV on day 8, 10, or 14 of life and then given endotoxin (0.5 mg/kg, 0.2 mg/kg, 0.1 mg/kg, or 0.05 mg/kg) intraperitoneally 2 days later. RAIV-naive pups were given endotoxin in the same doses and time interval as the first group. Additional controls were treated with RAIV only on PND 8, 10, or 14. Pups were examined every 4 h for morbidity and mortality, and deceased animals were removed for necropsy.83 Adult (8-week-old) rats were similarly challenged with the established adult dose of 100 µl of RAIV intranasally and endotoxin (2 mg/kg, 0.5 mg/kg, 0.2 mg/kg, 0.1 mg/kg) intraperitoneally 2 days post-RAIV treatment.85 There was no morbidity or mortality in the adult animals.83 Interestingly, Liu and Wong-Riley showed developmental changes in the respiratory center in the brainstem of rats in the susceptible period.86–88 On PND 12 rat pups had an abrupt switch in the expression of GABAA receptor subunits in the preBotzinger complex in the rat brain, the key area for respiratory control in the brainstem. The transitory period of imbalance between inhibitory and excitatory drives may leave the respiratory system vulnerable to respiratory insults, including an immune response to a significant infectious stimulus.86–88 These experiments have helped us to establish a model of sudden unexplained death in neonatal rats. Influenza administration on PND 10 followed by an endotoxin challenge of 0.2 mg/kg on PND 12 caused the highest mortality and the lowest morbidity in these animals. The model was used in all subsequent experiments, described below.

MATERIALS AND METHODS ANIMALS Fischer-344 rat dams were obtained from Charles River Laboratories (Raleigh, NC). Procedure 1. Animal cages were placed in Illinois isolation cubicles for all of the studies. 2. Dams were housed in individual flat bottom plastic cages at 72ºF ± 2, humidity 50% ± 10, in with 12-h light–dark cycles. 3. All animals received food and water ad libitum. 4. Within 48 h of birth, rat pups were randomly crossfostered and litters standardized to eight pups per dam.

5. A vigorous sentinel animal program was in place to evaluate viral spread. However, it has been shown that there is no between-cage contamination and little within-cage cross-infection using RAIV.89 6. In developmental studies each litter is considered a single group (n = 1); therefore for the experiments, only one pup was removed from each cage for each treatment group. INFLUENZA We developed our model based on work done by Burleson, who showed that an influenza strain could be adapted to induce an immune response in rats without causing signs of illness.89 RAIV was developed from Influenza A/PortChalmers/1/73 (H3N2) virus. It had been initially purchased from American Type Culture Collection and adapted by 10 successive passages in allantoic fluid of chicken eggs.89,90 A detailed description of this model and methods can be found in Methods in Immunotoxicology.89 For our studies, the virus was a gift from Gary Burleson (Burleson Research Technologies, Raleigh, NC). The virus was passaged in adult male F-344 rats using isoflurane anesthesia, as described by Burleson.89 One day after infection the lung tissues were removed and homogenized in Eagle’s minimal essential medium (EMEM) solution diluted to a 10% weight/volume suspension. The virus was washed and frozen in 0.5-ml aliquots at −70ºC.89 Viral content was analyzed using the Madin–Darby canine kidney plaque assay.89 Prior to use, the virus was deemed clear of mycoplasma, bacterial, viral, and fungal contaminants. Procedure 1. On PND 10, stock virus was mixed with phosphatebuffered saline (PBS) in a 1:100 dilution and held on ice until used. The viral content of the 1:100 diluted solution was 1.9 × 104 plaque-forming units (PFU)/ml. 2. Pups were removed from the dam and given 25 µl of RAIV intranasally using a pipetteman. The viral solution was dripped on the nare and inspired by the pup. 3. Pups were allowed to recover before they were placed back with the dam. 4. All pups in five cages were RAIV treated and those in another five cages were treated with PBS. This was done to ensure five subjects in each treatment group. ENDOTOXIN E. coli endotoxin 0127:B8 was obtained from Sigma (St Louis, MO). Procedure 1. On PND 12, endotoxin was diluted with PBS to a concentration of 0.02 mg/ml. 2. Individual pups were weighed, and the average weight for these studies was 20 ± 2 g. 3. Endotoxin dilution was given in a single intraperitoneal dose of 0.2 ml. 4. At 2, 4, 6, or 8 h postendotoxin administration, five pups were removed from five dams in each treatment group and sedated using a 2:10 dilution of Nembutal 50 mg/ml mixed in PBS (0.2 ml intraperitoneally = 0.01 ml/g). 5. Dams were kept in cages with the pups until the end of the experiment for pup warmth and feeding. COLLECTION OF SERUM FROM A CARDIAC PUNCTURE Procedure 1. After establishing that the animal was adequately sedated, a 25-gauge needle and syringe were used to collect blood

CHAPTER 60 / ANIMAL MODELS OF SUDDEN INFANT DEATH SYNDROME

2.

3. 4. 5.

from the heart. Prior to starting, the plunger of the syringe was pulled to the 0.05 ml position. Placing the needle at the zyphoid process, the needle was inserted, then angled at 15–20º and advanced slowly toward the left axillae until blood appeared in the syringe. Blood was slowly removed and placed in a serum separator collection tube. The tubes were spun according to the package directions. The serum was collected, placed in a cryotube, and frozen at −70ºC.

HARVESTING OF ORGANS FROM EACH PUP Procedure 1. The spleen was removed and placed in RPMI to be processed for cell phenotyping. 2. One kidney with adrenal gland, one lobe of lung, one lobe of liver, one half thymus, one half heart, and a small piece of spleen were placed in cassettes for histology. 3. The other half of each organ was placed in a cryotube, flash frozen in liquid nitrogen, then stored at −70ºC to be used for reverse transcriptase polymerase chain reaction (RT-PCR) assay. 4. All remaining animal tissues were placed in biohazard bags and treated as infectious waste.

CHARACTERISTICS OF THE MODEL Characteristic findings reflected increased inflammation and the release of nitric oxide, stimulated by the dual infectious challenge. At each time point following endotoxin, the RAIV-treated animals were more severely affected than the RAIV-untreated animals. At 6 h postendotoxin, the pups were mildly cyanotic and cool, and adequate cardiac blood samples were difficult to obtain. These animals have been shown to be hypotensive and bradycardic using a tail cuff measuring device (Hatteras Instruments, Inc., Raleigh, NC). Following cardiac puncture at 6 h, RAIVtreated pups continued to bleed without clotting, suggesting disorder of the clotting mechanisms, often seen with severe infection. Gross examination revealed petechiae in the lungs, subendocardial hemorrhage, lymphoid cell necrosis of the thymic cortex, and pale kidneys in endotoxin-treated RAIV-treated animals. All pups still had milk in their stomachs, suggesting a rapid progression to death. The RAIV-treated animals died between 8 and 10 h postendotoxin. No other animals died. HISTOLOGY Tissues (liver, spleen, thymus, heart, lung, kidney) were processed, embedded in paraffin, sectioned at 5– 6 µm, and stained with hematoxylin and eosin (H&E) for microscopic examination, as described by Blood-Siegfried.83,91 All RAIV-treated animals at 6 and 8 h postendotoxin injection had signs of inflammation and oxidative stress. The inflammatory component was minor compared to the prominent necrotic process. In addition, there were signs of altered clotting processes.83 CELL PHENOTYPING (FLUORESCENCE-ACTIVATED CELL SORTER) Cells were double stained for surface antigen expression, as described in Blood-Siegfried.83,92 The macrophage cell population started migrating out of the spleen by 2 h after endotoxin administration in the RAIV-treated animals. This occurred in RAIV-untreated animals between 2 and 4 h posten-

587

dotoxin administration. A similar change also occurred in Tlymphocyte populations, both CD4 and CD8. Migration out of the spleen started at 2 h postendotoxin administration in the RAIVtreated animals, which responded 2 h before the RAIV-untreated animals.83 BRONCHEOALVEOLAR LAVAGE FLUID Alveolar cells and fluid were harvested by lavaging the lungs of five animals per group in situ at least five times with 2 ml of ice-cold Ca2+, Mg2+free PBS as described in Blood-Siegfried.83,85 Cell populations identified on Wright stain from broncheoalveolar fluid (BALF) showed a predominance of alveolar macrophages with little fluctuation in cell numbers across time or between groups. RAIV treatment did not appear to increase inflammatory changes in the lungs of these animals.83 ENZYME-LINKED IMMUNOASSAY Commercial rat cytokine enzyme-linked immunosorbent assay (ELISA) kits (Biosource, Camarillo, CA) were used to measure tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-12 in serum, BALF samples, and liver homogenates.83,84 The only cytokine showing a statistically significance change was IFN-γ with RAIV-untreated pups demonstrating a 2-fold greater increase at 6 h than RAIV-treated animals. There was no significant difference in TNF- α, IL-12, and IFN-γ in BALF or liver tissue cytokines across time or between groups; however, there was an increase in inflammatory cytokines in all endotoxin-exposed animals.83,84 REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION ASSAYS Total RNA was isolated from flash frozen liver and lung samples according to the methods described by the manufacturer for the RNeasy kit (Quiagen, Valencia, CA).84 Endotoxin administration stimulated a brisk upregulation of inflammatory cytokine mRNA in RAIV-treated animals and this occurred several hours earlier than in the animals not exposed to RAIV. The same pattern occurred with nitric oxide synthase mRNA. RAIV treatment thus appears to prime the immune system in these animals to respond very quickly to the endotoxin challenge.84 IMMUNOHISTOCHEMISTRY Tissues were fixed in 10% neutral buffered formalin, processed routinely, and embedded in paraffin. Serial sections (6 µm) of liver were stained for NF-κB, COX-2, or inducible nitric acid synthase (iNOS).84,93 The same pattern of rapid response of iNOS was verified by immunohistochemical staining at 6 h postendotoxin; higher levels of iNOS staining were found in liver samples in animals not treated with RAIV, substantiating the PCR findings. No differences in immunostaining for NF-κB or COX-2 were noted between liver sections from RAIV-treated or untreated animals at any time points examined.84

CONCLUSIONS Animal models provide a useful tool for evaluating potential mechanisms of sudden unexplained death in infants. Indeed, examination of specific pathology may be possible only in an animal model. Comparisons between species may be difficult, but some characteristics of SIDS can be evaluated in these systems. In our model, RAIV treatment appears to prime the immune response in 12-day-old rat pups, and the response is specific to this age group. Our model thus offers a unique way to evaluate the role of immune development and its relationship to potential pathogens in the etiology of SIDS.

588

SECTION VI / MODELS OF OTHER HUMAN DISEASES

REFERENCES 1. Blackwell CC, Weir DM. The role of infection in sudden infant death syndrome. FEMS Immunol Med Microbiol 1999;25:1–6. 2. Willinger M, James LS, Catz C. Defining the sudden infant death syndrome (SIDS): Deliberations of an expert panel convened by the National Institute of Child Health and Human Development. Pediatr Pathol 1991;11:677–684. 3. Rognum TO. Definition and pathologic features. In: Byard RW, Krouse HF, Eds. Sudden Infant Death Syndrome: Problems, Progress and Possibilities. London: Hodder Arnold, 2001:4–30. 4. American Academy of Pediatrics. The changing concept of sudden infant death syndrome: Diagnostic coding shifts, controversies regarding the sleeping environment, and new variables to consider in reducing risk. Pediatrics 2005;116:1245–1255. 5. Alm B, Milerad J, Wennergren G, et al. A case-control study of smoking and sudden infant death syndrome in the Scandinavian countries, 1992 to 1995. The Nordic Epidemiological SIDS Study. Arch Dis Child 1998;78:329–334. 6. Klonoff-Cohen HS, Edelstein SL, Lefkowitz ES, et al. The effect of passive smoking and tobacco exposure through breast milk on sudden infant death syndrome. JAMA 1995;273:795–798. 7. MacDorman MF, Cnattingius S, Hoffman HJ, Kramer MS, Haglund B. Sudden infant death syndrome and smoking in the United States and Sweden. Am J Epidemiol 1997;146:249–257. 8. Duke SG, Postma GN, McGuirt WF, Ririe D, Averill DB, Koufman JA. Laryngospasm and diaphragmatic arrest in immature dogs after laryngeal acid exposure: A possible model for sudden infant death syndrome. Ann Otol Rhinol Laryngol 2001;110:729–733. 9. Gershan WM, Becker CG, Forster HV, Besch NS, Lowry TF. Apnea and bradycardia due to anaphylaxis to tobacco glycoprotein in the infant rabbit. Environ Res 2004;94:152–159. 10. Huang YH, Brown AR, Costy-Bennett S, Luo ZL, Fregosi RF. Influence of prenatal nicotine exposure on postnatal development of breathing pattern. Respir Physiol Neurobiol 2004;143:1–8. 11. Kahraman L, Thach BT. Inhibitory effects of hyperthermia on mechanisms involved in autoresuscitation from hypoxic apnea in mice: A model for thermal stress causing SIDS. J Appl Physiol 2004;97: 669–674. 12. Kinney HC, Filiano JJ, Harper RM. The neuropathology of the sudden infant death syndrome. A review. J Neuropathol Exp Neurol 1992;51:115–126. 13. Harper RM, Kinney HC, Fleming PJ, Thach BT. Sleep influences on homeostatic functions: Implications for sudden infant death syndrome. Respir Physiol 2000;119:123–132. 14. Abdel-Rahman A, Dechkovskaia A, Mehta-Simmons H, Guan X, Khan W, Abou-Donia M. Increased expression of glial fibrillary acidic protein in cerebellum and hippocampus: Differential effects on neonatal brain regional acetylcholinesterase following maternal exposure to combined chlorpyrifos and nicotine. J Toxicol Environ Health 2003;66:2047–2066. 15. Abdel-Rahman A, Dechkovskaia AM, Mehta-Simmons H, et al. Maternal exposure to nicotine and chlorpyrifos, alone and in combination, leads to persistently elevated expression of glial fibrillary acidic protein in the cerebellum of the offspring in late puberty. Arch Toxicol 2004;78:467–476. 16. Abdel-Rahman A, Dechkovskaia AM, Sutton JM, et al. Maternal exposure of rats to nicotine via infusion during gestation produces neurobehavioral deficits and elevated expression of glial fibrillary acidic protein in the cerebellum and CA1 subfield in the offspring at puberty. Toxicology 2005;209:245–261. 17. Huang ZG, Wang X, Evans C, Gold A, Bouairi E, Mendelowitz D. Prenatal nicotine exposure alters the types of nicotinic receptors that facilitate excitatory inputs to cardiac vagal neurons. J Neurophysiol 2004;92:2548–2554. 18. Lichtensteiger W, Ribary U, Schlumpf M, Odermatt B, Widmer HR. Prenatal adverse-effects of nicotine on the developing brain. Prog Brain Res 1988;73:137–157. 19. Blackwell CC, Moscovis SM, Gordon AE, et al. Cytokine responses and sudden infant death syndrome: Genetic, developmental,

20. 21. 22. 23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35. 36.

37. 38. 39.

40.

41.

and environmental risk factors. J Leukoc Biol 2005;78:1242– 1254. Harper RM. Autonomic control during sleep and risk for sudden death in infancy. Arch Ital Biol 2001;139:185–194. Berry PJ. Pathological findings in SIDS. J Clin Pathol 1992;45: 11–16. Krous HF. Sudden infant death syndrome: Pathology and pathophysiology. Pathol Annu 1984;19:1–14. Harrison M, Curran C, Gillan JE. Mast-cell degranulation suggests nonimmune anaphylaxis as a cause of deaths in SIDS—an electronmicroscopy study. Lab Invest 1992;66:P5. Holgate ST, Walters C, Walls AF, et al. The anaphylaxis hypothesis of sudden infant death syndrome (SIDS): Mast cell degranulation in cot death revealed by elevated concentrations of tryptase in serum. Clin Exp Allergy 1994;24:1115–1122. Rambaud C, Guibert M, Briand E, Grangeot-Keros L, CoulombL’Hermin A, Dehan M. Microbiology in sudden infant death syndrome (SIDS) and other childhood deaths. FEMS Immunol Med Microbiol 1999;25:59–66. Toth LA, Chaudhary MA. Developmental alterations in auditory arousal from sleep in healthy and virus-infected cats. Sleep 1998;21: 143–152. Harper RM, Woo MA, Alger JR. Visualization of sleep influences on cerebellar and brainstem cardiac and respiratory control mechanisms. Brain Res Bull 2000;53:125–131. Sayers NM, Drucker DB, Morris JA, Telford DR. Lethal synergy between toxins of staphylococci and enterobacteria: Implications for sudden infant death syndrome. J Clin Pathol 1995;48:929– 932. Sayers NM, Drucker DB, Morris JA, Telford DR. Significance of endotoxin in lethal synergy between bacteria associated with sudden infant death syndrome: Follow up study. J Clin Pathol 1996;49: 365–368. Drucker DB, Aluyi HA, Morris JA, Telford DR, Oppenheim BA, Crawley BA. Possibility of separating toxins from bacteria associated with sudden infant death syndrome using anion exchange chromatography. J Clin Pathol 1992;45:802–805. Drucker DB, Aluyi HS, Morris JA, Telford DR, Gibbs A. Lethal synergistic action of toxins of bacteria isolated from sudden infant death syndrome. J Clin Pathol 1992;45:799–801. Jakeman KJ, Rushton DI, Smith H, Sweet C. Exacerbation of bacterial toxicity to infant ferrets by influenza virus: Possible role in sudden infant death syndrome. J Infect Dis 1991;163:35– 40. Lundemose JB, Smith H, Sweet C. Cytokine release from human peripheral blood leucocytes incubated with endotoxin with and without prior infection with influenza virus: Relevance to the sudden infant death syndrome. Int J Exp Pathol 1993;74:291–297. Bonham AC, Chen CY, Mutoh T, Joad JP. Lung C-fiber CNS reflex: Role in the respiratory consequences of extended environmental tobacco smoke exposure in young guinea pigs. Environ Health Perspect 2001;109:573–578. Nakamura A, Fukuda Y, Kuwaki T. Sleep apnea in mice. FASEB J 2003;17:A1212. Nakamura A, Fukuda Y, Kuwaki T. Sleep apnea and effect of chemostimulation on breathing instability in mice. J Appl Physiol 2003;94:525–532. Nakamura A, Fukuda Y, Kuwaki T. Mouse as an animal model of sleep apnea. Sleep 2003;26:A232–A233. Hendrickx AG, Tarantal AF. Infant-mortality—the role of the macaque as a model for human disease. Am J Primatol 1994;34:35–40. Emery MJ, Hlastala MP, Matsumoto AM. Depression of hypercapnic ventilatory drive by testosterone in the sleeping infant primate. J Appl Physiol 1994;76:1786–1793. Post EJ, Wood AK, Page M, Jeffery HE. A method for simultaneous physiological and radiographic recordings from sleeping neonatal piglets. Sleep 1995;18:309–316. Lavoue S, Dagorne M, Morvan H, Madec F, Durigon M. Is the piglet a useful animal-model of sudden-infant-death-syndrome. Biol Neonate 1994;65:310–316.

CHAPTER 60 / ANIMAL MODELS OF SUDDEN INFANT DEATH SYNDROME

42. Galland BC, Peebles CM, Bolton DPG, Taylor BJ. Sleep state organization in the developing piglet during exposure to different thermal stimuli. Sleep 1993;16:610–619. 43. Galland BC, Peebles CM, Bolton DPG, Taylor BJ. Oxygenconsumption in the newborn piglet during combined cold face hot body exposure. J Paediatr Child Health 1992;28:S33–S35. 44. Galland BC, Peebles CM, Bolton DPG, Taylor BJ. The microenvironment of the sleeping newborn piglet covered by bedclothes— gas-exchange and temperature. J Paediatr Child Health 1994;30: 144–150. 45. Elder DE, Bolton DP, Dempster AG, Taylor BJ, Broadbent RS. Pathophysiology of overheating in a piglet model: Findings compared with sudden infant death syndrome. J Paediatr Child Health 1996;32:113–119. 46. Voss LJ, Bolton DPG, Galland BC, Taylor BJ. Effects of prior hypoxia exposure, endotoxin and sleep state on arousal ability to airway obstruction in piglets: Implications for sudden infant death syndrome. Biol Neonate 2005;88:145–155. 47. Curran AK, Xia L, Leiter JC, Bartlett D. Elevated body temperature enhances the laryngeal chemoreflex in decerebrate piglets. J Appl Physiol 2005;98:780–786. 48. Xia L, Leiter JC, Bartlett D. Laryngeal water receptors are insensitive to body temperature in neonatal piglets. Respir Physiol Neurobiol 2006;150:82–86. 49. Jeffery HE, Page M, Post EJ, Wood AKW. Physiological-studies of gastroesophageal reflux and airway protective responses in the young animal and human infant. Clin Exp Pharmacol Physiol 1995;22: 544–549. 50. McKelvey GM, Post EJ, Wood AK, Jeffery HE. Airway protection following simulated gastro-oesophageal reflux in sedated and sleeping neonatal piglets during active sleep. Clin Exp Pharmacol Physiol 2001;28:533–539. 51. Richardson MA, Adams J. Fatal apnea in piglets by way of laryngeal chemoreflex: Postmortem findings as anatomic correlates of sudden infant death syndrome in the human infant. Laryngoscope 2005;115: 1163–1169. 52. Togari H, Kato I, Yamaguchi N. Reduction of cerebral blood flow produces respiratory arrest in hypercarbic newborn piglets: A possible pathogenesis of sudden infant death syndrome. Dev Brain Dysfunct 1996;9:253–257. 53. Froen TF, Aker H, Stray-Pedersen B, Saugstad OD. Adverse effects of nicotine and interleukin-1 beta on autoresuscitation after apnea in piglets: Implications for sudden infant death syndrome. Pediatrics 2000;105:E52. 54. Froen JF, Amerio G, Stray-Pedersen B, Saugstad OD. Detrimental effects of nicotine and endotoxin in the newborn piglet brain during severe hypoxemia. Biol Neonate 2002;82:188–196. 55. Tong SW, Ingenito S, Frasier ID, Sica AL, Gootman N, Gootman PM. Effects of cardiac autonomic imbalance on postnatal changes in the electrocardiographic Q-T interval in conscious swine. J Auton Nerv Syst 1997;64:162–165. 56. Tong SW, Frasier ID, Ingenito S, Sica AL, Gootman N, Gootman PM. Age-related effects of cardiac sympathetic denervation on the responses to cardiopulmonary receptor stimulation in piglets. Pediatr Res 1997;41:72–77. 57. Tong SW, Ingenito S, Anderson JE, Gootman N, Sica AL, Gootman PM. Development of a swine animal-model for the study of suddeninfant-death-syndrome. Lab Anim Sci 1995;45:398–403. 58. Gingras JL, Weesemayer D. Maternal cocaine addiction. 2. An animal-model for the study of brain-stem mechanisms operative in sudden-infant-death-syndrome. Med Hypotheses 1990;33: 231–234. 59. Weesemayer DE, Klemkawalden LM, Gingras JL, Brouillette RT. Effects of in utero cocaine exposure on postnatal respiration in the newborn rabbit. Pediatr Res 1989;25:A331. 60. Weesemayer DE, Klemkawalden LM, Barkov GA, Gingras JL. Effects of prenatal cocaine on the ventilatory response to hypoxia in newborn rabbits. Dev Pharmacol Ther 1992;18:116–124. 61. Pickering LK, Snyder JD, Behrman RE, Kliegman RM, Jenson HB. Gastroenteritis. In: Behrman RE, Kliegman RM, Jenson HB, Eds.

62.

63.

64.

65.

66.

67.

68.

69. 70.

71.

72. 73.

74.

75. 76.

77.

78.

79.

80.

589

Nelson Textbook of Pediatrics, 16th ed. Philadelphia, PA: W.B. Saunders, 2000:765–768. Siarakas S, Damas E, Murrell WG. Is cardiorespiratory failure induced by bacterial toxins the cause of sudden infant death syndrome? Studies with an animal model (the rabbit). Toxicon 1995; 33:635–649. Wetmore RF. Effects of acid on the larynx of the maturing rabbit and their possible significance to the sudden-infant-death-syndrome. Laryngoscope 1993;103:1242–1254. Kulangara AC, Schechtman AM. Passage of heterologous serum proteins from mother to foetal compartments in the rabbit. Am J Physiol 1962;203:1071–1080. Siarakas S, Damas E, Murrell WG. The effect of enteric bacterial toxins on the catecholamine levels of the rabbit. Pathology 1997;29:278–285. Lee S, Barson AJ, Drucker DB, Morris JA, Telford DR. Lethal challenge of gnotobiotic weanling rats with bacterial isolates from cases of sudden infant death syndrome (SIDS). J Clin Pathol 1987;40: 1393–1396. Orienstein DM. Aspiration pneumonias and gastroesophageal refluxrelated respiratory disease. In: Behrman RE, Kliegman RM, Jenson HB, Eds. Nelson Textbook of Pediatrics, 16th ed. Philadelphia, PA: W.B. Saunders, 2000:1288–1291. Pattison CP, Marshall BJ, Scott LW, Herndon B, Willsie SK. Proposed link between Helicobacter pylori (HP) and sudden infant death syndrome (SIDS): Possible pathogenic mechanisms in an animal model. I. Effects of intratracheal urease. Gastroenterology 1998;114: A900. Elitsur Y. Helicobacter pylori and SIDS: The jury is in at last! Am J Gastroenterol 2002;97:1576–1577. Ho GY, Windsor HM, Snowball B, Marshall BJ. Helicobacter pylori is not the cause of sudden infant death syndrome (SIDS). Am J Gastroenterol 2001;96:3288–3294. Blair PS, Fleming PJ, Bensley D, et al. Smoking and the sudden infant death syndrome: Results from 1993–1995 case-control study for confidential inquiry into stillbirths and deaths in infancy. Confidential enquiry into stillbirths and deaths regional coordinators and researchers. BMJ 1996;313:195–198. Slotkin TA. Fetal nicotine or cocaine exposure: Which one is worse? J Pharmacol Exp Ther 1998;285:931–945. Fewell JE, Smith FG, Ng VKY. Prenatal exposure to nicotine impairs protective responses of rat pups to hypoxia in an age-dependent manner. Respir Physiol 2001;127:61–73. Slotkin TA, Saleh JL, McCook EC, Seidler FJ. Impaired cardiac function during postnatal hypoxia in rats exposed to nicotine prenatally: Implications for perinatal morbidity and mortality, and for sudden infant death syndrome. Teratology 1997;55:177– 184. St John WM, Leiter JC. Maternal nicotine depresses eupneic ventilation of neonatal rats. Neurosci Lett 1999;267:206–208. Evans C, Wang J, Neff R, Mendelowitz D. Hypoxia recruits a respiratory-related excitatory pathway to brainstem premotor cardiac vagal neurons in animals exposed to prenatal nicotine. Neuroscience 2005;133:1073–1079. Neff RA, Simmens SJ, Evans C, Mendelowitz D. Prenatal nicotine exposure alters central cardiorespiratory responses to hypoxia in rats: Implications for sudden infant death syndrome. J Neurosci 2004; 24:9261–9268. Huang ZG, Griffioen KJS, Wang X, et al. Differential control of central cardiorespiratory interactions by hypercapnia and the effect of prenatal nicotine. J Neurosci 2006;26:21–29. Huang ZG, Wang X, Dergacheva O, Mendelowitz D. Prenatal nicotine exposure recruits an excitatory pathway to brainstem parasympathetic cardioinhibitory neurons during hypoxia/hypercapnia in the rat: Implications for sudden infant death syndrome. Pediatr Res 2005;58:562–567. Abreu-Villaca Y, Seidler FJ, Slotkin TA. Does prenatal nicotine exposure sensitize the brain to nicotine-induced neurotoxicity in adolescence? Neuropsychopharmacology 2004;29:1440– 1450.

590

SECTION VI / MODELS OF OTHER HUMAN DISEASES

81. Gleeson M, Cripps AW. Development of mucosal immunity in the first year of life and relationship to sudden infant death syndrome. FEMS Immunol Med Microbiol 2004;42:21–33. 82. Dye JA, Morgan KT, Neldon DL, Tepper JS, Burleson GR, Costa DL. Characterization of upper respiratory disease in rats following neonatal inoculation with a rat-adapted influenza virus. Vet Pathol 1996;33:43–54. 83. Blood-Siegfried, Nyska A, Lieder H, et al. Synergistic effect of influenza A virus on endotoxin-induced mortality in rat pups: A potential model for sudden infant death syndrome. Pediatr Res 2002;52:481–490. 84. Blood-Siegfried, Nyska A, Geisenhoffer K, et al. Alteration in regulation of inflammatory response to influenza A virus and endotoxin in suckling rat pups: A potential relationship to sudden infant death syndrome. FEMS Immunol Med Microbiol 2004;42:85–93. 85. Ehrlich JP, Burleson GR. Enhanced and prolonged pulmonary influenza virus infection following phosgene inhalation. J Toxicol Environ Health 1991;34:259–273. 86. Liu QL, Wong-Riley MTT. Postnatal developmental expressions of neurotransmitters and receptors in various brain stem nuclei of rats. J Appl Physiol 2005;98:1442–1457. 87. Liu QL, Wong-Riley MTT. Developmental changes in the expression of GABA(A) receptor subunits alpha 1, alpha 2, and alpha 3 in the rat pre-Botzinger complex. J Appl Physiol 2004;96:1825–1831.

88. Wong-Riley MTT, Liu QL. Neurochemical development of brain stem nuclei involved in the control of respiration. Respir Physiol Neurobiol 2005;149:83–98. 89. Burleson G. Influenza virus host resistance model for assessment of imunotoxicity, immunostimulation, and antiviral compounds. In: Burleson G, Dean JH, Munson AE, Eds. Methods in Immunotoxicology, Vol. 2. New York: Wiley-Liss, 1995:181–202. 90. Burleson G. Pulmonary immunocompetence and pulmonary immunotoxicology. In: Smialowicz RJ, Holsapple MP, Eds. Experimental Immunotoxicology. Boca Raton, FL: CRC Press, 1996:119–141. 91. Lomnitski L, Nyska B, Ben-Shaul V, et al. Effects of antioxidants apocynin and the natural water-soluble antioxidant from spinach on cellular damage induced by lipopolysaccaride in the rat. Toxicol Pathol 2000;28:580–587. 92. Gehrs BC, Riddle MM, Williams WC, Smialowicz RJ. Alterations in the developing immune system of the F344 rat after perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin: II. Effects on the pup and the adult. Toxicology 1997;122:229. 93. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981;29:577–580.

61

Animal Models of Posttraumatic Stress Disorder HAGIT COHEN, MICHAEL A. MATAR, AND JOSEPH ZOHAR

ABSTRACT

studies report that the rate of lifetime exposure to at least one “serious” traumatic event (excluding grief and mourning) is quite high; a conservative estimate reported 61% among men and 51% among women.8 Other studies have found similar rates.9–14 The lifetime prevalence of PTSD in the general population reaches about 7% overall,15 suggesting that about 20–30% of individuals exposed to severe stressors will develop PTSD.9 This figure varies depending on the type of trauma studied, where male rape victims suffer very high rates and populations exposed to natural disasters significantly less.16 The discrepancy between the proportion of the general population exposed to PTE and those who eventually fulfill criteria for the disorder suggests qualitative differences in vulnerability and/or resilience. After extensive study over the past two decades, PTSD was established as a disorder and listed in the Diagnostic and Statistic Manual (DSM). A diagnosis of PTSD is made if the required symptoms are present 1 month or more after exposure to a triggering event: (1) intrusive reexperiencing of the traumatic event in the form of nightmares and flashbacks, with an exaggerated response to trauma-related reminders/cues; (2) persistent avoidance of stimuli associated with the trauma and emotional numbing; and (3) persistent symptoms of exaggerated startle response, increased physiological arousal, and sustained preparedness for an instant alarm response.1 PTSD has severe effects on widespread areas of the individual’s functioning, severely compromising their quality of life and affecting their workplace, family, and social life. Moreover, PTSD is also often comorbid with other disorders such as depressive and anxiety disorders, drug and alcohol abuse, cognitive and memory impairments, and sexual dysfunction.1 The development of PTSD is often a gradual process and extends over time through a series of stages ranging from relatively contained distress to severe disability.17 As the disorder evolves over time, pathological changes and debilitating comorbidity may become fixed and irreversible. Unlike processes in which exposure to repeated stimuli induces a process of learning or conditioning, implying an increased efficiency in processing of data to produce the required response, the psychopathology underlying PTSD produces a paradoxical vulnerability to negative sequelae upon subsequent exposure to stress.17 The sequelae of exposure to a traumatizing stressor are subject to extensive clinical study. Clinical research often gives rise to important questions or hypotheses as to the pathogenesis, clinical course, and outcomes of such events. Among the issues raised are

Posttraumatic stress disorder (PTSD) affects about 20–30% of exposed individuals. Clinical studies of PTSD generally employ stringent criteria for inclusion in study populations, and yet in animal studies the data collection and analysis are generally expressed as a function of “exposed” versus “nonexposed” populations, regardless of individual variation in response. There is a need for a better parallel between the approach to understanding animal behavioral models and the contemporary understanding of the clinical conditions. We conceived an approach to understanding the consequences of stress exposure in a manner that would enable us to segregate the study animals into groups according to the degree of their response to a stressor, i.e., the degree to which their behavior is altered or disrupted. The idea was to set apart the most clearly affected, i.e., “extreme behavioral response” group from the “minimal behavioral response” at the extremes, and to establish a middle group of partial responders, groups that better reflect behavioral criteria akin to clinical symptoms already defined. This chapter presents an overview of a series of studies, examining the contribution, validity, and practicality of this animal model. Key Words: Posttraumatic stress disorder, Animal model, Hypothalamic–pituitary–adrenal axis, Early-life stress, Juvenile stress, Corticosterone, Brain-derived neurotrophic factor.

POSTTRAUMATIC STRESS DISORDER Posttraumatic stress disorder (PTSD) is an incapacitating chronic syndrome reflecting a disorder of cognitive, emotional, and physiological processing and/or recovery from the initial reaction to exposure to a potentially traumatic experience (PTE).1 In the first hours to days following the experience the majority of individuals exposed to an extreme event will demonstrate, to a varying degree, symptoms such as intense fear, helplessness, or horror followed by anxiety, depression, agitation, shock, or dissociation, and may have trouble functioning in their usual manner for a while.2–7 Retrospective and prospective epidemiological studies indicate that most individuals affected by a PTE will adapt within a period of 1–4 weeks following exposure,2,6 and only a small proportion will develop long-term psychopathology.2,6 In the United States, From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

591

SECTION VI / MODELS OF OTHER HUMAN DISEASES

The basis of PTSD as a clinical entity rests upon etiology and requires exposure to a severely stressful experience or traumatic event. Thus, exposure to a stressogenic/traumatic experience has formed the basis for animal models of PTSD. There are as yet no uniformly accepted animal models for PTSD. A number of animal models have been developed in which intense stressful experiences, aversive challenges, and situational reminders of a traumatic stressor have been shown to have longterm effects on psychological/behavioral and physiological functioning, reflected in biobehavioral paradigms and biophysiological tests, e.g., hypothalamic–pituitary–adrenal (HPA) axis assays and electrophysiological studies.18–30 Irrespective of the study design or of the stress paradigm, animal studies have generally included the entire stress-exposed population as the study population and the results discussed and conceptualized as involving this population versus “others.” However, in practice there is considerable heterogeneity in the degree of behavioral disruption or response to the stressor and animals tend to demonstrate considerable individual differences, certainly in the degree to which their behavior is disrupted.

THE CUT-OFF BEHAVIORAL CRITERIA MODEL One way to reconcile this disparity is to regard the heterogeneity of animal responses to stress paradigms as modeling the heterogeneity of human responses to traumatic stress, and to develop means by which the degree of the individual’s behavioral response forms the basis for a system of classification, akin to the inclusion and exclusion criteria employed in clinical studies. Since the scope of animal models is limited to observable and measurable parameters, the behavioral criteria for the model of stress-induced conditions developed by this study group are based on two well-established paradigms that measure anxiety-like, fearful, avoidant and hypervigilant/hyperalert behaviors, paralleling aspects of traumatic stress-induced human behaviors.25,31 Exploratory behavior on the elevated plus maze (EPM) serves as the main platform for the assessment of these behaviors and the acoustic startle response (ASR) paradigm allows for a precise quantification of hyperalertness, in terms of magnitude of response and habituation to the stimulus. In our model, we exposed adult rats to a predator scent (cat urine) stressor (PSS) for 10 min as previously described by Adamec et al.23,25,26 and others.28–30,32–35 Regarding the conceptual

2000

1500

1000

100 75 50 25

500

ation

ANIMAL MODELS OF POSTTRAUMATIC STRESS DISORDER

validity of the model itself, “predator exposure trauma” is a potentially life-threatening situation (criteria A) and it may represent a more ‘‘natural’’ setting than other types of stressors on a teleological level,23,24 such as electrical tail shocks and restraint which might possibly be related to extreme conditions like torture. Seven days after a single 10-min PSS exposure, the exposed rats showed significantly decreased time spent in the open arms of the maze and increased levels of avoidance and anxiety-like behavior in the EPM, and higher mean startle responses as compared to control rats. However, the animals’ behavior was not uniformly disturbed, but rather demonstrated a broad range of variation in severity of anxiety-like behaviors (Figure 61–1). Quantitative and qualitative assessment of individual animal behavioral responses to stress exposure provides a degree of standardization that enables a greater degree of resolution. For instance, groups of subjects can be defined by their response pattern or magnitude and selectively focused on for study or followed up over time; the prevalence of defined patterns of response may be assessed and compared, allowing the “potency” of different stressors to be compared, behaviors to be correlated to physiological parameters, and so on. Since partial responses are far more difficult to quantify and interpret, in animal models more so than in human subjects, the studies carried out according to this approach to date have preferred specificity to sensitivity, and have sought to create clearly defined groups of behaviors for comparison. By means of well-defined cut-off behavioral criteria (CBCs), the studies have focused on animals displaying an extreme behavioral response (EBR) on both the EPM and the ASR paradigms performed consecutively, compared to those displaying a minimal

0 0

1

Time s pe

2

nt in th

3

e open

4

arms (m i n)

St ar tle H abitu

those relating to factors that may confer risk or resilience for the development of more severe stress-induced clinical outcomes, such as PTSD. By their nature, clinical studies raise issues concerning premorbid factors largely by means of extrapolating retrospectively. Prospective studies are almost impossible to conceive and would most probably be prohibitively expensive to put into practice. An animal model can give a good approximation of certain aspects of the complex clinical disorder at best, enabling the study of questions raised in clinical research in a prospective study design and under far more controllable conditions. To maximize its validity as an extension of clinical research, the design of the animal model must strive to conform as closely as possible to the clinical entity and the contemporary conception of the disorder.

Startle Amplitude

592

0 -25 -50 5

Figure 61–1. The effect of single PSS exposure versus unexposed control on rat anxiety-like behavior and acoustic startle response and habituation. The representation of the data from both paradigms (EPM and ASR) shows two obvious and rather distinct features. First, it is clear that PSS exposure alters the response of the majority of individuals to at least some degree. Second, the cluster of individuals that forms in the upper left hand corner of the graph (i.e., had the more extreme responses to exposure) is quite distinct from the majority of individuals.36,48 (See color insert.)

CHAPTER 61 / ANIMAL MODELS OF POSTTRAUMATIC STRESS DISORDER

behavioral response (MBR) to both, and compared to controls. “Maladaptive” responses to both of these serial CBCs were required for “inclusion” into the EBR group, whereas the reverse was required for inclusion in the MBR group.36 The criteria for each behavioral paradigm were as follows: 1. Extreme behavioral response: 5 min (entire session) spent in the closed arms and no entries into the open arms on the EPM; the mean amplitude of the startle response (at 110 dB) exceeds 800 units and the startle response shows no habituation over time. 2. Minimal behavioral response: 0–1 min spent in the closed arms and eight or more open arm entries on the EPM; the mean amplitude of the startle response (at 110 dB) does not exceed 700 units and habituation is demonstrated. 3. Partial behavioral response: animals neither EBR nor MBR are termed “partial behavioral responders” (PBR). When behavioral data for the entire exposed population were examined in this way, based on the segregation of animals according to the CBCs, it was demonstrated that only about 25% develop significant extreme behavioral disruptions as the result of exposure to the stressor (Figure 61–2A), as compared to 1.3% in controls (Figure 61–2B). Similarly, the prevalence of MBR rats in the exposed groups was 24.7% (Figure 61–2A) and in the control group was 80.0% (Figure 61–2B).36 These results are in agreement with trends in the literature on human studies,9 mentioned above, in which about 15–35% fulfill criteria and another 20–30% are considered as PTSD spectrum disorders.9,10,13 It was therefore suggested that application of CBCs is both feasible and valid. The above-mentioned approach to the analysis of animal data is conceptually equivalent to DSM criteria for PTSD and the criteria proposed by Yehuda and Antelman.37 The demonstrated reduction in the time spent by rats in the open arms of the plus maze may parallel avoidance behavior seen in human PTSD (Criteria C). Since the total number of entries into any arms of the plus maze is the same, except that the EBR rats keep to the closed arms only, the results are consistent with anxiety, and not with nonspecific impairment of locomotion. DSM-IV defines this symptom as a persistent avoidance of reminders of the trauma and numbing of responsiveness. Since the exposure took place in an open space it is in keeping with the described definition.

PSS-exposure

A

593

The assessment of rats after 7 days has previously been established by Adamec et al.,25 and is taken to refer to PTSD and not to acute stress reaction. Our results have since shown repeatedly that as of day 7, the prevalence rate remains constant at about 25% until day 30. The DSM-IV defines PTSD as lasting more than a month after the exposure. So the persistence of anxiety symptoms on day 7 after the exposure fulfills the time element of PTSD.25 In the next step, we examined the validity of applying the CBC concept in animal studies, using dynamic unsupervised fuzzy clustering. Unsupervised fuzzy clustering (UFC) analysis is a mathematical tool that attempts to assess the relationships among patterns of a data set by organizing the patterns into groups or clusters, such that patterns within one cluster are more similar to each other than patterns belonging to different clusters.38–42 The main goal of most clustering methods is to supply useful information by grouping data in clusters, where within each cluster the data exhibit similarity. Similarity is defined by a distance measure, and global objective functional or regional graph theoretical criteria are optimized to find the optimal partition of data. The partition approach defines the cluster to which each data element belongs by using the elements of the membership matrix.38–42 The full range of the dataset was classified into three (C = 3) clusters. The data in the clusters that the model yielded corresponded to the data for the animals designated behaviorally by means of the CBCs as clearly affected, partially affected (including some unexposed animals), and clearly unaffected (Figure 61–3). The UFC analysis yielded, in addition to the “anxious” and the “baseline” clusters, a third group corresponding to an extreme pathological response (see Figure 61–3). According to this analysis, about 22% develop extreme behavioral disruptions as the result of exposure to the stressor, as compared to 0.7% of the unexposed control group. This extreme response group (i.e., maladapted rats) exhibits significantly higher startle response and significantly more avoidance behavior in the EPM than other animals. Again, these proportions are in agreement with current trends in the literature on PTSD studies in human subjects.9 Taken together, the UFC analysis supports the conceptual contention of the CBCs and thus lends validity to our previous studies. Moreover, the clustering process may help us to find and define the characteristics of the subgroup of the unexposed population that has a higher risk of developing PTSD.43

Un-exposed control

B 50%

PBR 80.01%

MBR 24.7%

PBR

18.66%

EBR

25.3%

Figure 61–2.

MBR

1.33% EBR

Reanalysis of data applying cut-off behavioral criteria. (A) PSS exposure. (B) Unexposed control.

594

A C T I V I T Y

SECTION VI / MODELS OF OTHER HUMAN DISEASES

2

25

3

20 15 10 5 0 5 4

2000 3

1

2

TIME SPENT IN THE CLOSED ARMS

1500

1

1000 500

ACOUSTIC STARTLE AMPLITUDE

In the next section, we present a series of studies that were constructed to examine the prevalence of EBR and MBR responses over time, in an attempt to map the transition from the immediate reaction, concomitant with an acute stress response, to more enduring and chronic changes, representing PTSD, under different conditions.44 By means of singling out the clearly affected individuals labeled as EBR from among the entire population of rats exposed to the stress paradigms, and focusing on them, a time curve could be constructed to reflect the prevalence of clearly “symptomatic” behavior over time (Figure 61–4). Immediately following the traumatic stimulus, most of the animals (90%) manifested a pattern of behaviors that fulfills criteria for EBR status for the first few hours. This can be regarded as being equivalent to the initial response observed in human subjects in the first hours or days after an extreme event. The prevalence of rats fulfilling the CBC criteria for EBR decreased rapidly, and subsequent sampling revealed that at the

Figure 61–3. Unsupervised fuzzy clustering: Partition of four behavioral parameters into three clusters. The ellipses represent the standard error of each characteristic in each cluster. The first cluster (C1) is related to baseline behavior associated with unstressed animals and is characterized by very low levels of anxiety (MBR). The middle cluster (cluster C3) represents the subclinical or partially affected animals and is characterized by some degree of anxiety and stress levels (PBR). Cluster C2 corresponds to the exposed animals and is characterized by higher levels of anxiety and stress, namely the extremely affected animals (EBR).43 The behavioral parameters were acoustic startle response amplitude, time spent in the closed arms on the EPM, number of entries to the closed arms, and the total number of entries to the open and the closed arms of the EMP (total activity).43

end of a week only 25% of the total exposed population had PTSD-like symptoms. The time curve appears to indicate that the rats responded initially with a ubiquitous, probably normative, acute response, but only about 15% went on to suffer from enduring behavioral effects. The curve representing these responses, although gleaned from different groups of rats, very clearly resembles, both in term of time and of prevalence, the curve of human responses to exposure to a significant stressor and the proportions affected.45 This pattern of symptoms has been demonstrated across the different kinds of traumatic events, both in human PTSD and in the different models in this study. We submit that the distinction between the acute phase of the stress response seen in most subjects immediately after exposure and the chronic enduring effects associated with stress found after several days or weeks is important in the study of animal models dealing with the behavioral and physiological consequences of stress.44

Prevalence of EBR rats

100 90 80 70 60 50 40 30 20 10 0

1

3

5 7 14 D ays after P S S -ex p o su re

30

90

Figure 61–4. The prevalence of EBR rats after single exposure to a predator stimulus along the time axis. A steady drop in prevalence of EBR rats from 90% on day 1 to 15% by day 90 is shown. As of day 7, this rate remains constant until day 30. Our results have shown a progressive, timedependent decline in the prevalence of EBR rats during the first few weeks that follow a trauma.44

CHAPTER 61 / ANIMAL MODELS OF POSTTRAUMATIC STRESS DISORDER

BIOLOGICAL STRESS RESPONSE It is well known that the biological stress response is key to understanding the pathophysiology of PTSD and its associated symptoms. Thus, the primary aim of the following study was to analyze separately various biobehavioral parameters of the animals that responded in a more marked manner to exposure to a stressful, potentially traumatic, event and to compare these to the parameters of rats that demonstrated no such response. We submit that this approach would better approximate the individual variance seen in posttraumatic stress responses in humans (i.e., the clinical syndrome) in a more valid manner. In a series of studies this approach has elicited highly significant overlap between animals showing EBR and those with extreme biophysiological measures, i.e., HPA axis assays (corticosterone and neuroactive steroids), heart rate variability (reflecting autonomic nervous system activity), and BDNF mRNA and protein levels much more clearly than when the exposed group was analyzed as a whole.

HYPOTHALAMIC–PITUITARY–ADRENAL AXIS Exposure to stress or a threat to homeostasis triggers activation of the parvicellular neurons of the paraventricular nucleus in the hypothalamus to release corticotropin-releasing factor and vasopressin. These hormones stimulate synthesis of polypeptide precursor proopiomelanocortin products, which include adrenocorticotrophic hormone (ACTH), melanocyte-stimulating hormone cells, and β-endorphin, that have been shown to interact with the immune system.46,47 In the adrenal gland ACTH stimulates the release of glucocorticoids, typically cortisol in man and corticosterone in rodents. The responses of the HPA axis are regulated by an efficient negative feedback system, exerted by glucocorticoids. Taken together, the HPA stress hormones play

a major role in mediating both adaptive and maladaptive responses. We evaluated plasma corticosterone and ACTH levels 7 days after PSS exposure. Throughout the study data were analyzed for exposed versus unexposed populations as a whole, and then reanalyzed according to the CBC classification separately for EBR, MBR, and PBR versus unexposed controls, and finally these groups were compared to each other (Figure 61–5). Our results have shown that a single 10-min exposure to a PSS significantly increased plasma corticosterone (Figure 61–5A) and plasma ACTH concentrations (Figure 61–5B) only in EBR rats, but not in MBR individuals or in nonexposed controls.36,48 The fact that there is a clear-cut involvement of the HPA axis in the physiological component of the syndrome has been quite firmly established by studies in PTSD patients and animals, despite conflicting data reported by different groups.49–52 The results of the present study are in keeping with those of certain groups of investigators.53 Moreover, Adamec and colleagues24–26 have found evidence of differential dysregulation of the HPA axis in certain strains of rats exposed to cats. One week after cat exposure, Hooded rats show elevations in basal plasma corticosterone, whereas Wistar rats do not. This difference in strain reactivity was proposed to be related to the higher basal anxiety in Hooded rats. We may speculate that greater basal anxiety could even stem from factors such as response to the blood sampling procedure, or possibly that different strains possess different vulnerability factors. The fact that EBR rats show a dysfunction of the HPA axis, whereas MBR rats do not, indicates quite clearly that the population of exposed animals is not a homogeneous cohort, and that it is important to distinguish between affected and unaffected subjects in the analysis and interpretation of data from animal studies.36,48

A

B

p