Indwelling Neural Implants: Strategies for Contending with the In Vivo Environment (Frontiers in Neuroengineering Series)

INDWELLING NEURAL IMPLANTS STRATEGIES for CONTENDING with the INmnnVIVOENVIRONMENT © 2008 by Taylor & Francis Group, LL...

Author: William M. Reichert

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INDWELLING NEURAL IMPLANTS STRATEGIES for CONTENDING with the INmnnVIVOENVIRONMENT

© 2008 by Taylor & Francis Group, LLC

FRONTIERS IN NEUROENGINEERING Series Editors Miguel A.L. Nicolelis, M.D., Ph.D. Sidney A. Simon, Ph.D.

Published Titles Indwelling Neural Implants: Strategies for Contending with the In Vivo Environment William M. Reichert, Duke University, Durham, North Carolina

Electrochemical Methods for Neuroscience Adrian C. Michael, University of Pittsburg, Pennsylvania Laura M. Borland, University of Pittsburg, Pennsylvania


INDWELLING INDWELLING NEURAL NEURAL IMPLANTS IMPLANTS STRATEGIES for CONTENDING with the STRATEGIES for CONTENDING with the INVIVOENVIRONMENT ENVIRONMENT Edited by

William M. Reichert Duke Duke University University North North Carolina Carolina

CRC Press Taylor & Francis Croup Boca Raton London New York

CRC CRC Press Press is is an imprint of the the Taylor & Francis Francis Group, an an informa business business


CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2008 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-13: 978-0-8493-9362-4 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www. copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Indwelling neural implants : strategies for contending with the in vivo environment / editor, William M. Reichert. p. ; cm. -- (Frontiers in neuroengineering) “A CRC title.” Includes bibliographical references and index. ISBN 978-0-8493-9362-4 (hardbook : alk. paper) 1. Implants, Artificial. 2. Brain. 3. Electrodes. 4. Foreign-body reaction. 5. Nerve tissue. 6. Wound healing. I. Reichert, William M. II. Series: Frontiers in neuroengineering (Boca Raton, Fla.) [DNLM: 1. Central Nervous System--physiopathology. 2. Wound Healing--physiology. 3. Electrodes, Implanted--adverse effects. 4. Foreign-Body Reaction--pathology. WL 300 I421 2007] RD594.I515 2007 617.9’5--dc22 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com


2007029372

To Shirley Sue Reichert. Mother. Advisor. Friend.


Contents Series Preface............................................................................................................ix Preface ......................................................................................................................xi Acknowledgments.................................................................................................. xiii Editor ....................................................................................................................... xv Contributors ...........................................................................................................xvii

PART I Chapter 1

Overview of Wound Healing in Different Tissue Types......................3 John D. Stroncek and W. Monty Reichert

PART II Chapter 2

Signal Considerations for Chronically Implanted Electrodes for Brain Interfacing ................................................................................ 41 Warren M. Grill

Chapter 3

Thermal Considerations for the Design of an Implanted Cortical Brain–Machine Interface (BMI).......................................... 63 Patrick D. Wolf

PART III Chapter 4

In Vitro Models for Neuroelectrodes: A Paradigm for Studying Tissue–Materials Interactions in the Brain ........................ 89 Vadim Polikov, Michelle Block, Cen Zhang, W. Monty Reichert, and J. S. Hong

Chapter 5

In Vivo Solute Diffusivity in Brain Tissue Surrounding Indwelling Neural Implants ............................................................. 117 Michael J. Bridge and Patrick A. Tresco

vii © 2008 by Taylor & Francis Group, LLC

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Indwelling Neural Implants

PART IV Chapter 6

A Molecular Perspective on Understanding and Modulating the Performance of Chronic Central Nervous System (CNS) Recording Electrodes ....................................................................... 151 Wei He and Ravi V. Bellamkonda

Chapter 7

Soft, Fuzzy, and Bioactive Conducting Polymers for Improving the Chronic Performance of Neural Prosthetic Devices .................. 177 Dong-Hwan Kim, Sarah Richardson-Burns, Laura Povlich, Mohammad Reza Abidian, Sarah Spanninga, Jeffrey L. Hendricks, and David C. Martin

Chapter 8

Strategies for Regeneration and Repair in the Injured Central Nervous System................................................................................ 221 Molly S. Shoichet, Ciara C. Tate, M. Douglas Baumann, and Michelle C. LaPlaca


Series Preface The Frontiers in Neuroengineering series presents the insights of experts on emerging experimental techniques and theoretical concepts that are or will be at the vanguard of neuroscience. Books in the series cover topics ranging from methods to investigate apoptosis to modern techniques for neural ensemble recordings in behaving animals. The series also covers new and exciting multidisciplinary areas of brain research, such as computational neuroscience and neuroengineering, and describes breakthroughs in fields such as insect sensory neuroscience, primate audition, and biomedical engineering. The goal is for this series to be the reference that every neuroscientist uses to become acquainted with new methodologies in brain research. These books can be used by graduate students and postdoctoral fellows who are looking for guidance to begin a new line of research. Each book is edited by an expert and consists of chapters written by the leaders in a particular field. Books are richly illustrated and contain comprehensive bibliographies. Chapters provide substantial background material relevant to the particular subject. Hence, the books in this series are not the usual type of method books. They contain detailed “tricks of the trade” and information as to where particular methods can be safely applied. In addition, they include information about where to buy equipment and Web sites helpful in solving both practical and theoretical problems. Finally, they present detailed discussions of the present knowledge of the field and where it should go. We hope that, as the volumes become available, that our efforts as well as those of, the publisher, the book editors, and the individual authors will contribute to the further development of brain research. The extent to which we achieve this goal will be determined by the utility of these books. Sidney A. Simon, Ph.D. Miguel A.L. Nicolelis, M.D., Ph.D. Series Editors

ix © 2008 by Taylor & Francis Group, LLC

Preface In the quarter century since the idea of using neural signals to control an external effector prosthetic first surfaced, enormous strides have been made in understanding the neuronal circuitry of the relevant brain structures, developing the computer hardware and software algorithms capable of transforming the neuronal potentials into control signals, and engineering high-density recording arrays. In contrast, a number of fundamental and unresolved questions remain as to the source of signal degradation in chronically implanted neuroelectrodes. Is it the result of insertion trauma, micromotion, mechanical mismatch, or simply the consequences of glial scar formation arising from a normal chronic foreign body response? What roles do electrode size, shape, surface chemistry, mechanical impedance, and insulating material play? What is the fate of neurons adjacent to the recording site as the signal degrades? Are they silenced, killed, or just pushed out of the way by the glial scar? Do these effects arise from inhibitory cues expressed in the glial scar or by the inflammatory molecules released into the vicinity of the electrode? What happens when one introduces devices that stimulate the surrounding brain tissue? What is the impact on the surrounding tissue from telemetric communication and on-chip processing? What are the best methods for intervening in the repair of trauma caused by device implantation? At this point, nobody really knows; however, if answers are not found, then the field will never progress from its current state of the occasional well-publicized success to widespread use in humans. This text entitled Indwelling Neural Implants: Strategies for Contending with the In Vivo Environment is a compendium of contributions from a number of noted experts who are working in the field of neuroprosethetics and tissue repair. These experts were requested to contribute chapters that focused on this general theme, but I also encouraged them to elaborate on specific aspects important to future therapies for the response of central nervous system (CNS) tissue to implants. Combined, this text is a unique contribution to the CNS literature and one that certainly fits the theme of Taylor & Francis/CRC’s Frontiers in Neuroengineering series. The central feature of these chapters is the impact, characterization, and mitigation of the healing of the tissue that surrounds the implant. By and large, these chapters speak for themselves; however, the text can be divided thematically into four parts. Part I reviews and highlights the differences between wound healing in the CNS, peripheral nervous system, subcutaneous tissue, and bone. While healing in these tissues shares a number of molecular and cellular phenomena, differences arise rapidly as one progresses from the initial trauma to inflammation to repair. This chapter serves as the phenomenological basis for the subsequent contributions. Part II by Warren Gill and Patrick Wolf of Duke University is composed of two chapters that present the performance of implanted neuroprosthetics from the components perspective. Compared to the earliest “passive” recording electrodes, neuroprosthetics are increasingly “active” implants that impact the surrounding tissue xi © 2008 by Taylor & Francis Group, LLC

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chemically, mechanically, thermally, and electrically. These chapters present performance issues that arise from chronic tissue stimulation and heating effects from on-chip and telemetric processing. In Part III, Jau-Shyong Hong of the National Institute of Environmental Health Sciences and Patrick Tresco of the University of Utah contribute, respectively, in vitro and in vivo approaches to assessing the CNS wound healing response to materials. Dr. Hong’s chapter compares the complexity and the level of information obtained from the different cell culture and organotypic models. Dr. Tresco describes the application and numerical modeling of his chronic hollow-fiber-membrane in vivo implant model for assessing molecular transport to the surrounding brain tissue. Part IV consists of three chapters that describe molecular and materials strategies for intervening in CNS wound repair and enhancing the electrical communication between the electrode surface and the surrounding tissue. The chapter by Ravi Bellamkonda of the Georgia Institute of Technology stresses molecular approaches for biasing cellular migration during the various stages of healing around implanted electrodes to encourage greater and more specific cell–surface interaction. The chapter by David Martin of the University of Michigan presents the deployment and characterization of novel conducting polymers to facilitate the communication between the electrode surface and the adjacent neurons. Finally, the chapter by Molly Shoichet of the University of Toronto and Michelle LaPlaca of the Georgia Institute of Technology takes the broader view of addressing the sequelae of CNS traumatic injury (of which neuroprosthesis insertion is a specific example) and presents novel strategies for nerve regeneration and repair. William M. Reichert, Ph.D. Duke University Durham, North Carolina


Acknowledgments I extend my sincere appreciation to the lead authors and their coauthors for the hard work in preparing their fine contributions. I also thank series editors Sidney Simon and Miguel Nicolelis of Duke University for the opportunity to compile this text.


Editor William M. Reichert, Ph.D., was born in San Francisco, California, grew up in Ann Arbor, Michigan, and lives in Hillsborough, North Carolina, with his wife Kate, his son Stephen, and three dogs and a cat. He also has a daughter Elizabeth and a stepdaughter Miranda. He graduated with a B.A. in Biology and Chemistry from Gustavus Adolphus College in 1975, was a part-time student, a hospital technician and bartender for a couple of years, and received a doctorate in Macromolecular Science and Engineering from the University of Michigan in 1982. He was a National Institutes of Health (NIH) National Research Service Award Postdoctoral Fellow, a Whitaker Fellow, and a NIH New Investigator Fellow at the University of Utah in the Department of Bioengineering, where he “learned the ropes” from Professors Joe Andrade and Art Janata. He joined the Department of Biomedical Engineering at Duke University in 1989 and is currently Professor of Biomedical Engineering and Chemistry, and Director of the Center for Biomolecular and Tissue Engineering. He is a fellow of the American Institute of Medical and Biological Engineering and is on the editorial boards for the Journal of Biomedical Materials Research and Langmuir. He is program director of an NIH predoctoral training grant that supports graduate fellowships in biotechnology and has been a steadfast advocate for promoting underrepresented minorities in engineering graduate education, receiving the Catalyst for Institutional Change from Quality Education for Minorities (QEM) Network, Pioneer Award from the Samuel DuBois Cook Society, and the Dean’s Award for Excellence in Mentoring. His current research interests are wound healing-related implant failure, biosensors, vascular graft endothelialization, and cytokine profiling. He has trained a number of doctoral and postdoctoral students now working in academics and industry, he has published nearly 100 scientific papers, and he holds patents for multianalyte waveguide sensors and protein detection arrays.

xv © 2008 by Taylor & Francis Group, LLC

Contributors Mohammad Reza Abidian Department of Biomedical Engineering University of Michigan Ann Arbor, Michigan

Jeffrey L. Hendricks Department of Biomedical Engineering University of Michigan Ann Arbor, Michigan

M. Douglas Baumann Department of Chemical Engineering and Applied Chemistry University of Toronto Toronto, Ontario, Canada

J. S. Hong Neuropharmacology Section National Institute of Environmental Health Sciences National Institutes of Health Research Triangle Park, North Carolina

Ravi V. Bellamkonda Coulter Department of Biomedical Engineering Georgia Institute of Technology/Emory University Atlanta, Georgia

Dong-Hwan Kim Department of Biomedical Engineering Duke University Durham, North Carolina

Michelle Block Neuropharmacology Section National Institute of Environmental Health Sciences National Institutes of Health Research Triangle Park, North Carolina Michael J. Bridge Department of Bioengineering University of Utah Salt Lake City, Utah Warren M. Grill Department of Biomedical Engineering Duke University Durham, North Carolina Wei He Coulter Department of Biomedical Engineering Georgia Institute of Technology/Emory University Atlanta, Georgia

Michelle C. LaPlaca Coulter Department of Biomedical Engineering Georgia Institute of Technology/Emory University Atlanta, Georgia David C. Martin Department of Biomedical Engineering Biomedical Engineering and Macromolecular Science and Engineering University of Michigan Ann Arbor, Michigan Vadim Polikov Department of Biomedical Engineering Duke University Durham, North Carolina Laura Povlich Macromolecular Science and Engineering University of Michigan Ann Arbor, Michigan xvii


xviii

Sarah Richardson-Burns Department of Biomedical Engineering University of Michigan Ann Arbor, Michigan Molly S. Shoichet Department of Chemical Engineering and Applied Chemistry University of Toronto Toronto, Ontario, Canada Sarah Spanninga Macromolecular Science and Engineering University of Michigan Ann Arbor, Michigan John D. Stroncek Department of Biomedical Engineering Duke University Durham, North Carolina



Ciara C. Tate Coulter Department of Biomedical Engineering Georgia Institute of Technology/Emory University Atlanta, Georgia Patrick A. Tresco Department of Bioengineering University of Utah Salt Lake City, Utah Patrick D. Wolf Department of Biomedical Engineering Duke University Durham, North Carolina Cen Zhang Neuropharmacology Section National Institute of Environmental Health Sciences National Institutes of Health Research Triangle Park, North Carolina

Part I


11/7/2007 6:27:39 AM

1

Overview of Wound Healing in Different Tissue Types John D. Stroncek and W. Monty Reichert

CONTENTS Introduction and Overview3 +HPRVWDVLV6HFRQGVWR+RXUV 6 ,QÀDPPDWLRQ+RXUVWR'D\V 6 ,QLWLDl Events 6 /DWHU(YHQWV&HQWUDO1HUYRXV6\VWHP>&16@YHUVXV1RQ&16 8 5HSDLU'D\VWR:HHNV 1 1RQ&167LVVXH 13 3ULPDU\5HSDLU1R([WUDFHOOXODU0DWUL[>(&0@ 3URGXFWLRQ 13 6HFRQGDU\5HSDLU(&03URGXFWLRQ 17 &165HSDLU*OLRVLV 0 3XUSRVHRIWKH*OLDO6FDU 1 *OLDO6FDU,QGXFWLRQ 5HPRGHOLQJ:HHNVWR0RQWKV 1RQ&165HPRGHOLQJ 3ULPDU\5HPRGHOLQJ 6HFRQGDU\5HPRGHOLQJ 7 &165HPRGHOLQJ 9 &RQFOXVLRQ 30 5HIHUHQFHV 31

1.1 INTRODUCTION AND OVERVIEW The inevitable response to any implant is wound healing comprised of hemostasis, LQÀDPPDWLRQUHSDLUDQGUHPRGHOLQJ)RUQRQGHJUDGDEOHVPRRWKVXUIDFHGLPSODQWV UHSDLUDQGUHPRGHOLQJOHDGVWRLVRODWLRQRIWKHLPSODQWWKURXJKWLVVXHHQFDSVXODWLRQ The nature of the encapsulation tissue and the cellular participants in the immune reaction leading to this outcome varies depending on the site of implantation and the W\SHRIWLVVXHWKDWKRVWVWKHLPSODQWQRWWRPHQWLRQWKHVNLOORIWKHVXUJHRQ ,WLVQRZZHOOHVWDEOLVKHGWKDWWKHZRXQGKHDOLQJSURFHVVKDVVXEVWDQWLDOGHO HWHULRXVHIIHFWVRQWKH¿GHOLW\DQGUHOLDELOLW\RILPSODQWHGVHQVRUVDQGHOHFWURGHV$ number of reviews [1–6] and a multitude of primary articles address this issue for 3 © 2008 by Taylor & Francis Group, LLC

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implanted sensors, primarily glucose electrodes; however, there has been only one review [7] and a limited number of primary articles regarding electrodes implanted LQFHQWUDOQHUYRXVV\VWHP&16 WLVVXH>±@7KLVFKDSWHUZLOOSURYLGHDVXPPDU\ RIWKHZRXQGKHDOLQJSURFHVVLQ&16WLVVXHDQGFRPSDUHLWWRWKHZRXQGKHDOLQJ SURFHVVLQWKHSHULSKHUDOQHUYRXVV\VWHP316 ERQHDQGVNLQ 5HFHQW UHSRUWV KDYH VKRZQ WKDW LPSODQWHG HOHFWURGHV FDQ PRQLWRU QHXUDO VLJ QDOVLQWKH&16DQGWKHUHIRUHFDQEHXVHGIRUWKHFRQWURORISURVWKHWLFVLQSDWLHQWV VXIIHULQJIURPIXOORUSDUWLDOSDUDO\VLV>@:KLOHLQLWLDOVWXGLHVDUHSURPLVLQJ FKURQLFDOO\LPSODQWHGVHQVRUVDQGHOHFWURGHVIUHTXHQWO\DQGXQSUHGLFWDEO\H[SHUL ence component failure or complications arising from the wound healing response >@7KHZRXQGKHDOLQJSURFHVVEHJLQVDVVRRQDVWKHHOHFWURGHVDUHLQVHUWHGLQWRWKH EUDLQLQHYLWDEO\GLVUXSWLQJWKHEORRG±EUDLQEDUULHU>@%UHDFKLQJRIWKHEORRG± EUDLQ EDUULHU OHDGV WR KHPRVWDVLV DQG WKH LQLWLDO VWDJHV RI LQÀDPPDWLRQ LQ &16 WLVVXH WKDW DUH W\SLFDO RI WKRVH VHHQ LQ DOO YDVFXODUL]HG WLVVXH +RZHYHU GLVWLQFW GLIIHUHQFHVDULVHLQ&16WLVVXHGXULQJWKHODWHUVWDJHVRILQÀDPPDWLRQUHSDLUDQG remodeling ()LJXUH 7KH ¿UVW GLIIHUHQFH LV WKH EORRG±EUDLQ EDUULHU 7KH &16 LV KLJKO\ YDVFXODU ized; however, maintaining a barrier between capillaries and the surrounding tissue is important for regulating the concentration of ions and other molecules in both WKH YDVFXODU DQG H[WUDYDVFXODU VSDFH RI WKH &16 >@ (QGRWKHOLDO FHOOV IRUPLQJ FDSLOODULHVLQWKH&16GLIIHUIURPWKRVHLQWKHUHVWRIWKHERG\LQWZRUHVSHFWV)LUVW WKH\DUHDEOHWRIRUPWLJKWMXQFWLRQVUHVWULFWLQJSDUDFHOOXODUÀX[DQGVHFRQGWKH\ have very few endocytotic vesicles, limiting transcellular solute movement from the EORRGWRWKHEUDLQLQWHUVWLFHVUHYLHZHGE\5XELQDQG6WDGGRQ>@ $VDUHVXOWWKH EORRG±EUDLQ EDUULHU LPSHGHV HQWU\ RI YLUWXDOO\ DOO EORRG PROHFXOHV H[FHSW WKRVH WKDWDUHVPDOODQGOLSRSKLOLFVXFKDVVWHURLGV+RZHYHUVRPHODUJHUPROHFXOHVVXFK as glucose and certain amino acids are still able to move into brain tissue through VSHFL¿FWUDQVSRUWHUSURWHLQV>@$GHQVHEDVHPHQWPHPEUDQHDQGDVWURF\WHSUR FHVVHVWHUPHGHQGIHHWVXUURXQGFDSLOODU\HQGRWKHOLDOFHOOVIXUWKHUFRQWULEXWLQJWR WKHEORRG±EUDLQEDUULHU8QGHUQRUPDOQRQSDWKRORJLFDOFRQGLWLRQVWKH&16YDVFX lar endothelium with its tight junctions completely separates brain tissue from cir culating blood, isolating the brain’s unique cell types while protecting neural tissue from potentially harmful immune responses occurring outside of the neural tissue LQWKHERG\>@7KLVLVLPSRUWDQWEHFDXVHXQOLNHPRVWSHULSKHUDOWLVVXHVWKH&16 functions through a network of neurons that is generally incapable of proliferation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neural support functions but are notable for forming the glial boundary between &16 DQG QRQ&16 VWUXFWXUHV WR FUHDWH WKH EORRG±EUDLQ EDUULHU PLFURJOLD VKDUH PDQ\SKHQRW\SLFDODQGIXQFWLRQDOFKDUDFWHULVWLFVZLWKEORRGGHULYHGPDFURSKDJHV


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Overview of Wound Healing in Different Tissue Types 5

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Primary Repair (No Extracellular Matrix [ECM] Production)

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proliferation continue until keratinocytes receive a stop signal, likely due to contact LQKLELWLRQ>@$WWKLVWLPH003H[SUHVVLRQLVLQWHUUXSWHGDQGDQHZEDVHPHQW PHPEUDQHLVSURGXFHGZKHUHE\QHZFHOOPDWUL[DGKHVLRQVDUHHVWDEOLVKHG>@ 1.4.1.1.2 Stabilized Bone Repair :KHQPRWLRQDWWKHIUDFWXUHVLWHLVSUHYHQWHGDQGERQHIUDFWXUHGHQGVDUHULJLGO\ KHOG LQ SODFH LPPHGLDWHO\ DIWHU LQMXU\ SULPDU\ PLQHUDOL]HG WLVVXH UHSDLU RFFXUV ,QSULPDU\ERQHUHSDLUWKHUHLVQRQHHGIRU(&0ULFKVWUXFWXUDOVXSSRUWEHFDXVH WKH WLVVXH LV DOUHDG\ VWDELOL]HG 7ZR W\SHV RI SULPDU\ PLQHUDOL]HG WLVVXH KHDOLQJ FDQRFFXUJDSUHSDLUZKHUHWKHUHLVDOHVVWKDQPPEUHDFKVHSDUDWLQJVWDEL lized bone fragments, and contact repair, where the fractured ends are held in direct DSSRVLWLRQ>@ ,QJDSUHSDLUKHDOLQJEHJLQVDVEORRGYHVVHOVDQGORRVHFRQQHFWLYHWLVVXH¿OOWKH ZRXQG$IWHUZHHNVSOXULSRWHQWPHVHQFK\PDOFHOOVGHULYHGIURPWKHERQHPDUURZ DUULYHDWWKHVLWHRILQMXU\DQGGLIIHUHQWLDWHLQWRERQHSURGXFLQJFHOOVFDOOHGRVWHR EODVWV2VWHREODVWV¿OODQ\JDSVLQWKHWLVVXHE\VHFUHWLQJOD\HUVRIXQPLQHUDOL]HG ERQHPDWUL[FDOOHGRVWHRLGDWDUDWHRIWRPSHUGD\ZKLFKEHFRPHVPLQHUDOL]HG DIWHUDSSUR[LPDWHO\GD\V>@7KHQHZERQHIRUPHGLQWKLVVWDJHLVSHUSHQGLFXODU WRWKHORQJD[LVRIWKHERQH)LJXUH $ $VRVWHRLGEHFRPHVPLQHUDOL]HGVRPH RVWHREODVWVUHPDLQHPEHGGHGLQWKHPDWUL[DQGEHFRPHHQWUDSSHGDVWKHWLVVXHLV PLQHUDOL]HG7KHVHRVWHREODVWVEHFRPHRVWHRF\WHVDQGDUHUHVSRQVLEOHIRUPHFKDQR WUDQVGXFWLRQZLWKLQWKHWLVVXH2VWHREODVWVDUHOLNHO\DFWLYDWHGE\SURWHLQVEHORQJ LQJWRWKH7*)ȕIDPLO\VXFKDVERQHPRUSKRJHQLFSURWHLQV%03V >@7KHQHZ ERQH¿OOVWKHJDSEXWGRHVQRWFRPSOHWHO\XQLWHWKHIUDFWXUHHQGV$WWKLVSRLQWWKH LQWHUIDFHEHWZHHQWKHQHZDQGRULJLQDOERQHLVWKHZHDNHVWOLQNLQWKHXQLRQ>@ This newly formed bone acts as a scaffold for future remodeling by osteoclasts and RVWHREODVWV &RQWDFW UHSDLU RFFXUV ZKHQ WKHUH LV GLUHFW FRQWDFW EHWZHHQ ERQH HQGV 7KLV DOORZV RUJDQL]HG ODPHOODU ERQH WR IRUP GLUHFWO\ DFURVV WKH IUDFWXUH OLQH +RZ ever, before new bone can be deposited to repair the wound, necrotic bone must EHUHPRYHG2VWHRFODVWVODUJHPXOWLQXFOHDWHGFHOOVWKDWGHULYHIURPKHPRSRLHWLF progenitor cells in the bone marrow and are closely related to macrophages, tun nel across the fracture line and remove old bone and necrotic tissue through the release of proteases and lysosomal enzymes)LJXUH% 7KH\DWWDFKWRWKHWLVVXH PDWUL[WKURXJKȕLQWHJULQVFUHDWLQJDUXIÀHGFHOOERUGHUEHWZHHQWKHFHOODQGWKH ERQHVXUIDFH>@+\GURJHQLRQVDUHSXPSHGWRZDUGWKHRVWHRFODVWPHPEUDQH WRFUHDWHDQDFLGLFPLFURHQYLURQPHQWWRGLJHVWWKHERQH¶VPLQHUDOFRPSRQHQW7KH ERQH¶VRUJDQLFPDWUL[LVGHJUDGHGE\O\VRVRPDOSURWHDVHVDQGLVWKHQHQGRF\WRVHG E\ RVWHRFODVWV >@ 7KH UDWH RI ERQH GHJUDGDWLRQ E\ RVWHRFODVWV GHSHQGV RQ WKH RULHQWDWLRQRIWKHWLVVXH2VWHRFODVWVDUHFDSDEOHRIFXWWLQJERQHDWDVSHHGRI WRȝPSHUGD\SDUDOOHODQGWRȝPSHUGD\SHUSHQGLFXODUWRWKHERQH¶VORQJ D[LV>@&DSLOODULHVIRUPZLWKLQWKHVH³FXWWLQJFRQHV´SURYLGLQJQXWULHQWVWRWKH WLVVXH EHLQJ UHSDLUHG >@ %HKLQG WKH FXWWLQJ FRQH URZV RI RVWHREODVWV OLQH WKH UHVRUSWLYH FKDQQHO GHSRVLWLQJ OD\HUV RI RVWHRLG 7KLV UHJLRQ LV UHIHUUHG WR DV WKH ³FORVLQJFRQH´ZKHUHRVWHREODVWVDWWHPSWWRUHSODFHDSSUR[LPDWHO\DVPXFKERQH DVKDGEHHQUHPRYHG>@)LJXUH% /D\HUV of osteoid are initially made up of


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Secondary Repair (ECM Production)

1.4.1.2.2 Full-Thickness Cutaneous Tissue Repair If the basement membrane is damaged in a full thickness skin wound and substan WLDOGHUPLVLVORVWWKHZRXQGFDQQRWKHDOE\UHHSLWKHOLDOL]DWLRQDORQH(&0SUR GXFLQJGHUPDO¿EUREODVWVDGMDFHQWWRWKHZRXQGVLWHDUHDFWLYDWHGDQGSUROLIHUDWH PLJUDWLQJLQWRWKHZRXQGKHPDWRPDZLWKLQWRGD\V>@ ,Q VHFRQGDU\ UHSDLU GXULQJ DSSUR[LPDWHO\ WKH VDPH WLPH SHULRG WKDW NHUDWL QRF\WHVDWWHPSWWRUHHSLWKHOLDOL]HWKHZRXQGDVSUHYLRXVO\LOOXVWUDWHGLQSULPDU\ VNLQ UHSDLU ¿EUREODVWV PLJUDWH WKURXJK WKH SURYLVLRQDO PDWUL[ E\ FRQWUDFWLRQ RI WKHLUDFWLQRP\RVLQF\WRVNHOHWRQ0LJUDWLQJ¿EUREODVWVLQLWLDOO\V\QWKHVL]H¿EURQHF


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Bone Repair

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FIGURE 1.6 +LVWRORJLFDODQDO\VLVRIVHFRQGDU\IUDFWXUHKHDOLQJLQERQHVKRZLQJWKHSURJUHVVLRQRIUHSDLURQGD\VDQG)UDFWXUHGERQH DSSHDUVGHQVHUWKDQWKHVXUURXQGLQJWLVVXH2QGD\H[WHQVLYHVRIWFDOOXVLVVHHQIRUPLQJDURXQGWKHLQMXUHGERQH$WGD\WKHVRIWFDOOXVEHFRPHV PLQHUDOL]HGWRIRUPQHZERQHDQGDFKLHYHXQLRQE\GD\DQG+ (VWDLQ[ $GDSWHGIURPJ. Bone Miner. Res., ±:LWK SHUPLVVLRQRIWKH$PHULFDQ6RFLHW\IRU%RQHDQG0LQHUDO5HVHDUFK

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gen mineralization to form woven bone is different from osteoid mineralization to IRUPODPHOODUERQH,QFROODJHQPLQHUDOL]DWLRQFKRQGrocytes stop their production RIFROODJHQHORQJDWHDQGUHOHDVHSURWHDVHV>@*O\FRVDPLQRJO\FDQVZLWKLQWKH FROODJHQPDWUL[LQKLELWPLQHUDOL]DWLRQDQGPXVWEHUHPRYHGE\FKRQGURF\WHSUR WHRJO\FDQDVHVIRUPLQHUDOL]DWLRQWRRFFXU>@$VWKHFDUWLODJHPDWUL[LVGHJUDGHG FKRQGURF\WHVGLIIHUHQWLDWHDQGVHFUHWHDQJLRJHQLFIDFWRUVVXFKDV9(*)WRLQGXFH FDSLOODU\LQJURZWKIURPDGMDFHQWWLVVXH>@2VWHREODVWVOLQLQJWKHERQHVXUIDFH WKHQ VHFUHWH FROODJHQIUHH RUJDQLF PDWUL[HV VXFK DV RVWHRQHFWLQ DQG RVWHRSRQWLQ providing nucleation sites for the initiation of nanocrystaline calcium phosphate PLQHUDOL]DWLRQ%RQHIRUPHGE\WKLVPHWKRGZKHUHFROODJHQLVPLQHUDOL]HGWRIRUP ERQHLVWHUPHGHQGRFKRQGUDORVVL¿FDWLRQ %\ WKH WKLUG ZHHN WKH PDMRULW\ RI WKH FDUWLODJH KDV EHFRPH ERQH DQG XQLRQ is achieved ()LJXUH GD\ >@ $W WKLV SRLQW WKH KHDOLQJ ERQH LV JHQHUDOO\ DEOHWRVXSSRUWORDGV+RZHYHUHYHQDIWHUVWDELOL]DWLRQWKHQHZO\IRUPHGERQHLV VWLOOZHDNHUWKDQQRUPDOXQLQMXUHGERQH2QO\DIWHUWKHUHPRGHOLQJSKDVHLQZKLFK woven bone becomes remodeled to more compact laminar bone, does the tissue DFKLHYHIXOOVWUHQJWK>@

1.4.2

CNS REPAIR (GLIOSIS)

7KHUHSDLURIDZRXQGLQWKH&16LVQRWIROORZHGE\QHXURQDOUHJHQHUDWLRQ8QOLNHWKH UHVSRQVHVHHQLQWKH316ZKHUHGHJHQHUDWHGD[RQVFDQUHJHQHUDWHGDPDJHGD[RQV RIWKH&16LQLWLDOO\VSURXWEXWUHJHQHUDWLRQLVLPSHGHGDVWKHJURZWKFRQHVFROODSVH ZLWKLQDGD\&16WLVVXHUHSDLUEHJLQVKRXUVDIWHULQMXU\DVDVWURF\WHVRXWVLGHWKH OHVLRQFRUHDQGWKHVXUURXQGLQJDUHDDUHDFWLYDWHG7KLVPDUNHGJOLDOUHVSRQVHFRP monly called gliosis, is made up of a multilayered sheet of activated astrocytes that form a boundary around areas of tissue damage ()LJXUH $ /LNHWKHUHVSRQVHVHHQ LQXQPLQHUDOL]HGWLVVXHUHSDLUDVWURF\WHVDOWHUWKHLULQWHJULQH[SUHVVLRQPLJUDWLQJ WRZDUGWKHOHVLRQZKLOHDOVRVHFUHWLQJ003VIRU(&0GHJUDGDWLRQ003H[SUHV VLRQE\DVWURF\WHVDQGQHXURQVWRZHHNVDIWHULQMXU\FDQDOVRSURPRWHUHSDLUE\ VWLPXODWLQJ9(*)SURGXFWLRQWRLQLWLDWHDQJLRJHQHVLV>@2QFHDVWURF\WHVDUULYH at the site of injury, their processes encircle the lesion and become tightly intertwined to give the glial barrier a highly disordered appearance, forming what is referred to DVWKHJOLDOVFDU7KHRYHUDOOPDJQLWXGHRIJOLDODFWLYDWLRQURXJKO\FRUUHODWHVZLWKWKH DPRXQWRIEORRG±EUDLQEDUULHUGLVUXSWLRQDQGWLVVXHGDPDJH>@ Inside the lesion and surrounded by reactive astrocytes, microglia and macro SKDJHV SHUVLVW LQ WKH DWWHPSW WR UHPRYH SRWHQWLDO SDWKRJHQV DQG GLJHVW WKH ¿EULQ FORW>@)LJXUH $ %HFDXVHRIWKHUREXVWLQÀDPPDWRU\UHVSRQVHSURGXFHGE\ PLFURJOLDDQGPDFURSKDJHVWKHUHDUHJHQHUDOO\QRQHXUR¿ODPHQWVDWWKHZRXQGVLWH GD\VDIWHULQMXU\>@:LWKRXWQHXURQDOYLDELOLW\WKH&16ORVHVLWVIXQFWLRQ DOLW\DWWKHVLWHRILQMXU\)URPDVWUXFWXUDOVWDQGSRLQWZKLOHVPDOOHUOHVLRQVLQWKH &16FDQEH¿OOHGE\UHDFWLYHDVWURF\WHV>@JOLDOK\SHUWURSK\DQGSUROLIHUDWLRQ FDQQRWFRPSHQVDWHIRUODUJHUDPRXQWVRIWLVVXHORVV7KHVHODUJHZRXQGVUHPDLQ FDYLWLHVZLWKUHDFWLYHDVWURF\WHVIRUPLQJDGHQVHEDUULHUDURXQGWKHOHVLRQ6LQFH WKH OHVLRQ LV QRW ¿OOHG ZLWK FHOOV RU (&0 VXUJHRQV FDQ YLVXDOL]H SDVW WUDXPDWLF brain injuries during imaging because of missing tissue architecture ()LJXUH $


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1.4.2.1

Purpose of the Glial Scar

6SLQDO FRUG LQMXU\ VWXGLHV ZKHUH DVWURF\WHV KDYH EHHQ LQDFWLYDWHG KDYH OHG WR D EHWWHU XQGHUVWDQGLQJ RI WKH SXUSRVH RI WKH JOLDO VFDU 6HOHFWLYH UHPRYDO RI UHDF WLYHDVWURF\WHVVKRZVQRJOLDOVFDUIRUPDWLRQDWZHHNVDIWHU VWDELQMXULHV>@ +RZHYHUDVWURF\WHUHPRYDOUHVXOWHGLQWKHORVVRILQMXU\FRQWDLQPHQWDVLQÀDPPD tory cells spilled into the tissues surrounding the initial wound, increasing neuronal GHJHQHUDWLRQQH[WWRWKHLQMXU\>@7KHJOLDOVFDULVEHOLHYHGWRSURWHFWQHXURQDO function following injury by repairing the blood–brain barrier and to subsequently OLPLWLQÀDPPDWRU\UHVSRQVHWRFHOOVRIWKH&16>@ 8QOLNHWKH316D[RQVZLWKLQWKH&16DUHXQDEOHWRUHJHQHUDWH:LWKLQWKHJOLDO VFDUHQYLURQPHQWWZRPDLQJURXSVRILQKLELWRU\PROHFXOHVLPSHGHD[RQDOUHJHQ HUDWLRQWKRVHDVVRFLDWHGZLWKWKHJOLDOVFDUDQGWKRVHDVVRFLDWHGZLWKP\HOLQ>@ ,QVLGHWKHJOLDOVFDUFKRQGURLWLQVXOIDWHSURWHRJO\FDQV&63*V DUHXSUHJXODWHGE\ DVWURF\WHVROLJRGHQGURF\WHSUHFXUVRUVDQGPHQLQJHDO¿EUREODVWV&36*VDUHPDGH up of a core protein to which a variable number of repeating disaccharide chondroi WLQVXOIDWHFKDLQVDWWDFK>@&63*VDUHVHFUHWHGE\UHDFWLYHDVWURF\WHVZLWKLQ KRXUVDIWHULQMXU\DQGFDQFRQWLQXHIRUPRQWKVWKHUHDIWHU>@3URWHRJO\ FDQH[SUHVVLRQLVKLJKHVWLQWKHFHQWHURIWKHOHVLRQDQGGLPLQLVKHVRXWZDUG>@ 0\HOLQZLWKLQWKH&16DOVRFRQWDLQVJURZWKLQKLELWRU\OLJDQGVWKDWDUHUHOHDVHG ORFDOO\IROORZLQJD[RQDOWUDXPD7KHVHLQKLELWRU\PROHFXOHVDUH1RJRP\HOLQDVVR ciated glycoprotein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FIGURE 1.7 :RXQGUHPRGHOLQJRIGLIIHUHQWWLVVXHW\SHV $ $WWKHVLWHRILQMXU\WKH¿EULQFORWDQGQHFURWLFWLVVXHLVUHPRYHGE\PLFURJOLDDQGPDF URSKDJHV8QOLNHQRQQHXUDOWLVVXHORVWWLVVXHLVQRWUHSODFHGOHDYLQJDOHVLRQZLWKDFHUHEUDOVSLQDOÀXLG±¿OOHGF\VW$VWURF\WHVEHFRPHPRUHGHQVH VXUURXQGLQJWKHF\VWWRIRUPDJOLDOVFDUSURWHFWLQJWKHXQLQMXUHGWLVVXHIURPIXUWKHULQMXU\% ,QSDUWLDOWKLFNQHVVZRXQGVOLWWOHRUQRUHPRGHOLQJ LVUHTXLUHGEHFDXVHQR(&0LVSURGXFHGGXULQJUHSDLU& ,QWKH316WKHWLVVXHUHJDLQVIXQFWLRQDVWKHD[RQVXFFHVVIXOO\LQQHUYDWHVLWVWDUJHWZKLOH 6FKZDQQFHOOVSURGXFHQHZP\HOLQWRLQVXODWHWKHUHJHQHUDWHGD[RQV' 5HPRGHOLQJRIFXWDQHRXVWLVVXHLQYROYHVFRQWUDFWLRQE\¿EUREODVWVWRFORVH WKHZRXQGVLWHDOLJQLQJWKHFROODJHQPDWUL[LQUHVSRQVHWROLQHVRIVWUHVVDQGUHHSLWKHOLDOL]DWLRQRIWKHHSLGHUPLVE\NHUDWLQRF\WHV$IWHUFRQWUDFWLRQ ¿EUREODVWVZLWKLQJUDQXODWLRQWLVVXHXQGHUJRDSRSWRVLVOHDYLQJDQDFHOOXODUFROODJHQRXVVFDU(See color insert.)


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1.5 REMODELING (WEEKS TO MONTHS) 7KH XOWLPDWH HQGSRLQW IROORZLQJ UHPRGHOLQJ GHSHQGV RQ WKH WLVVXH W\SH ,Q QRQ &16WLVVXHWKDWXQGHUJRHVSULPDU\KHDOLQJYHU\OLWWOHUHPRGHOLQJRFFXUVEHFDXVH RIWKHODFNRI(&0SURGXFHGGXULQJUHSDLU6HFRQGDU\KHDOLQJLQFRQWUDVWLQYROYHV ¿EHUDOLJQPHQWDQGFRQWUDFWLRQWRUHGXFHWKHZRXQGVL]HDQGWRUHHVWDEOLVKWLVVXH VWUHQJWK&RPSOHWHUHFRYHU\RIRULJLQDOWLVVXHVWUHQJWKLVUDUHO\REWDLQHGLQVHFRQG ary healing because repaired tissue remains less organized than noninjured tissue, ZKLFKUHVXOWVLQVFDUIRUPDWLRQ&ROODJHQULFKVFDUVDUHFKDUDFWHUL]HGPRUSKRORJL cally E\DODFNRIVSHFL¿FRUJDQL]DWLRQRIFHOOXODUDQGPDWUL[HOHPHQWVWKDWFRPSULVH WKHVXUURXQGLQJXQLQMXUHGWLVVXH,Q&16WLVVXHZKHUHWKHUHLVQRUHSDLURUUHJHQ eration of injured neurons, there is also relatively little reestablishment of structural LQWHJULW\LQWKHUHJLRQ,QVWHDGGXULQJ&16UHPRGHOLQJWKHJOLDOVFDUDURXQGWKH lesion becomes denser as astrocytic processes become more intertwined and more or less isolates but dRHVQRWUHSDLUWKHLQMXUHGUHJLRQ

1.5.1

NON-CNS REMODELING

1.5.1.1 Primary Remodeling 1.5.1.1.1 Partial-Thickness Cutaneous Tissue Remodeling ,QVXSHU¿FLDOLQMXULHVZRXQGVFDQKHDOE\HSLWKHOLDOL]DWLRQDORQHZLWKOLWWOHRUQR DGGLWLRQDO(&0UHTXLUHGWR¿OOLQWKHWLVVXH%HFDXVHWKHVHSULPDU\WLVVXHLQMXULHV FDQ KHDO E\ NHUDWLQRF\WH UHJHQHUDWLRQ DQG PLQLPDO (&0 SURGXFWLRQ YHU\ OLWWOH additional remodeling is required ()LJXUH % $V D UHVXOW WKHUH LV QR VFDUULQJ DQGWKHUHSDLUHGWLVVXHLVYLUWXDOO\LQGLVWLQJXLVKDEOHIURPXQLQMXUHGWLVVXH In the clinical setting, when there is little contamination or necrotic debris, phy VLFLDQVXVHVXWXULQJWREULQJGHUPDOHGJHVLQGLUHFWDSSRVLWLRQ8SRQFDUHIXODOLJQ ment and elimination of tension, the epidermal and dermal layers can heal primarily E\HSLWKHOLDOL]DWLRQZLWKLQWKHHSLGHUPLVDQGZLWKOLPLWHG(&0SURGXFWLRQLQWKH GHUPLV OHDGLQJ WR OLPLWHG VFDUULQJ >@ 2YHU D SHULRG RI ZHHNV WR PRQWKV WKH


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Stabilized Bone Remodeling

0LQHUDOL]HGWLVVXHUHPRGHOLQJLVDQDFWLYHDQGG\QDPLFSURFHVV%RQHLVXQLTXHLQ that remodeling occurs throughout the life of the tissue as mechanical stress induces bone to reorient itself and produce new bone to better handle the demands that are SODFHG RQ LW $FFRUGLQJ WR :ROII¶V ODZ WKH WLVVXH DGDSWV WR WKH HQYLURQPHQW E\ EHFRPLQJRULHQWHGDORQJOLQHVRIPD[LPXPVWUHVV>@ 5HPRGHOLQJ DIWHU SULPDU\ IUDFWXUH UHSDLU ZKHUH ERQH LV VWDELOL]HG LV VLPLODU to the remodeling response that occurs over the life of the tissue, lasting up to sev HUDO \HDUV EHIRUH IXOO SUHLQMXU\ VWUHQJWK LV UHVWRUHG >@ ,QSULPDU\JDSKHDOLQJ UHPRGHOLQJLVLPSRUWDQWIRUUHVWRULQJWLVVXHVWUHQJWK+RZHYHULQSULPDU\FRQWDFW KHDOLQJUHPRGHOLQJLVFRXSOHGWRWKHUHSDLUSURFHVV'XULQJFRQWDFWUHPRGHOLQJWKH FXWWLQJFRQHVPDWXUHGHSRVLWLQJODPHOODUERQHFHQWULSHWDOO\WRIRUPULQJVKDSHG VWUXFWXUHVZLWKDFHQWHUEORRGYHVVHOFRQWDLQLQJFDQDO)LJXUH% In primary gap remodeling, lamellar bone deposited perpendicular to the long D[LVZLWKLQWKHLQMXU\GXULQJUHSDLULVXVHGDVDVFDIIROG)LJXUH$ 7KLVSURFHVV LVFRPPRQO\UHIHUUHGWRDV+DYHUVLDQUHPRGHOLQJZKHUHERQHLVUHPRGHOHGLQVPDOO SDFNHWVRIFHOOVFDOOHGEDVLFPXOWLFHOOXODUXQLWV%08V >@7KLVLVWKHVDPHSUR cess that occurs during primary contact repair where osteoclasts form cutting cones DOORZLQJWKHLQÀX[RIHQGRWKHOLDOFHOOVWKDWIRUPFDSLOODULHV)LJXUH% 7KHEXG ding capillaries bring in pluripotent mesenchymal cells that differentiate into osteo EODVWV DIWHU DWWDFKLQJ WR WKH VXUIDFH RI WKH LQWHUQDO FXWWLQJ FRQH 2VWHREODVWV DUH activated and synthesize new lamellar bone concentrically, gradually closing the diameter of the tunnel ()LJXUH $ $IWHUDERXWIRXUZHHNVERQHSURGXFWLRQVWRSV as the tunnel is closed, leaving behind a vascularized cavity called an osteon that UXQVSDUDOOHOWRWKHORQJD[LV>@,WLVEHOLHYHGWKDWWKHJUHDWHUQXPEHURIRVWHRQV WKDWFURVVWKHVLWHRILQMXU\WKHJUHDWHUWKHXOWLPDWHVWUHQJWK>@ 1.5.1.1.3

PNS Remodeling

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FIGURE 1.8 3ULPDU\ YHUVXV VHFRQGDU\ ERQH UHPRGHOLQJ $ /DPHOODU ERQHGHSRVLWHGSHUSHQGLFXODUWRWKHERQH¶VORQJD[LVGXULQJSULPDU\UHSDLULV XVHGDVDVFDIIROGIRUFXWWLQJFRQHV2VWHRFODVWVFUHDWHWXQQHOVWKURXJKZKLFK new blood vessels follow, stimulating osteoblasts to produce new osteoid that EHFRPHV PLQHUDOL]HG WR IRUP ODPHOODU ERQH $ PDWXUH RVWHRQ IRUPV ZKHQ RVWHRFODVWDQGRVWHREODVWDFWLYLW\EHFRPHVTXLHVFHQW% ,QSULPDU\FRQWDFW remodeling the cutting cones initiated during repair mature, forming “mature RVWHRQV´7KHODUJHUWKHQXPEHURIRVWHRQVWKHJUHDWHUWKHWLVVXHVWUHQJWKRI WKHUHSDLUHGWLVVXH& ,QVHFRQGDU\UHPRGHOLQJWKHPLQHUDOL]HGKDUGFDOOXV near the periosteum not needed for structural support is removed and remod HOHGE\RVWHRFODVWVDUULYLQJIURPWKHSHULRVWHXP:LWKLQWKHFRUWH[FXWWLQJ FRQHVDUHIRUPHGDVGHVFULEHGLQSULPDU\UHPRGHOLQJ/HVVRUJDQL]HGZRYHQ ERQHZLWKLQWKHZRXQGJDSLVUHSODFHGZLWKRULHQWHGODPHOODUERQH7KHXOWL mate endpoint of both primary and secondary bone healing is tissue with FRPSOHWHO\UHVWRUHGIXQFWLRQ


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$[RQDOUHSDLUDQGUHPRGHOLQJGHSHQGVRQWKHVHYHULW\RIWKHWUDXPD0RVWLQMX ULHV LQ WKH 316 DUH VWUHWFK UHODWHG ZKHUH WKH WHQVLOH IRUFH DSSOLHG WR WKH WLVVXH H[FHHGV WKH QHUYH¶V DELOLW\ WR VWUHWFK *HQHUDOO\ LQ WKHVH LQMXULHV WKH RXWHU FRQ QHFWLYHWLVVXHOD\HURIWKHQHUYHWKHHQGRQHXULXPLVLQWDFWDQGWKHD[RQLVDEOHWR regenerate as illustrated previously ()LJXUH & >@+RZHYHULQPRUHWUDXPDWLF LQMXULHVVXFKDVODFHUDWLRQVWKHHQGRQHXULXPDQGWKHD[RQDUHVHYHUHGDQGWKHWLVVXH XVXDOO\IDLOVWRUHJHQHUDWH$IWHUWUDXPDWLFLQMXULHVWKH¿EUREODVWVFRQWDLQHGZLWKLQ the endoneurium proliferate and produce a collagenous scar around the nerve trunk GXULQJWKHUHSDLUDQGUHPRGHOLQJSKDVHV7KLVFROODJHQRXVVFDUPLVGLUHFWVRUEORFNV D[RQDOUHJHQHUDWLRQ>@6SURXWLQJD[RQVWKDWFDQQRW¿QGWKHLUWDUJHWRUFRQWDFW ZLWKD6FKZDQQFHOOFRQGXLWDUHHLWKHUFOHDYHGRUJURZLQWRDGLVRUJDQL]HGPDVV UHVXOWLQJLQWKHIRUPDWLRQRIDQHXURPD0HDQZKLOHWKHWDUJHWPXVFOHUHPDLQVLQDF WLYHDQGQHXUDOFHOOERGLHVDWURSK\DQGHYHQWXDOO\GLH(YHQZKHQWKHHQGRQHXULXP is not severed, regeneration of the target site fails more often than not, and even in the best case, regeneration of a peripheral nerve does not fully restore the tissue back to its original status since there is inevitably inaccuracy during the attempted UHWXUQRIDQD[RQWRLWVRULJLQDOWDUJHW>@ 6XUJLFDOLQVHUWLRQRIDQDXWRORJRXVQHUYHJUDIWFDQEHXVHGWRUHSDLU316OHVLRQV WKDWDUHWRRODUJHWREHEULGJHGE\6FKZDQQFHOOV7KHSXUSRVHRIWKHJUDIWLVWR UHFRQQHFWGDPDJHGQHUYHVHQGWRHQGZLWKRXWFDXVLQJWHQVLRQ6XWXULQJLVXVHGWR FRQQHFWWKHFRQQHFWLYHWLVVXHVKHDWKRIWKHGDPDJHGQHUYHWRWKHDXWRORJRXVJUDIW )UHVKO\LQMXUHGWLVVXHVKHDWKVGRQRWKROGVXWXUHVYHU\ZHOOVRVXUJLFDOUHSDLULV generally not performed until three weeks after injury, when the sheaths have had WLPHWRWKLFNHQ7KHPDMRUGUDZEDFNRIWKLVPHWKRGRIUHSDLULVWKDWKDUYHVWRIWKH DXWRORJRXVQHUYHHQWDLOVVDFUL¿FLQJRQHRUPRUHQHUYHV$QXPEHURIJURXSVDUH ZRUNLQJWRHQJLQHHUV\QWKHWLFQHUYHJUDIWVIRU316UHSDLU>@ 1.5.1.2 Secondary Remodeling 1.5.1.2.1 Full-Thickness Cutaneous Tissue Remodeling 7KHPDMRUJRDORIVHFRQGDU\ZRXQGUHPRGHOLQJLVWRUHGXFHWKHDPRXQWRIH[FHVV (&0DQGDOLJQWKH(&0WKURXJKFRQWUDFWLRQ,IWKHH[WHQWRIWKHZRXQGLVUHODWLYHO\ VPDOO DQG WKH UHRUJDQL]DWLRQ RI WKH (&0 LV HI¿FLHQW UHODWLYHO\ OLWWOH FRQWUDFWXUH RFFXUVDQGOLWWOHRUQRVFDUULQJLVREVHUYHG+RZHYHULQODUJHULQMXULHVZKHUHWKHUH LV PRUH H[WHQVLYH WLVVXH UHSDLU WKURXJK (&0 SURGXFWLRQ VLJQL¿FDQW UHPRGHOLQJ LVUHTXLUHGZKLFKUHVXOWVLQVFDUULQJ5HPRGHOLQJRFFXUVRYHUDORQJWLPHSHULRG This phase can overlap with the repair phase, as it can begin as early as 1 week after LQMXU\DQGFDQODVWDVORQJDV\HDUVGHSHQGLQJRQWKHZRXQG 'XULQJUHSDLU¿EUREODVWVPLJUDWHLQWRWKHVLWHRILQMXU\DQGSURGXFH(&0WR UHSODFHORVWWLVVXH7KHWUDFWLRQDOIRUFHVWKDW¿EUREODVWVFUHDWHDVWKH\PRYHWKURXJK the wounded tissue generate mechanical tension, which promotes wound closure >@ 7KH UHPRGHOLQJ SKDVH RI¿FLDOO\ EHJLQV ZKHQ 7*)ȕ DQG RWKHU F\WRNLQHV UHOHDVHGIURPSODWHOHWVDQGDFWLYDWHGPDFURSKDJHVFDXVH¿EUREODVWVWRGLIIHUHQWLDWH LQWRDPRUHFRQWUDFWLOHSKHQRW\SHFDOOHGP\R¿EUREODVWV0\R¿EUREODVWVDUHFKDUDF WHUL]HGE\WKHLUH[SUHVVLRQRIDOSKDVPRRWKPXVFOHDFWLQDQGSURGXFWLRQRIFROODJHQ ,>@2QFHDFWLYDWHGWKH\LQFUHDVHWKHLUF\WRVNHOHWDOVWUHVV¿EHUVDQGIRFDO


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adhesions, providing constant tension to contract the wound bed (for review see >@ 0\R¿EUREODVWFRQWUDFWLRQLVVLPLODUWRWKDWRIVPRRWKPXVFOHFHOOVWKRXJK WKHPRGHRIDFWLYDWLRQLVYDVWO\GLIIHUHQW6PRRWKPXVFOHFHOOVFRQWUDFWEHFDXVHRI HOHYDWLRQVRI&D6LQFH&D levels around cells can change rapidly, the contractil LW\RIVPRRWKPXVFOHcells can chaQJHTXLFNO\>@0\R¿EUREODVWVDUHEHOLHYHGWR EHUHJXODWHGE\WKH5KR±5KRNLQDVHSDWKZD\ZKLFKLVOHVVWUDQVLHQWDQGFDXVHVD ORQJHUODVWLQJFRQWUDFWLRQIRUFH&RQWUDFWXUHVRIWRPSHUGD\DUHSRVVLEOH without the need to generate large amounts of force because of the low basal tension OHYHO RI VNLQ >@ 0\R¿EUREODVWV FRQWLQXH WR VXSSRUW ORDGV LQ FRQWUDFWHG WLVVXH XQWLO(&0LVSURGXFHGDQGFURVVOLQNHGOHDGLQJWRVWUHVVVKLHOGLQJ>@&ROODJHQ ¿EHUVJUDGXDOO\WKLFNHQDQGDORQJZLWKP\R¿EUREODVWVWKH\EHFRPHRULHQWHGSDU allel to the wound bed along lines of stress, resulting in the appearance of striated scar tissue ()LJXUH ' 7KLVLVLQGLUHFWFRQWUDVWWRWKHEDVNHWZHDYHSDWWHUQVHHQ LQXQLQMXUHGVNLQ>@ 2QFH ZRXQG FRQWUDFWLRQ RFFXUV VWUHVV UHOD[DWLRQ FDXVHV P\R¿EUREODVWV WR UHWXUQ WR D TXLHVFHQW VWDWH 7KH FHOOV WKHQ UHFHLYH D VLJQDO WR XQGHUJR DSRSWRVLV WUDQVIRUPLQJ WKH ZRXQG IURP FHOOULFK JUDQXODWLRQ WLVVXH WR FHOOSRRU VFDU WLVVXH ZLWK DQ H[FHVV RI (&0 >@ $W WKH VDPH WLPH WKH FDSLOODU\ GHQVLW\ JUDGXDOO\ GLPLQLVKHVDQGWKHZRXQGORVHVLWVSLQNFRORUEHFRPLQJSURJUHVVLYHO\SDOHU>@ The ultimate end point of the remodeling process is the formation of acellular scar tissue that is poorly reorganized into dense parallel bundles, as opposed to the tightly woven meshwork of normal dermalWLVVXH)LJXUH' >@'HUPDOVWUXF tures such as hair follicles, sweat glands, and sebaceous glands that are lost during LQMXU\DUHQRWUHJHQHUDWHG>@$IWHUWKUHHPRQWKVXQPLQHUDOL]HGWLVVXHFDQKDYH DPD[LPXPRIRIWKHVWUHQJWKRIXQZRXQGHGWLVVXH>@7KLVLVJHQHUDOO\WKH KLJKHVWOHYHORIVWUHQJWKWKDWDKHDOHGWLVVXHFDQDFKLHYH 1.5.1.2.2

Unstabilized Bone Remodeling

The ultimate goal of secondary remodeling of mineralized tissue is full structural UHVWRUDWLRQ ZLWK OLWWOH WR QR VFDUULQJ )LJXUH( :KLOH UHPRGHOLQJ FDQ ODVW XS to 6 months, the endpoint is healed tissue that is remarkably similar to noninjured WLVVXHLQWHUPVRIUREXVWQHVVEXWPD\DSSHDUVOLJKWO\OHVVRUJDQL]HG>@7KHRQO\ major difference between primary and secondary mineralized tissue remodeling is WKHH[WHQVLYHERQHUHPRYDOUHTXLUHGWRUHPRYHWKHH[FHVVFDOOXVSURGXFHGGXULQJ ERQHUHSDLU In secondary healing, osteoclasts arrive at the site in need of remodeling via the FLUFXODWLRQ7KH\UHFRJQL]HDQGDWWDFKWRFHOODGKHVLRQSURWHLQVVXFKDVRVWHRSRQ WLQRVWHRFDOFLQDQGRVWHRQHFWLQ>@2VWHRFODVWVDUHSUHVHQWQRWRQO\WRUHPRYH H[FHVVERQHQRWQHHGHGIRUVWUXFWXUDOVXSSRUWEXWDOVRWRGLJHVWZRYHQERQHV\Q thesized from the soft callus during repair so it can be replaced with lamellar bone DOLJQHG LQ UHVSRQVH WR VWUHVV /LNH SULPDU\ UHPRGHOLQJ VHFRQGDU\ UHPRGHOLQJ RFFXUVPDLQO\WKURXJKFXWWLQJFRQHV:LWKLQWKHFRUWH[ZRYHQDQGQHFURWLFODPHOOD ERQHLVUHPRYHGE\RVWHRFODVWV2VWHREODVWVIROORZFORVHO\DQGSURGXFHQHZFRP pact lamellar bone, giving the tissue greater strength ()LJXUH & (DFKFRQFHQWULF PWKLFN ODPHOODU OD\HU LV GLUHFWHG LQ D VSHFL¿F PDQQHU WR EXWWUHVV IUDFWXUH IUDJPHQWV>@6HFRQGDU\UHPRGHOLQJLVDOWHUHGIURPSULPDU\UHPRGHOLQJEHFDXVH


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1.5.2

CNS REMODELING

5HPRGHOLQJLQWKH&16LVOLPLWHG%HFDXVHRIWKHQHHGWRSURWHFWWKH&16IURPWKH ERG\¶VUREXVWLQÀDPPDWRU\UHVSRQVHVUHDFWLYHDVWURF\WLFSURFHVVHVEHFRPHIXUWKHU intertwined, forming a dense sheath around the wound sitH)LJXUH$ 'XULQJ WKHUHSDLUVWDJHV*)$3UHDFWLYLW\LVVHHQDSSUR[LPDWHO\PRXWVLGHWKHOHVLRQ 'XULQJWKHUHPRGHOLQJVWDJHV*)$3UHDFWLYLW\LVRQO\REVHUYHGOHVVWKDQP IURPWKHVLWHRIWLVVXHGDPDJH>@*OLDOVFDUGHQVLW\LQFUHDVHVDVK\SHUWURSKLF DVWURF\WHVEHFRPHPRUHFRQGHQVHGDURXQGWKHVLWHRIGDPDJH)LJXUH >@ ,QVLGHWKHOHVLRQUHPRYDORIWKH¿EULQFORWDQGWKHQHFURWLFQHXURQVDQGVXS SRUWLQJJOLDOFHOOVLVFRPSOHWHGE\PLFURJOLDDQGPDFURSKDJHV)LJXUH$ >@

FIGURE 1.9 7LPHFRXUVHRIJOLDOVFDUIRUPDWLRQDWIRXUWLPHSRLQWVDVLPDJHGE\*)$3 VWDLQLQJ$WDQGZHHNWLPHSRLQWVWKHDVWURF\WLFSURFHVVHVIDOOEDFNLQWRWKHYRLGOHIW E\WKHSUREHH[WUDFWLRQEHIRUHWLVVXHSURFHVVLQJ%\ZHHNVWKHSURFHVVHVKDYHLQWHUZRYHQ WRIRUPDVWURQJHUPRUHGHQVHVKHDWKVXUURXQGLQJWKHLPSODQW0LQLPDOFKDQJHVEHWZHHQ WKHDQGZHHNWLPHSRLQWVLQGLFDWHWKHJOLDOVFDUFRPSOHWLRQZLWKLQZHHNV5HSULQWHG from Exp. Neurol.±:LWKSHUPLVVLRQIURP(OVHYLHU


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Once complete, microglia and macrophages undergo apoptosis, leaving a cerebral VSLQDOÀXLG±¿OOHGF\VWLQWKHFHQWHURIWKHOHVLRQZKHUHWKHLQLWLDOZRXQGRFFXUUHG >@7KHF\VWLVERUGHUHGE\DWKLQGHQVHOD\HURIUHDFWLYHDVWURF\WHVWKDWVHUYHDV a barrier between healthy and lost tissue and may help protect neurons outside the LQMXU\7KHD[RQVRIQHXURQVSURWHFWHGRXWVLGHWKHJOLDOVFDUFDQQRWUHJURZLQWRORVW tissue because of inhibitory molecules within the scar, and thus tissue function is QHYHUUHJDLQHG)LJXUH$

1.6

CONCLUSION

7KHFHOOXODUUHDFWLRQDIWHULQMXU\GHSHQGVRQWKHWLVVXHW\SHDVZHOODVWKHH[WHQW RI WKH ZRXQG ,Q LQMXU\ WR &16 WLVVXH WKDW GDPDJHV QHXURQV DQG WKH VXSSRUWLQJ glial cells, the body’s response is unforgiving, as regeneration of lost neurons is QRWSRVVLEOH$FWLYDWHGDVWURF\WHVZDOORIIWKHOHVLRQFUHDWLQJDJOLDOVFDU7KHVH DFWLYDWHG DVWURF\WHV PD\ SUHYHQW IXUWKHU WLVVXH GDPDJH DOWKRXJK QHXURQ D[RQDO UHJURZWKLVLQKLELWHG,QFRQWUDVWLQQRQ&16WLVVXHDVLQJOHWLVVXHW\SHFDQKDYH PXOWLSOHUHVSRQVHVGHSHQGLQJRQWKHPDJQLWXGHRILQMXU\)RUH[DPSOHDVXSHU¿FLDO VNLQZRXQGRIWHQKDVORZHUOHYHOVRILQÀDPPDWRU\LQ¿OWUDWHDQGFDQXQGHUJRSUL PDU\KHDOLQJZKLOHDGHHSHUZRXQGZLWKPRUHH[WHQVLYHWLVVXHGDPDJHDQGFHOOXODU ORVVZLOOXQGHUJRVHFRQGDU\KHDOLQJ7KLVFKDSWHUIRFXVHGRQWKHSULPDU\KHDOLQJ UHVSRQVH VHHQ DIWHU LQMXULHV LQ VNLQ ERQH DQG WKH 316 ZKHUH WKH JHQHUDO WLVVXH architecture is maintained after injury, allowing the tissues to undergo repair pre GRPLQDQWO\E\WKHUHJHQHUDWLRQRIORVWFHOOVWRUHVWRUHWKHWLVVXH¶VQRUPDOVWUXFWXUH In secondary healing, the wounds are often deeper and the general tissue structure LVFRPSURPLVHG7KHLQÀDPPDWRU\UHVSRQVHPD\EHPRUHLQWHQVHDQGSURORQJHG DVZHOOUHFUXLWLQJ¿EUREODVWVDQGHQGRWKHOLDOFHOOVWKDWSURGXFHJUDQXODWLRQWLVVXH :LWKLQ JUDQXODWLRQ WLVVXH WKH GHSRVLWLRQ RI FROODJHQ E\ ¿EUREODVWV FKDQJHV WKH structure and function of the tissue and eventually leads to scar formation upon the FRPSOHWLRQ RI WKH UHPRGHOLQJ VWDJH 6HFRQGDU\ KHDOLQJ LQ ERQH LV WKH H[FHSWLRQ DVERQHWLVVXHLVFDSDEOHRIUHPRYLQJWKHLQLWLDO¿EURXVFDOOXVDQGUHSODFLQJLWZLWK ODPHOODUERQH2WKHUH[DPSOHVRIVFDUULQJDIWHUVHFRQGDU\KHDOLQJRFFXULQWLVVXHV WKDWDUHQRWFDSDEOHRIXQGHUJRLQJUHJHQHUDWLRQVXFKDVWKHKHDUW ,Q VXPPDU\ ZKLOH WKH RYHUDOO RXWFRPH RI &16 DQG QRQ&16 WLVVXH ZRXQG KHDOLQJ KDV EHHQ GHVFULEHG IRU WKHUDSHXWLF SXUSRVHV LQ WKH &16 LW LV LPSRUWDQW to consider the different junctures within the wound healing process at which the UHVSRQVH FDQ EH PRGL¿HG WR SXVK WKH ERG\¶V ZRXQG KHDOLQJ UHVSRQVH WRZDUG D GHVLUHGRXWFRPH7KHVHSRLQWVRILQWHUYHQWLRQDUHPDLQO\ZLWKLQWKHVWDJHVKHPR VWDVLV LQÀDPPDWLRQ DQG UHSDLU :LWKLQ HDFK VWDJH WKHUH DUH SRLQWV WKDW PD\ EH XVHIXOIRUPRGXODWLQJWKHZRXQGKHDOLQJUHVSRQVH)RULQVWDQFHWROLPLWH[WHQVLYH clot formation, it has been suggested that there are three main stages within clot IRUPDWLRQIRUZKLFKWKHUDSHXWLFVFDQEHGHYHORSHGLQLWLDWLRQSODWHOHWDFWLYDWLRQ SURSDJDWLRQ FRDJXODWLRQ FDVFDGH DQG ¿EULQ IRUPDWLRQ WKURPELQ >@ 'XULQJ LQÀDPPDWLRQ PRGXODWLRQ VWUDWHJLHV FHQWHU DURXQG OLPLWLQJ LPPXQH FHOO DFWLYD WLRQDQGUHGXFLQJLQÀDPPDWRU\FHOOPLJUDWLRQLQWRWKHZRXQG>@6LQFHQHXURQV ZLWKLQWKH&16DUHHVSHFLDOO\VHQVLWLYHWRLQÀDPPDWRU\GDPDJH>@DQGEHFDXVH FKURQLFLQÀDPPDWLRQLVLQYROYHGLQPDQ\RWKHUGLVHDVHVRXWVLGHWKH&16WKHUHKDV


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REFERENCES

:LVQLHZVNL1DQG5HLFKHUW00HWKRGVIRUUHGXFLQJELRVHQVRUPHPEUDQHELRIRXO LQJColloids and Surfaces B: Biointerfaces *HUULWVHQ0DQG-DQVHQ-3HUIRUPDQFHRIVXEFXWDQHRXVO\LPSODQWHGJOXFRVHVHQ VRUVDUHYLHZJournal of Investigative Surgery :LOVRQ*6DQG*LIIRUG5%LRVHQVRUVIRUUHDOWLPHLQYLYRPHDVXUHPHQWVBiosensors and Bioelectronics 8SGLNH66KXOWV0DQG5KRGHV53ULQFLSOHVRIORQJWHUPIXOO\LPSODQWHGVHQVRUV with emphasis on radiotelemetric monitoring of blood glucose from inside a subcu WDQHRXVIRUHLJQERG\FDSVXOH)%& ,Q)UDVHU'HG Biosensors in the Body1HZ EHWD@FKHPRNLQHV 5$17(6 DQG 0,3>EHWD@ E\ KXPDQ EUDLQ PLFURYHVVHO HQGRWKHOLDO FHOOV LQ SULPDU\ FXOWXUH -RXUQDO1HXURSDWKRORJ\Experimental Neurology 6LPSVRQ'0DQG5RVV57KHQHXWURSKLOLFOHXNRF\WHLQZRXQGUHSDLUDVWXG\ZLWK DQWLQHXWURSKLOVHUXPJournal Clinical Investigation /HLERYLFK6-DQG5RVV57KHUROHRIWKHPDFURSKDJHLQZRXQGUHSDLU$VWXG\ZLWK K\GURFRUWLVRQH DQG DQWLPDFURSKDJH VHUXP Americal Journal of Pathology 78(1), 71, +LFNH\:%DVLFSULQFLSOHVRILPPXQRORJLFDOVXUYHLOODQFHRIWKHQRUPDOFHQWUDOQHU YRXVV\VWHPGlia 3HUU\9+%URZQ0&DQG*RUGRQ67KHPDFURSKDJHUHVSRQVHWRFHQWUDODQG SHULSKHUDOQHUYHLQMXU\$SRVVLEOHUROHIRUPDFURSKDJHVLQUHJHQHUDWLRQJournal of Experimental MedIcine $YHOOLQR$+DUW''DLOH\$0DF.LQQRQ0(OOHJDOD'DQG.OLRW0'LI ferential macrophage response in the peripheral and central nervous system during :DOOHULDQGHJHQHUDWLRQRI$[RQVExperimental Neurology %LVE\05HJHQHUDWLRQRISHULSKHUDOQHUYRXVV\VWHPD[RQV,Q:D[PDQ6.RFVLV -DQG6W\V3HGV The Axon: Structure, Function, and Pathophysiology1HZ@

ment curves of the number of neurons stimulated (or, equivalently, the amplitude of the response mediated by neuronal activation) as a function of the stimulation intensity are sigmoidal (Figure 2.4B). Damage or death of neurons around the electrode can give rise to changes in the input–output function (i.e., impact the response evoked by electrical stimulation). We conducted computer simulations to quantify the effects of neuronal death on the input–output properties of neural stimulation. Simulations were conducted under two conditions. First, we assessed changes that might result when neurons die as a function of their proximity to the stimulating electrodes. This would occur, for example, when the tissue reaction to the electrode causes neuronal death or if electrochemical by-products, produced at the electrode–tissue interface, were harmful to the surrounding neurons. Second, we assessed changes in input–output properties that occurred when neurons died as a function of their excitability, that is, when the PRVWH[FLWDEOHQHXURQVGLHG¿UVW7KLVVLWXDWLRQZRXOGRFFXUZKHQQHXURQDOGHDWK resulted from the induced activity, for example, as a result of metabolic overload. In all cases, neuronal death resulted in an increase in the threshold stimulation intensity as well as a reduction in the amplitude of the response (number of neurons activated) for a given stimulation current. These effects were observed whether considering a population of axons (Figure 2.4C) or a population of local cells (Figure 2.4D). Killing neurons according to their relative excitability produced proportionally larger shifts in threshold, and the foot of the input–output curve shifted to


46


FIGURE 2.4 Effect of neuronal death on stimulation input–output properties. (A) A population of neurons distributed at different distances and positions with respect to a stimulating electrode. (B) The distribution of neuronal positions and excitability yields a distribution of thresholds to excite individual neurons. The recruitment (input–output) curve describes the number of neurons stimulated as a function of the stimulus intensity, where the intensity can be modulated through changes in either the amplitude or duration of the stimulus pulse. The sigmoidal shape of the recruitment curve arises from cumulative integration of the distribution of thresholds as the stimulation intensity is increased. (C) Computation of WKHHIIHFWRQWKHLQSXW±RXWSXWSURSHUWLHVRIDSRSXODWLRQRIQHUYH¿EHUVIROORZLQJGHDWKRI different proportions of neurons according to either their proximity to the stimulating electrode or their excitability. (D) Computation of the effect on the input–output properties of a population of local cells following death of different proportions of neurons according to either their proximity to the stimulating electrode or their excitability. The simulations in (C) and (D) included 500 neurons distributed uniformly in a 600 µm x 600 µm x 600 µm cube ZLWKDSRLQWVRXUFHHOHFWURGHDWWKHFHQWHU7KHWLVVXHZDVDQLQ¿QLWHLVRWURSLFKRPRJHQHRXV YROXPH ZLWK UHVLVWLYLW\ FP 6WLPXOL ZHUH PRQRSKDVLF UHFWDQJXODU PV GXUDWLRQ cathodic pulses, and threshold was determined to ± 0.1 % by variable time-step numerical integration. The neuron models were based upon the S-type motoneuron model in McIntyre and Grill, 2000 [58].


Signal Considerations for Chronically Implanted Electrodes

47

the right. The difference between the control input–output curve and the curve with neurons killed according to their excitability was approximately constant over the full range of recruitment. In contrast, the input–output curves of neuronal populations with neurons killed according to their proximity to the electrode were closer to the control curve near the foot of the curve and deviated more as the activation level increased to converge with the curve of neurons killed by excitability. Overall, the input–output curves were very similar whether neurons died according to their proximity to the electrode or according to their excitability. These results suggest that neuronal death according to either criterion is likely to have a similar effect on device function and that measurement of changes in input–output curves is not likely to be a sensitive diagnostic for the characteristics of neuronal death.

2.2.3

IMPACT OF ELECTRODE TO NEURON DISTANCE ON RECORDED SIGNALS

As with stimulation intensity, the amplitude of the signal recorded from an active neuron (or neurons) varies inversely with the distance between the source (active neurons) and the recording electrode, r. For a monopolar source the potential varies as 1/r, while for a dipolar source, more typical of transmembrane currents, the potential varies as 1/r2. The historical rule of thumb is that to record isolated signals from single active neurons requires that the electrode lie within r = 100 µm of the active neuron. The relationship between the recorded signal amplitude and the estimated electrode to neuron distance obtained from two published studies were replotted. These data (Figure 2.5) are consistent with this rule of thumb and indicate that the recorded signal amplitude falls off rapidly as the electrode to neuron distance is increased.

2.3 MECHANISMS OF NEURONAL INJURY AND DEATH Given the clear performance consequences of increases in the electrode to neuron distance on both stimulation thresholds and recorded signal amplitudes, considerations of the underlying contributors to neuronal injury or death (Figure 2.1B) are of paramount importance in designing neural prosthetic interfaces. Most genHUDOO\ WKH WLVVXH UHVSRQVH FDQ EH FODVVL¿HG DV HLWKHU SDVVLYH UHVXOWLQJ IURP WKH presence of the electrode) or active (resulting from the passage of stimulus current), although it is not always possible to differentiate between these two contributions to the tissue response. The passive tissue response can result from surgical trauma and the mechanical and chemical properties of the implant. The active tissue response results from electrochemical reaction products formed at the tissue–electrode interface and from physiological changes associated with neural activity.

2.3.1

PASSIVE PROPERTIES

The tissue response to an implanted electrode depends on the physical attributes of WKHGHYLFH>@LQFOXGLQJLWVVL]HVKDSH>±@DQGVXUIDFHWH[WXUH>@,QDGGLtion, the chemical or material composition of the device has a strong impact on the tissue response to the material and ranges from the benign (so-called biocompatible materials) to those that are toxic and lead to cell death [19–21].


48


FIGURE 2.5 Effect of electrode to neuron distance on recorded potential amplitudes. Amplitude of extracellular action potentials recorded from single neurons as a function of the distance of the recording electrode from the point of maximum amplitude. Each line represents a different single unit recording. (Data extracted from Figure 7 in Drake et al., 1988 [59] and Figure 8 in Cham et al., 2005 [60].)

The “passive” tissue response to the residence of the electrode within the brain can result from ongoing mechanical injury to the tissue as a result of electrode PRWLRQ>@DVZHOODVIURPWKHHOHFWURGHPDWHULDOV(YHQLQTXLHVFHQWDQHVWKHWL]HG animals, intrinsic brain movement from respiration exceeds 10 µm [23], and brain displacement may be much larger during changes in posture and free movement. Such motion can lead to variability in the recorded signal (Figure 2.5) or stimulation-evoked response (Figure 2.3) and lead to exacerbation of the tissue response to the residence of the device. There is a substantial mismatch between the mechanical properties of electrode materials and brain tissue, and this leads to substantial strain at the electrode–brain interface [22]. The shear modulus of the brain is 200 to 1500 Pa [24,25], which is several orders of magnitude less than the shear modulus of silicon (~50 GPa) or tungsten (~130 GPa), two commonly used electrode materials. Recall that shear modulus, G, is related to Young’s modulus, E, and the Poisson ratio, v, by G = E/[2*(1 + v)]. Several efforts have been made to develop polymerbased electrodes, but there is still a substantial mechanical mismatch between the properties of the brain and the properties of commonly used polymeric substrates including polyimide (~1 GPa), liquid crystal polymer (~3 GPa), and parylene (~1 GPa). Computational studies suggest that tighter mechanical coupling between the electrode and the brain [22], for example, through the use of integrative coatings, can reduce the strain at the electrode–tissue interface [26].



2.3.2

49

ACTIVE PROPERTIES

The tissue response due to the passage of current as required for neuronal stimulation may arise from electrochemical reaction products formed at the electrode-tissue interface or from physiological changes in the tissue that are associated with neural H[FLWDWLRQ,WLVGLI¿FXOWWRJHQHUDOL]HWKHUHVXOWVREWDLQHGLQVWXGLHVRIWLVVXHGDPage resulting from electrical stimulation to new electrode geometries or materials, new stimulation patterns, or different anatomical regions. As described by Agnew and colleagues “the manner and degree to which this [neural injury] occurs depends upon the physical and pharmacological composition of the particular neural substrate, as well as the stimulus parameters” [27]. The tissue response to the active properties of a neural interface can result from electrochemical reaction products formed at the tissue–electrode interface and from physiological changes associated with neural activity, which include both transient depression of excitability and neuronal damage. Each of these three active effects is considered in turn. 2.3.2.1 Electrochemistry of the Electrode–Electrolyte Interface Electrical stimulation is typically delivered using metal electrodes, which carry FXUUHQW DV WKH ÀRZ RI HOHFWURQV LPSODQWHG LQ WKH ERG\ ZKLFK FDUULHV FXUUHQW DV WKH ÀRZ RI LRns (Figure 2.6A). Thus, there exists an interface between the metal electrode and the ionic conductor of the body. In general this interface has a nonlinear impedance that is a function of the voltage across the interface. This interface impedance can impact the properties of stimulation, and electrochemical reactions at the electrode–tissue interface can lead to electrode dissolution and production of chemical species that may be damaging to tissue. Reactions that take place at the electrode–tissue interface are dependent on the potential of the electrode as well as the time course of the stimulus pulse and may be accelerated by increased current density. The electrode interface can be modeled by the parallel combination of a capacitor (C), representing the double-layer capacitance, and nonlinear impedances representing faradaic electrochemical reactions (Figure 2.6B). The voltage developed across the electrode–tissue interface (V) determines which chemical reactions will take place and is dependent on the amount of charge in the stimulus pulse (Q = current intensity * pulse duration) and the electrode capacitance, since V = Q/C. The electrode capacitance is determined by the properties of the material and is proportional to the electrode area, A. Therefore, the potential developed across the interface is proportional to electrode area. This relationship is the basis for the correlation between charge density and tissue damage and the assertion that the charge density is an indirect measure of the electrochemical contribution to tissue damage (see below). If the voltage across the electrode–tissue interface is kept within certain limits, then chemical reactions can be avoided, and all charge transfer will occur by the charging and discharging of the double-layer capacitance [21,28]. However, in many LQVWDQFHVWKHHOHFWURGHFDSDFLWDQFHLVQRWVXI¿FLHQWWRVWRUHWKHFKDUJHQHFHVVDU\ for the desired excitation without the electrode voltage reaching levels where reactions will occur. The principal approach to control the interface voltage has been the use of charge-balanced biphasic stimuli that have two phases that contain equal and opposite charge [29,30]. However, even with charge-balanced pulses it is pos-


50


FIGURE 2.6 (A) Characteristics of waveforms typically used for electrical stimulation of the nervous system. Typical stimuli are biphasic-balanced charge pulses with a duration of WRPVSKDVHDQDPSOLWXGHRIWRP$DQGDIUHTXHQF\RIWR+]% Equivalent circuit models of a metal electrode in an ionic conducting medium. C dl is the double layer capacitance and the Zi represent potential-dependent electrochemical reactions that can occur at the electrode–tissue interface. (C) Relationship between charge per phase and charge density for damaging and nondamaging electrical stimulation. Charge is the product of current and pulse width, and charge density is obtained by dividing by the electrode area. The damaging and nondamaging regions are based upon previous experimental studies. (Data compiled from Figure 9.1 of Agnew et al., 1990 [11] and Figure 8 from Merrill et al., 2005 [21].) The line is the Shannon model with k = 1.75.

sible that the interface voltage may reach levels where electrochemical reactions can occur. 2.3.2.2 Activity-Dependent Changes in Neuronal Excitability 'XULQJ DUWL¿FLDO HOHFWULFDO VWLPXODWLRQ UHODWLYHO\ ODUJH QXPEHUV RI QHXURQV DUH active synchronously. This nonphysiological pattern of activation results in activity-



51

dependent changes in neuronal excitability [31], as well as apparent activity-dependent neuronal injury. &RQWLQXRXV VWLPXODWLRQ RI ODUJHGLDPHWHU SHULSKHUDO QHUYH ¿EHUV FDQ FDXVH D SURORQJHGLQFUHDVHLQWKHLUHOHFWULFDOWKUHVKROG(LJKWKRXUVRI+]VWLPXODWLRQRI the common peroneal nerve at suprathreshold intensities produced a reduction in the amplitude of the evoked compound nerve action potential in the large-diameter QHUYH¿EHUV>@,QVRPHLQVWDQFHVWKLVUHGXFWLRQLQH[FLWDELOLW\UHFRYHUHGDIWHU 7 days of no stimulation, even in nerves that exhibited axonal degeneration, while in others it did not. The reduction in excitability was mitigated by using intermittent GXW\F\FOHVDQGORZHUIUHTXHQFLHV+] RIVWLPXODWLRQ6LPLODUO\VWLPXODWLRQRI the cochlear nerve can produce reversible elevations in electrical thresholds [34]. Continuous stimulation of the cortex also produced reversible increases in the thresholds to produce both direct activation as well as trans-synaptic activation of FRUWLFDO S\UDPLGDO WUDFW QHXURQV >@ 6WLPXODWLRQ DW +] IRU K RU KGD\ for 1 week with suprathreshold pulses (40 to 80 µA, 200 µs per phase, 400 to 800 µC/cm 2) generated profound but reversible decreases in neuronal excitability. These elevations in threshold were not associated with damage or loss of neurons associated with the delivery of stimulation [36]. However, with the stimulation intensity increased to 320 µA (3200 µC/cm2), depression of excitability following 24 h of continuous stimulation was not reversed by 7 to 12 days after stimulation. In WZRLQVWDQFHVWKLVZDVDVVRFLDWHGZLWKKLJKO\ORFDOL]HGQHXURQDOGDPDJHDURXQG the tips of platinum–iridium electrodes but not around other platinum–iridium or activated iridium microelectrodes. Thus, there was not a clear correlation between either reversible or irreversible increases in electrical threshold and neuronal damage. Furthermore, stimulus levels that when applied to a single microelectrode did not produce depression of excitability, when applied simultaneously to multiple electrodes within an array did induce substantial depression of excitability. This supports for the concept that changes in excitability are driven through some “mass action” arising from the simultaneous activation of a critical number of neurons. Electrical microstimulation of the cochlear nucleus, as might be used for restoration of hearing, also produces transient reductions in neuronal excitability [37]. 2.3.2.3

Stimulation-Induced Neuronal Injury

The number and spatial extent of neurons activated are related to the charge injected in each stimulus pulse, while the charge density (charge per unit area of the stimulating contact) contributes to the type and rate of electrochemical reactions that RFFXUDWWKHHOHFWURGHWLVVXHLQWHUIDFH7KH¿QGLQJWKDWFKDUJHSHUSKDVHDQGFKDUJH density are both factors in determining the threshold for neural injury by stimulation in both the peripheral nervous system (PNS) [32] and the CNS [38] suggests that in addition to electrochemical mechanisms, physiological mechanisms also contribute to neural damage. The correlation of charge per phase and tissue damage is thought to result from physiological changes associated with neural excitation. Physiological changes resulting from synchronous activation of a population of neurons increase as the number of excited neurons increases. This relationship may create the correlation


52


between charge per phase and neural damage. There is substantial support for this hypothesis in the PNS and the CNS. In the PNS, peripheral nerve injury can still occur at very low charge densities [32], and anesthetic block of electrical activity occludes the damaging effect of stimulation [39]. In the CNS, equivalent tissue damage was seen under platinum electrodes and tantalum pentoxide capacitor-type electrodes (which were presumed not to have electrochemical reactions) [40]. These studies, which have attempted to differentiate electrochemically induced and activity-induced tissue damage, support thH¿QGLQJWKDWWLVVXHLQMXU\UHVXOWVIURPSK\VLological changes in the neural environment resulting from synchronous activation of populations of neurons. 2.3.2.4 The Shannon Model The experimental data on the role of charge and charge density in determining whether a stimulation condition is likely to cause tissue damage are well described by the quantitative model of Shannon [41]. This model was originally conceived to describe the empirical data of McCreery et al. [38] from cortical stimulation with circular disk electrodes, but the model is also consistent with other seemingly disparate data. The Shannon model is described using the equation, log(Q/A) = k íORJQ), where Q is the charge per phase of the stimulus pulse, A is the electrode area, and k LVDFRQVWDQWGHULYHGIURPHPSLULFDOGDWDWRGH¿QHWKHERXQGDU\EHWZHHQVWLPXOXV parameters that produced tissue damage and those that did not (Figure 2.6C).

2.4 A CASE STUDY: DEEP BRAIN STIMULATION Deep brain stimulation (DBS) has emerged rapidly as a successful treatment for the symptoms of movement disorders including Parkinson’s disease, essential tremor, and dystonia and is under investigation for treatment of epilepsy as well as psychiatric disorders including depression and obsessive–compulsive disorder. The DBS system includes an electrode that is surgically implanted within a brain target, according to the disease being treated, connected by a lead extension to a batterypowered pulse generator implanted in the chest. The electrode leads used for DBS (Model 3387 and 3389, Medtronic, Inc., Minneapolis, Minnesota) are cylindrical with macroscopically smooth texture. The four electrode conductors are platinum alloyed with iridium, and the insulating jacket is polyurethane (Figure 1.2C). The HIIHFWV RI WUHDWPHQW DUH W\SLFDOO\ RQO\ UHDOL]HG ZKHQ WKH GHYLFH LV RQ DQG WKXV this is a permanently implanted medical device intended to last the lifetime of the recipient.

2.4.1

PASSIVE PROPERTIES

There is evidence for a strong passive effect due to DBS electrode insertion in the brain. Immediately after implantation, in many cases the symptoms disappear because of a transient lesioning effect of device placement, most often attributed to edema around the electrode. There are also maintained insertional effects, apparently caused by the lesion made by the presence of the electrode. This has been



53

observed in pain treatment [42] as well as in epilepsy treatment [5]. However, for the treatment of movement disorders, the continued delivery of stimulation is required for the maintenance of effect, and the effect of the implantation-associated lesion is resolved within weeks following implantation.

2.4.2

ACTIVE PROPERTIES

The typical output parameters for DBS are 3 V stimulation amplitude (i.e., 3 mA LQWRDQRPLQDONORDG VSXOVHGXUDWLRQDQGWR+]VWLPXODWLRQIUHquency (Figure 1.6A), and the devices are typically on for 12 to 24 hours per day. 2.4.2.1 Preclinical Studies Preclinical studies were conducted by Medtronic, Inc. to evaluate the tissue response to DBS lead implantation and stimulation [43]. Acute studies were conducted in 10 pigs implanted bilaterally with Model 3387 leads and stimulated for 7 hours at supraFOLQLFDOSDUDPHWHUV9WR+]DQGSXOVHGXUDWLRQVIURPWR ms). Postmortem pathology revealed tissue damage around both unstimulated and stimulated leads, and the difference was detectable in only 3 of 9 cases. The tissue UHVSRQVHLQFOXGHGLQÀDPPDWLRQJOLRVLVDQGQHXURQDOGHJHQHUDWLRQ6XEVHTXHQWO\ a chronic study was conducted in 8 pigs implanted bilaterally with Model 3387 leads for 2 to 9 months of continuous stimulation with parameters more similar to those XVHG FOLQLFDOO\ +] SXOVH GXUDWLRQ RI PV DQG YROWDJH VHW WR PD[LPXP tolerable). From the 14 lead pairs that were evaluated, 11 (79%) showed differential effects of stimulation and 3 (21%) did not. The tissue response within 2 mm of the OHDGLQFOXGHGLQÀDPPDWLRQPLQHUDOL]DWLRQJOLRVLVDQGQHXURQDOGHJHQHUDWLRQ 2.4.2.2 Autopsy Findings A number of studies have examined the tissue response to chronically implanted DBS electrodes. This review is restricted to DBS for the treatment of movement GLVRUGHUVXVLQJHOHFWURGHV0RGHOV WKDWKDYHZHOOFKDUDFWHUL]HGPDWHULals and regulatory approval. Caparros-Lefebvre reported on a patient who was discontinuously stimulated over the course of 43 months [44] with DBS of the Vim thalamus to treat ParkinsoQLDQWUHPRU*OLRVLVDQGYDFXROL]DWLRQZHUHREVHUYHGZLWKLQPPRIWKHHOHFWURGH and adjacent to polyurethane insulation macrophages and giant cells were observed. The authors concluded that the “lesion” resulting from implantation and application of stimulation was smaller than functional thalamic lesions placed to treat motor symptoms. Haberler and colleagues [45] reported on a series of 8 patients with Parkinson’s disease who had received either thalamic (6 cases) or subthalamic nucleus (STN) (2 cases) DBS for up to 70 months. The brain areas around 11 leads were examined following between 3 and 70 months of stimulation, and the response to a single lead ZDVH[DPLQHGGD\VDIWHULPSODQWDWLRQ7KHOHDGVZHUHVXUURXQGHGE\D¿EURXV WLVVXHFDSVXOHWRPWKLFNIROORZHGE\DULPRIDJOLDO¿EULOODU\DFLGLFSURWHLQ


54


(GFAP)-positive gliosis about 500 µm thick, followed by a 1-mm thick annulus of scattered GFAP-reactive astrocytes. Furthermore, mononuclear leukocytes, macrophages, and multinucleated giant cells were observed in the track and surrounding tissue. In one patient the giant cells were seen to have engulfed fragments of foreign material. No loss of axons or demyelination was observed, and healthy neurons were found immediately adjacent to the electrode tracks. Two observations from this study suggest that stimulation per se did not contribute to the observed tissue response. First, the tissue characteristics were similar across cases, independent of the duration of stimulation. Second, the tissue characteristics were the same in the vicinity of insulated and on stimulated regions of the lead as over the stimulated portions of the lead. The authors concluded that “chronic DBS does not cause damage to adjacent brain parenchyma” [45]. In a case report, Henderson et al. [46] reported the presence of a thalamic lesion following implantation and stimulation for 2 years to treat Parkinson’s disease. The electrode tract was surrounded by a glial sheath and tissue vacuoles, and the tip was located within the centromedian–parafascicular nucleus of thalamus (CM/Pf). The tip was surrounded by an apparent lesion and increased astrocytosis, and there was extensive neuronal loss within the CM/Pf. The extensive lesion present in this case differs from the benign tissue response described for most other cases, although the stimulus parameters were quite similar, and the authors conclude that DBS may be associated with thalamic damage. It is noteworthy that this electrode was implanted to replace a lead initially placed within the Vim thalamus and forcibly removed by the patient. In a second case report, Henderson et al. [47] described the tissue response to STN DBS in a patient with Parkinson’s disease who died 2 months after implantaWLRQ $Q LQÀDPPDWRU\ UHDFWLRQ ZDV SUHVHQW ZLWKLQ PP RI WKH HOHFWURGH WUDFW FKDUDFWHUL]HG E\ DVWURJOLRVLV WKH DSSHDUDQFH RI PDFURSKDJHV DQG PLFURJOLD DQG GFAP staining. Furthermore, there was apparent “minor cell loss” within the thalamus through which the tract passed as well as in the STN. The authors concluded that little tissue damage is associated with STN DBS. In another case report, Burbaud et al. [48] reported on the tissue response to 2 years of DBS in the Vop thalamus in a patient with chorea–acanthocytosis. The right electrode was forcibly removed by the patient and was subsequently replaced at the same coordinates. The corresponding electrode tract exhibited astrocytic JOLRVLVLQ¿OWUDWLRQRIO\PSKRF\WHVDQGLURQGHSRVLWV7KHSUHVHQFHRILURQDURXQG the implant site is somewhat perplexing, but this could be the result of hemosiderin containing macrophages [45]. The left electrode tract induced less tissue response, DQGDWWKHWLSRIERWKHOHFWURGHVWKHUHZDVIRFDOJOLRVLVDQGOHXNRF\WHLQ¿OWUDWLRQ The authors concluded that DBS induced only minor changes in the tissue in the vicinity of the electrode. Henderson and colleagues reported on a third case of a patient with Parkinson’s disease who received DBS of the Vim thalamus for 7 years [49]. A region of astrocytosis surrounded the electrode track. Within 0.5 mm of the stimulated electrode and the electrode tip there was astrocytosis, an apparent reduction in the density RIP\HOLQDQGVLJQL¿FDQWORVVRIQHXURQVORVVFRPSDUHGWRWKHFRQWUDODWHUDO Vim), and the remaining neurons appeared pyknotic. However, there were no signif-



55

icant changes in neuronal density near the electrodes not used for stimulation or in the region further (0.5 to 1.5 mm) from the active electrode. The authors also elaborated on their earlier report [47] to reveal that neuronal loss within the STN was approximately 30% and was noted within 0.5 mm of the stimulated contact but not near the unstimulated contacts. These results suggest, and the authors concluded, that the neuronal loss may have been due to stimulation, rather than just the surgical introduction of the electrode. Importantly, even with these levels of neuronal loss, WKHUHVXOWLQJ³OHVLRQ´ZDVLQVXI¿FLHQWWRWUHDWWKHPRYHPHQWGLVRUGHUDQGHI¿FDF\ required the continued delivery of stimulation. In an abstract reporting a single case of DBS in the STN to treat Parkinson’s disease, Larsen et al. [50] described that the electrode track was surrounded by a GFAP-positive “capsule” approximately 150 µm thick constituted of a “thin collagen layer lining the lumen of the tract” surrounded by a region of decreased neuronal density. There were no apparent differences in tissue in regions receiving stimulation and regions of no stimulation. Moss et al. [51] reported on the characteristics of the tissue that was adhered to DBS electrodes removed after 3 to 31 months for clinical reasons including suboptimal positioning (14/21 leads), scalp infection (2/21), increased impedance (2/21), fracture of the electrode (1/21), damaged connection (1/21), or displacement of the electrode (1/21). Scanning and transmission electron microscopy revealed the presence of multinucleated giant cells and mononuclear macrophages. Electron-dense material was present within and around both cell types, suggesting active phagocytosis, and although it was suggested that this material might be polyurethane particles, subsequent microscopy of polyurethane insulation did not support this conclusion. 2.4.2.3 Charge Density Limits The Shannon model, and the data on which it was based, provided the rationale for the manufacturer of DBS systems to recommend that stimulus parameters be selected not to exceed a charge density of 30 µC/cm 2. Since the electrode area is ¿[HGFP 2 for the 3387 and 3389 leads), this leads to a line relating charge per phase to charge density (Figure 2.7). However, several factors must be considered when evaluating this recommendation, and the human postmortem studies that reported substantial loss of neurons in proximity to the active electrode [49] employed parameters well below this limit. First, all human postmortem studies to date are from cases where stimulus parameters were set well under the proposed limit, so the lack of pathology in these cases does not necessarily support this limit. Second, the stimulation frequency used to generate the data from which the limit was established, was in some cases ORZHUWKDQWKH+]IUHTXHQF\W\SLFDOO\HPSOR\HGZLWK'%6DQGWKHDSSHDUance of neural damage appears to have a frequency-dependent threshold. Finally, the distribution of charge across the electrode contact is highly nonuniform [52],


56


FIGURE 2.7. Relationship between charge per phase and charge density for damaging and nondamaging electrical stimulation, as relates to deep brain stimulation. The damaging and nondamaging regions are based upon previous experimental studies and described using the Shannon model with k = 1.5. The combinations of charge and charge density from postmortem studies of deep brain stimulation (DBS) are indicated by the circles. The clinical DBS electrode has an area of 0.059 cm 2 and results in the gray line.

and the peak charge density is likely to exceed the recommended limit, even when the average charge density does not. 2.4.2.4

Performance Considerations

These studies indicate that the implantation and residence of DBS electrodes evokes a classic chronic foreign body response characteri]HGE\WKHSUHVHQFHRID¿EURXV tissue capsule, often termed a “glial scar,” surrounded by a region of gliosis, as demonstrated by upregulation of GFAP. The spatial extent of the resulting scar, and in some cases loss of neurons, extended further than 1 mm from the electrode. This apparent increase in the electrode to neuron distance is expected to increase the stimulation intensity required for effect (Figure 2.3) relative to that which would be required with a less pronounced tissue response. However, in all cases the resultLQJ OHVLRQ ZDV LQVXI¿FLHQW WR WUHDW WKH PRYHPHQW GLVRUGHU DQG HI¿FDF\ UHTXLUHG



57

continued stimulation, and there are no reported decreases in device performance as a result of tissue damage. The lack of correlation between the level of stimulation and the tissue response [45] and the lack of difference in tissue adjacent to stimulated and unstimulated electrodes [50] appear at odds with the reports of Henderson and colleagues [46, @LQGLFDWLQJVLJQL¿FDQWQHXURQDOORVVQHDUWKHVWLPXODWHGHOHFWURGHVEXWQRW near unstimulated electrodes. Two factors may contribute to these disparate results. First, the quantitative neuronal analysis used by Henderson et al. was likely more sensitive than the qualitative observations of others, and the latter may have missed reductions in neuronal density. Second, some neuronal loss may have been mechanical in nature and occurred preferentially at the end of the electrode [53].

2.5 FUTURE CONSIDERATIONS The chronic application of electrical stimulation of the brain or spinal cord, as required for neural prosthetic devices, can cause damage to the electrode or the underlying tissue. Such damage may render the device ineffective, and thus prevention of such damage is of paramount concern. Clinical implementation of DBS, for example, has not revealed apparent decreases in device performance as a result of tissue damage. However, continued use of these devices in a nondamaging manner requires an understanding of the mechanisms responsible for tissue damage, especially in light of reports of neuronal loss in proximity to active electrodes. As electrodes are made smaller, for example, to improve the selectivity of stimulation, or as stimulus parameters are increased, for example, in the treatment of obsessive compulsive disorder, larger voltages are used more than in the treatment of movement disorders, with the potential risks of tissue damage increase. The lack of knowledge about the fundamental causes of neuronal injury makes LWGLI¿FXOWWRGHWHUPLQHa priori whether a proposed electrode design or stimulation paradigm will be damaging or not. For example, changes in the shape of an electrode will result in changes in the current density distribution on the surface of electrodes. The change in current (or, equivalently, charge) density distribution may affect the propensity for stimulation to generate either tissue damage or electrode damage. Charge density is a factor in determining whether stimuli are damaging or not [38], and electrode corrosion appears to occur preferentially at the edges where the current density is highest [52]. When commenting on studies on neural damage with disk electrodes on the cortical surface [53], Larson [54] pointed out that “…current density and charge density is not uniform beneath the electrode…[and] consequently it is not possible to extend these observations to other electrodes.” Thus, until the fundamental causes of neuronal injury are determined, design changes QHFHVVLWDWHHPSLULFDOVWXGLHVWRGHWHUPLQHWKHSURSHQVLW\RIDVSHFL¿FJHRPHWU\RU set of parameters to produce neural damage.

ACKNOWLEDGMENTS Preparation of this chapter was supported by grants R01 NS040894 and R21 16IURPWKH1DWLRQDO,QVWLWXWHVRI+HDOWK7KDQN\RXWR'U'XVWLQ-7\OHU


58


Case Western Reserve University, for information on the mechanical properties of brain tissue and electrode materials, and to Amin Mahnam for conducting the simulations reported in Figure 2.4.

REFERENCES

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1LFROHOLV 0 $ / 'LPLWURY ' &DUPHQD - 0 &ULVW 5 /HKHZ * .UDOLN - D., and Wise, S. P. Chronic, multisite, multielecrode recording in macaque monkeys. Proc. Natl. Acad. Sci. 100, 11041, 2003. 1RUPDQQ5$0D\QDUG(05RXVFKH3-DQG:DUUHQ'-$QHXUDOLQWHUIDFH for a cortical vision prosthesis. Vision Res. 39, 2577, 1999. 5DQFN-%-U:KLFKHOHPHQWVDUHH[FLWHGLQHOHFWULFDO VWLPXODWLRQRIPDPPDOLDQ central nervous system: a review. Brain Res. 98, 417, 1975. 58. McIntyre, C. C., and Grill, W. M. Selective microstimulation of central nervous system neurons. Ann. Biomed. Eng. 28, 219, 2000. 'UDNH./:LVH.')DUUD\H-$QGHUVRQ'-DQG%H0HQW6/3HUIRUPDQFH of planar multisite microprobes in recording extracellular single-unit intracortical activity. IEEE Trans Biomed. Eng. 35, 719, 1988. &KDP-*%UDQFKDXG($1HQDGLF=*UHJHU%$QGHUVHQ5$DQG%XUGLFN -:6HPLFKURQLFPRWRUL]HGPLFURGULYHDQGFRQWURODOJRULWKPIRUDXWRQRPRXVO\LVRlating and maintaining optimal extracellular action potentials. J. Neurophysiol. 93, 570, 2005.


3

Thermal Considerations for the Design of an Implanted Cortical Brain–Machine Interface (BMI) Patrick D. Wolf

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3.2.2

SPIKE ACQUISITION AND PROCESSING

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3.2.3

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3.2.4

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3.3.1

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3.4.1

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3.5.1

RECOMMENDATION 1: DISTRIBUTE THE THERMAL LOAD

,QWHUSUHWDWLRQRIWKHVWXGLHVE\'DYLHVHWDO>@DQGFRQVLGeration of the bioheat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¿[HGWKHUPDOEXUGHQRYHUDJUHDWHUVXU IDFH DUHD ZLOO UHGXFH WKH WHPSHUDWXUH LQFUHDVHV DVVRFLDWHG ZLWK WKH ORDG :KLOH WKHUHLVWUHPHQGRXVSUHVVXUHWRPLQLPL]HWKHVL]HRIQHXUDOLPSODQWVWKHGLI¿FXOW\ RIPDQDJLQJWKHWKHUPDOEXUGHQLQDVPDOOSDFNDJHRQO\LQFUHDVHVDVWKHSDFNDJH VL]HGHFUHDVHV )LJXUH VKRZVRQHZD\VXFKDGLVWULEXWHGV\VWHPFRXOGEHLPSOHPHQWHG7KH DPSOL¿HUDUUD\VDUHORFDWHGRQWKHKHDGQHDUWKHHOHFWURGHVIRUPD[LPXP6157KH DUUD\VDUHFRQWDLQHGLQWKUHHSDFNDJHVIRUH[DPSOH WRIXUWKHUGLVWULEXWHWKHKHDW DFURVVWKHVFDOS7KHGDWDSURFHVVLQJDQGWHOHPHWU\DUHORFDWHGLQDQLPSODQWVLWH LQWKHWKRUD[7KLVLVDVXEVWDQWLDOGLVWDQFHIURPWKHVFDOSIDUHQRXJKIRUWKHWZR WKHUPDOORDGVWREHFRQVLGHUHGLQGHSHQGHQW 7KHFRLOIRUWKH7(76LVORFDWHGDVKRUWGLVWDQFHIURPWKHHOHFWURQLFVWRVHSDUDWH WKHVHWKHUPDOVRXUFHV7KHWUDQVPLWWLQJDQWHQQDLVORFDWHGVHYHUDOFHQWLPHWHUVIURP WKHLPSODQWVRWKDWWKHKHDWJHQHUDWHGLQWKHH[WUHPHQHDU¿HOGRIWKHDQWHQQDGRHV QRWDGGWRWKHWKHUPDOEXUGHQJHQHUDWHGE\WKHGHYLFH

3.5.2

RECOMMENDATION 2: USE A LOW TRANSMISSION FREQUENCY FOR TELEMETRY

7KHDEVRUSWLRQFRHI¿FLHQWĮ IRU5)VLJQDOVLQWLVVXHLVVWURQJO\IUHTXHQF\GHSHQ GHQW7LVVXHDEVRUSWLRQRI5)HQHUJ\FDQEHUHGXFHGE\XVLQJDORZHUWUDQVPLWWLQJ IUHTXHQF\7\SLFDOO\WKHKLJKHUWKHEDQGZLGWKUHTXLUHPHQWVRI DWUDQVPLWWHUWKH KLJKHUWKHFDUULHUIUHTXHQF\1HZKLJKEDQGZLGWK±ORZFDUULHUIUHTXHQF\WUDQVPLW WHUVPD\QHHGWREHGHVLJQHGWRDFFRPPRGDWHWKLVQHHG>@

3.5.3

RECOMMENDATION 3: SEPARATE POWER TRANSMISSION COILS FROM OTHER IMPLANTED METALLIC OBJECTS

7KHLQGXFWLRQRIHGG\FXUUHQWVLQDQLPSODQWHGPHWDOREMHFWFDQEHPLQLPL]HGE\ LQFUHDVLQJWKHGLVWDQFHEHWZHHQWKHPDJQHWLF¿HOGDQGWKHPHWDOREMHFWV


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3.5.4

RECOMMENDATION 4: MINIMIZE THE POWER CONSUMPTION IN DEVICES IN CLOSE PROXIMITY TO THE BRAIN

5HJLRQDOEUDLQWHPSHUDWXUHLQUDWVLQFUHDVHVE\XSWRGHJUHHVIRUKRXUVDWDWLPH LQUHVSRQVHWRLQFUHDVHGPHWDEROLFGHPDQG$WKHUPDOEXUGHQRIRQHRUWZRGHJUHHV RQWRSRIWKLVLQFUHDVHFRXOGSXWWKHWRWDOWHPSHUDWXUHLQFUHDVHLQWKHUDQJHZKHUHD WKHUPDOUHVSRQVHLVSURYRNHGRULQMXU\PD\RFFXU7KHVHIDFWVLQGLFDWHWKDWLI\RX DUHJRLQJWRLQFUHDVHWKHWKHUPDOEXUGHQLQWKHERG\LWLVSUREDEO\EHVWQRWWRGRLW LQWKHEUDLQ $VGHVFULEHGDERYHWKHDFTXLVLWLRQDQGWUDQVPLVVLRQRIQHXUDOVLJQDOVIURPWKH FRUWH[LVDFRPSOH[WDVNLQYROYLQJVHYHUDOSURFHVVLQJVWHSV1HXUDODPSOL¿HUVQHHG WREHSODFHGLQFORVHSUR[LPLW\WRHOHFWURGHVWRPDLQWDLQDQDGHTXDWH615+RZHYHU WKH DPSOL¿HUV FDQ EH VHSDUDWHG IURP WKH HOHFWURGHV E\ D VKRUW GLVWDQFH DERYH WKH ERQHSURYLGLQJDFULWLFDOLQVXODWLRQEDUULHUDQGOLPLWLQJWKHGLUHFWKHDWLQJRIEUDLQ WLVVXH



3.6

83

CONCLUSION

7KHDFTXLVLWLRQSURFHVVLQJDQGWHOHPHWU\RIKXQGUHGVRUWKRXVDQGVRIQHXUDOVLJ QDOVUHTXLUHVVLJQL¿FDQWSRZHU7KHKHDWIURPWKHLPSODQWHGV\VWHPLPSOHPHQWLQJ WKHVHIXQFWLRQVPXVWEHGLVVLSDWHGDGHTXDWHO\WRSUHYHQWDQDGYHUVHWLVVXH response WRWKHLPSODQWHGGHYLFH 7KHLQGLFDWLRQVDUHWKDWWKHOLPLWVRID&WHPSHUDWXUHLQFUHDVHRIP:FP KHDWÀX[DQGRIP:JRI6$5DUHYDOLGIRUPRVWWLVVXHVLQWKHERG\7KHDSSDU HQW VHQVLWLYLW\ RI &16 WLVVXH WR VPDOO WHPSHUDWXUH LQFUHDVHV WKH LQGLFDWLRQV WKDW regional metabolic heat generation in the brain may exceed the clearance capacity IRUXQNQRZQUHDVRQVDQGWKHIDFWWKDWWKHUHLVOLWWOHUHVHDUFKUHJDUGLQJYHU\ORQJ WHUPK\SRWKHUPLDLQQHXUDOWLVVXHVXJJHVWVWKDWXVLQJWKHEUDLQWLVVXHDVDVLQNIRU GHYLFHUHODWHGKHDWPD\EHTXHVWLRQDEOH

REFERENCES

'RQRJKXH-3&RQQHFWLQJFRUWH[WRPDFKLQHVUHFHQWDGYDQFHVLQEUDLQLQWHUIDFHV Nat. Neurosci.6XSSO ± 1LFROHOLV0$%UDLQPDFKLQHLQWHUIDFHVWRUHVWRUHPRWRUIXQFWLRQDQGSUREHQHXUDO FLUFXLWVNat. Rev. Neurosci. ± 1LFROHOLV0$DQG&KDSLQ-.&RQWUROOLQJURERWVZLWKWKHPLQGSci. Am. ± 6FKZDUW]$%&RUWLFDOQHXUDOSURVWKHWLFVAnnu. Rev. Neurosci.± :ROSDZ-5%LUEDXPHU10F)DUODQG'-3IXUWVFKHOOHU*DQG9DXJKDQ70 %UDLQFRPSXWHULQWHUIDFHVIRUFRPPXQLFDWLRQDQGFRQWUROClin. Neurophysiol. ± /HXWKDUGW ( & 0LOOHU . - 6FKDON * 5DR 5 3 DQG 2MHPDQQ - * (OHFWUR FRUWLFRJUDSK\EDVHGEUDLQFRPSXWHULQWHUIDFH²7KH6HDWWOHH[SHULHQFHIEEE Trans. Neural. Syst. Rehabil. Eng. ± /HXWKDUGW ( & 6FKDON * :ROSDZ - 5 2MHPDQQ - * DQG 0RUDQ ' : $ EUDLQ±FRPSXWHU LQWHUIDFH XVLQJ HOHFWURFRUWLFRJUDSKLF VLJQDOV LQ KXPDQV J. Neural Eng. ± &DUPHQD-0/HEHGHY0$&ULVW5(2¶'RKHUW\-(6DQWXFFL'0'LPL WURY')3DWLO3*+HQULTXH]&6DQG1LFROHOLV0$/HDUQLQJWRFRQWUROD EUDLQPDFKLQHLQWHUIDFHIRUUHDFKLQJDQGJUDVSLQJE\SULPDWHVPLoS Biol ( &KDSLQ-.0R[RQ.$0DUNRZLW]56DQG1LFROHOLV0$5HDOWLPHFRQWURO RIDURERWDUPXVLQJVLPXOWDQHRXVO\UHFRUGHGQHXURQVLQWKHPRWRUFRUWH[Nat. Neurosci. ± 7D\ORU'07LOOHU\6,DQG6FKZDUW]$%'LUHFWFRUWLFDOFRQWURORI'QHXUR SURVWKHWLFGHYLFHVScience ± :HVVEHUJ - 6WDPEDXJK & 5 .UDOLN - ' %HFN 3 ' /DXEDFK 0 &KDSLQ - ..LP-%LJJV6-6ULQLYDVDQ0$DQG1LFROHOLV0$5HDOWLPHSUHGLFWLRQ RI KDQG WUDMHFWRU\ E\ HQVHPEOHV RI FRUWLFDO QHXURQV LQ SULPDWHV Nature ± 'RQRJKXH-31XUPLNNR$%ODFN0DQG+RFKEHUJ/5$VVLVWLYHWHFKQRORJ\ DQG URERWLF FRQWURO XVLQJ PRWRU FRUWH[ HQVHPEOHEDVHG QHXUDO LQWHUIDFH V\VWHPV LQ KXPDQVZLWKWHWUDSOHJLDJ. Physiol.


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(OZDVVLI00.RQJ49D]TXH]0DQG%LNVRQ0%LRKHDWWUDQVIHUPRGHORI GHHSEUDLQVWLPXODWLRQLQGXFHGWHPSHUDWXUHFKDQJHVJ. Neural Eng± %X]VDNL*/DUJHVFDOHUHFRUGLQJRIQHXURQDOHQVHPEOHVNat. Neurosci. 7(5), 446– +ROW*5DQG.RFK&(OHFWULFDOLQWHUDFWLRQVYLDWKHH[WUDFHOOXODUSRWHQWLDOQHDUFHOO ERGLHVJ. Comput. Neurosci. ± 1LFROHOLV 0 $ *KD]DQIDU $ $ )DJJLQ % 0 9RWDZ 6 DQG 2OLYHLUD / 0 5HFRQVWUXFWLQJWKHHQJUDPVLPXOWDQHRXVPXOWLVLWHPDQ\VLQJOHQHXURQUHFRUGLQJV Neuron ± 6HUUX\D0'+DWVRSRXORV1*3DQLQVNL/)HOORZV05DQG'RQRJKXH-3 ,QVWDQWQHXUDOFRQWURORIDPRYHPHQWVLJQDONature ± +DUULVRQ55DQG&DPHURQ&$ORZSRZHUORZQRLVH&026DPSOL¿HUIRUQHXUDO UHFRUGLQJDSSOLFDWLRQVIEEE J. Solid-State Circuits ± 1HLKDUW 1 0 DQG +DUULVRQ 5 5 0LFURSRZHU FLUFXLWV IRU ELGLUHFWLRQDO ZLUHOHVV WHOHPHWU\LQQHXUDOUHFRUGLQJDSSOLFDWLRQVIEEE Trans. Biomed. Eng. ± 2OVVRQ5+UG%XKO'/6LURWD$0%X]VDNL*DQG:LVH.'%DQGWXQ DEOH DQG PXOWLSOH[HG LQWHJUDWHG FLUFXLWV IRU VLPXOWDQHRXV UHFRUGLQJ DQG VWLPXODWLRQ ZLWKPLFURHOHFWURGHDUUD\VIEEE Trans. Biomed. Eng. ± 6RQJ@ ,QFRQWUDVWDGLIIHUHQWJURXSXVLQJDGLIIHUHQW¿EUREODVWFHOOOLQHGHULYHGIURPUDW VNLQ &5/ $PHULFDQ 7\SH &XOWXUH &ROOHFWLRQ >$7&&@ GLG VKRZ D VLJQL¿FDQWGLIIHUHQFHLQDGKHVLYHSURSHUWLHVEHWZHHQWKH¿EUREODVWDQGJOLDOFHOOOLQHVRQ 5*'6PRGL¿HG VXUIDFes [12], underscoring the variability in responses between different cell lines. 4.2.1.2 Neuronal Cell Lines 2QH OLQH RI QHXURHOHFWURGH UHVHDUFK KDV IRFXVHG RQ ¿QGLQJ PDWHULDOV WKDW DWWUDFW neuronal processes or stimulate neurite growth. For these experiments, neuronal cell lines have often been used and their responses to various materials compared to other cell types found in the brain. The most widely used neuronal cell line in the neurobiology community is the PC-12 cell line, which exhibits many neuronal


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FIGURE 4.2 Photomicrograph depicting the morphology of adherent 3T3 cells on a PI electrode shank and surrounding wafer surface (scale bar = 100 ȝm). (Adapted from Lee, K. K., He, J. P., Singh, A., Massia, S., Ehteshami, G., Kim, B., and Raupp, G., J. Micromech. Microeng., 14, 32–37, 2004. With permission.) (See color insert following page 110.)

behaviors, especially reversible differentiation and neurite growth in response to NGF. A typical experiment with this cell line involves coating the substrate of interest with laminin or another molecule to promote adhesion, seeding the cells on the substrate, waiting for cell growth and differentiation (typically 1 to 3 days), and WKHQ¿[LQJWKHFHOOVDQGXVLQJLPPXQRVWDLQLQJWRLPDJHFHOOERGLHVDQGQHXULWHV 7RPHDVXUHDGKHVLRQWKHQXPEHURIFHOOVLVFRXQWHGLQUDQGRPPLFURVFRSH¿HOGV to obtain a cell number or cell coverage ratio. A more sensitive measure to look at how the electrode material (cell culture substrate) affects neuronal growth involves counting the neurites and even measuring average neurite length as the immortalized neuronal cells differentiate. In two such experiments, PC-12 cell neurite growth was found to be more robust on textured silicon neuroelectrodes than on nonporous silicon by Moxon et al. [13], and PC-12 cells were found to adhere to I.9$9SHSWLGHPRGL¿HGSRO\LPLGHDQGVLOLFRQR[LGHVXUIDFHVWRD PXFKJUHDWHU H[WHQWWKDQ¿EURblast or glial cell lines [11]. Martin’s group is attempting to integrate a conducting polymer grown from the neuroelectrode recording sites with neurons around the electrode to try to establish a more reliable and robust signal. They found that after seeding the cells on top of several different substrates, the human neuroblastoma SH-SY5Y cell line preferentially grew on top of the polypyrrole/CDPGYIGSR polymer/peptide blend “grown” from the recording sites of a silicon electrode [14]. Over a dozen well-characterized neuronal cell lines are available as a result of the decades of cell line experiments in the neurosciences [8].


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In Vitro Models for Neuroelectrodes

97

4.2.1.3 Astrocyte Cell Lines Because of the widespread understanding that the biocompatibility of neuroelectrodes is a function of the glial response to the implanted material, the majority of cell line–based experiments have been conducted using cell lines that approximate microglial or astroglial function. None of the astrocyte cell lines are perfect in recUHDWLQJSULPDU\DVWURF\WHEHKDYLRUEXWWKH\DUHVHHQDVPRUHUHOHYDQWWKDQ¿EURblast cell lines in studying brain biocompatibility. As with neuronal cell lines, the PRVWFRPPRQH[SHULPHQWPHDVXUHVFHOODGKHVLRQDQGJURZWKRIFHOOVRQDVSHFL¿F substrate. Cells are seeded in tissue culture plates containing the substrate of interHVWDQGDOORZHGWRDGKHUHDQGJURZIRUWRGD\V7KHFHOOVDUHWKHQ¿[HGULQVHG and stained to assess the number of cells adhering to the substrate (as compared to other substrates or tissue culture plastic control). Cell spreading can also be meaVXUHGWRLGHQWLI\PDWHULDOVWKDWSURPRWHDVSHFL¿FW\SHRIEHKDYLRUZKHWKHURQH desires spreading or no spreading depends on the objectives of the experiment). One of the most common astroglial cell lines is the C6 line derived from a rat N-nitrosomethylurea-induced glioma. The cell line expresses one stereotypical astrogliDOPDUNHU6EXWQRWWKHPRVWVSHFL¿FDVWURF\WHPDUNHUJOLDO¿EULOODU\ acid protein (GFAP). C6 cells were used to show preferential glial cell adhesion to SRO\S\UUROHVLONOLNH KDYLQJ ¿EURQHFWLQ IUDJPHQWV 6/3) SRO\PHUSHSWLGH EOHQG on a silicon electrode [14] (Figure 4.3). The DITNC1 astrocyte cell line (ATCC CRL-2005) expresses GFAP and appears to be similar in phenotype to type 1 astrocytes in culture (type 1 astrocytes are more physiologically relevant, while type 2 astrocytes are thought to be an in vitro phenomenon). This cell line showed lower adhesion to textured neuroelectrodes than to smooth silicon electrodes [13], which together with the neurite outgrowth data presented above suggests texturing as a possible strategy to improve biocompatibility. Massia’s group uses another Fischer rat glioma line, F98 (ATCC #CRL-1690) in experiments testing astrocyte adhesion to different surfaces [10,11]. Several groups working on neuroelectrode biocompatibility at Cornell University, the Wadsworth Center, and Rensselaer Polytechnic Institute have used the /50DVWURJOLDOFHOOOLQHWRWHVWQHZPDWHULDOVDQGPRGL¿FDWLRQV$WUDQVIRUPHG rat glioma cell line originally developed by Martin and Shain [15], this cell type has been shown to exhibit several important astrocyte metabolic features. Investigators have used this cell line to show adhesion and spreading on micropatterned hydrophilic hexagonal trimethoxysilyl-propyl-diethylene triamene (DET)A surfaces of varying sizes [16], an LRM55 preference for microfabricated silicon pillars and wells [17], the cell line preference for microcontact printed surfaces over traditional photolithographic patterned surfaces [18], and a preference for smooth chemically etched regions over roughened “silicon grass” regions [19] (Figure 4.4). Contradictory behavior from primary astrocytes, which favor roughened silicon JUDVVUHJLRQVXQGHUVFRUHVWKHOLPLWHGDELOLW\WRPDNHGH¿QLWLYHFRQFOXVLRQVIURP cell line experiments [17]. 4.2.1.4 Microglia Cell Lines 0LFURJOLDEDVHG FHOO OLQHV DUH FRPPRQO\ XVHG WR VWXG\ QHXURLQÀDPPDWRU\ SURcesses, but only recently have biocompatibility studies recognized the dominant


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98


FIGURE 4.3 PPy/SLPF-coated 4-shank, 16-channel neural probe cultured with C6 cells. Cells were stained using Hoechst 33342, and the blue spots correspond to individual cells. (Adapted from Cui, X. Y., Lee, V. A., Raphael, Y., Wiler, J. A., Hetke, J. F., Anderson, D. J., and Martin, D. C., J. Biomed. Mater. Res. 56, 261–272, 2001. With permission.) (See color insert.)

role of microglial cells. Often cell lines are employed to better understand processes already known to occur in vivo or in primary cells. For example, Tzeng and Huang used the immortalized mouse microglial BV-2 cell line to probe the effect of neuroWURSKLQ17 RQWKHF\WRNLQHVDQGLQÀDPPDWRU\PROHFXOHVUHOHDVHGE\PLFURJlia after an LPS immune challenge [20], a process suggested by in vivo observations. Kremlev et al. used the BV-2 cell line as well as the HAPI microglial-like rat cell line to assay for the release of chemokines and the expression of chemokine receptors after an immune challenge after in vivo data suggested the involvement of chePRNLQHVLQLQÀDPPDWLRQPHGLDWHGEUDLQLQMXU\>@%RWKWKH%9DQG+$3,FHOO lines are commonly used in the Hong lab to further explore behaviors observed in vivo or in primary cultures [22]. Once the cell lines show the same behavior as we earlier observed in vivo (i.e., release of a certain cytokine after immune challenge), we can then use the cell lines to examine the details of that behavior (i.e., the signal transduction pathway of cytokine expression). Another example where microglial cell lines were advantageous over primary culture is in the case of studying LPS internalization in microglia. All microglia, including cell lines, phagocytose LPS, and it was simpler to use the cell line, because they were less sensitive than primary cultures to the lower-density seeding that was necessary to get single cell images for confocal microscopy (Figure 4.5) [23].


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99

FIGURE 4.4 Time course of LRM55 astroglial cell attachment to surfaces patterned by PLFURFRQWDFWSULQWLQJ&HOOVZHUHSODWHGDQG¿[HGDIWHUKRXUV$DQG' KRXUV%DQG( DQGKRXUV&DQG) %DUPPIRUERWKORZPDJQL¿FDWLRQ× objective, A through & DQGKLJKPDJQL¿FDWLRQ× objective, D through F) images. Dark regions are a permissive DETA (a hydrophobic organosilane self assembled monolayer), and the light stripes are layers of inhibitory OTS (a hydrophobic organosilane). (Adapted from St. John, P. M., Kam, L., Turner, S. W., Craighead, H. G., Issacson, M., Turner, J. N., and Shain, W., J. Neurosci. Methods 75, 171–177, 1997. With permission.)

4.2.2

PRIMARY CELLS

7KH VWHS XS LQ FRPSOH[LW\ IURP FHOO OLQHV WR SULPDU\ FXOWXUHV LV VLJQL¿FDQW 3ULmary cells are genetically stable and are often the actual cells taking part in the in vivo process, so the results from primary culture experiments are much more trustZRUWK\+RZHYHULQH[FKDQJHIRUWKHLQFUHDVHLQUHOHYDQFHFRPHVVLJQL¿FDQWQHZ challenges, including additional costs of animal purchase and husbandry, isolation GLI¿FXOWLHV DQG LQFUHDVHG FXOWXUH YDULDELOLW\ 2QH DOVR ORVHV WKH DELOLW\ WR HDVLO\ culture and proliferate cells through many generations. As primary cells proliferate and are passaged, the phenotype may change to a point where the cells no longer behave in the same way that the initially isolated cells behaved. For this reason, only WKH¿UVWRUWKH¿UVWIHZSDVVDJHVRISULPDU\FHOOVFDQEHXVHGDQGWKHQXPEHURI valid passages depends on the cell type and behavior being investigated. Even with greater relevance to the in vivo situation, primary cell culture is still not a perfect model of the in vivo environment, as the isolation procedure often injures or activates the cell, resulting in an altered phenotype, and the two-dimensional culture system without vasculature, extracellular matrix (ECM), and other supporting cells further alters cellular behavior. Furthermore, as with any phenotypically diverse biological system, the results obtained with primary cultures will show a higher degree of variability than the results from genetically identical cell line cultures. Primary cultures can be subdivided further into increasing levels of complexity. The least complex cultures are single cell type primary cultures. These are the primary cell equivalent of cell lines, where the researcher is interested in the response


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FIGURE 4.5 Internalized LPS is localized in the golgi. HAPI microglial cells were exposed WRJPOODEHOHG/36DQGȝ01%'FHUDPLGHIRUKRXUDW&3HULQXFOHDUFRORFDOL]Dtion of NBD-ceramide (A) and Alexa568-LPS (B) in HAPI microglia. (C) The transmitted OLJKWLPDJHDQG' WKHRYHUOD\RIWUDQVPLWWHGOLJKWDQGÀXRUHVFHQFHLPDJHV7KHVFDOHEDUV indicate 20 µm. (See color insert.)

of a single cell type to the experimental conditions. By combining two independently isolated sets of primary cells (i.e., primary astrocytes and primary neurons), a more complex coculture can be used, thus allowing analysis of cell–cell interactions. Finally, if two or more cell types are isolated together from the same tissue in the same procedure (i.e., a culture of astrocytes and microglia from postnatal rat cortex), this is an additional step toward the more complex conditions of the in vivo environment. 4.2.2.1 Single Cell Type Primary Cultures: Primary Neurons Because of their unique electrical properties relative to other cells, and their varied phenotypes within the CNS and peripheral nervous system (PNS), neurons are not


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well represented by cell lines in culture. While a few neuronal cell lines, such as the PC-12 line, exhibit limited neuronal behavior, the vast majority of neurobiological studies in the past several decades have used primary neuronal cultures. Neurons are highly varied in form and in behavior depending on their age, location, and function in the brain, but commonly share negligible to no proliferative potential, PDNLQJFHOOFXOWXUHTXLWHGLI¿FXOW6WLOOVHYHUDOLVRODWLRQSURWRFROVKDYHEHHQHVWDElished that consistently produce highly pure, electrophysiologically active neuronal cultures. Because of the limited proliferative potential of adult neurons and their relative sensitivity to injury, neurons are usually isolated from embryonic or early postnatal animals, while they are still differentiating from the hardier, dividing neuronal precursor cells. Cells are mechanically or enzymatically dissociated, plated on SRO\'/\VLQHRUODPLQLQFRDWHGSRO\VW\UHQHDQGPDLQWDLQHGLQVSHFLDOO\GH¿QHG serum-free media. Dobbertin et al. used a standard cortical neuronal culture from embryonic day 17 (E17) Sprague–Dawley rats to study the growth of neurites on 5373ȕSKRVSKDFDQDFKRQGURLWLQVXOSKDWHSURWHRJO\FDQXSUHJXODWHGDIWHULQMXU\WR the CNS [24]. After removal of the striatum and hippocampus, the cerebral cortices were freed of meninges and cut into small pieces before enzymatic treatment with trypsin. By seeding at low density (104 cells/cm2DQGXVLQJVSHFLDOO\GH¿QHGJURZWK media (1:1 mixture of DMEM and Ham’s F12 with 1% N2 supplement), the investigators were able to get a cell culture that was 98% pure neurons. Ravenscroft et al. used a similar procedure to isolate E18-19 hippocampal neurons in their studies and SDWWHUQHGWKHPRQVLODQHPRGL¿HGVXUIDFHV>@7KH\ZHUHWKHQDEOHWRHQJLQHHU FLUFXLWVRIDOLJQHGQHXURQVRQJODVVFRDWHGZLWKDSDWWHUQHGVLODQH¿OP,QWKLVFDVH cells were dissociated with papain and layered over a step gradient to remove cellular debris before resuspension in serum-free neurobasal medium supplemented with B27, glutamine, and glutamate. Postnatal-derived neuronal cultures have a lower neuronal yield since most neurons perish in the isolation process and require more stringent precautions for removing nonneuronal cells, such as a nylon mesh to remove meninges and treatment with mitotic inhibitors to prevent the culture from being overgrown by proliferating glia [26]. Other neuronal cultures include embryonic spinal motorneuron cultures, embryonic or perinatal rodent sympathetic ganglia cultures, and embryonic dorsal root ganglion cultures [8]. If neurons are cultured alone without glia, they will become spontaneously electrophysiologically active within a week of the dissection and can remain viable and active in culture with the proper maintenance for many weeks to months. Potter’s group and others have cultured neurons on microelectrode array (MEA) recording systems for over a year by maintaining the osmolality of the culture within a narrow range [27]. However, while much of the neuroscience community uses primary neuronal cultures, neuroelectrode engineers have not used them for biocompatibility studies because there is little need for accurate electrophysiological behavior when straightforward cytotoxicity and cell attachment assays are being performed. As greater emphasis is placed on more complex in vitro models (i.e., simultaneous observation of signal degradation and glial scar formation in vitro), and electrically


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active electrodes are used in culture, primary neurons will need to replace neuronal cell lines in future biocompatibility studies. 4.2.2.2 Single Cell Type Primary Cultures: Primary Glia Primary glial cultures are easier to generate than primary neuronal cultures because glia have much higher proliferative abilities and are more likely to be part of the approximately 1% of cells that survive the isolation procedure [8]. Furthermore, since the diversity of astrocytes, microglia, and oligodendrocytes is not well understood, unlike the much greater appreciation for neuronal phenotype differences (i.e., dorsal root ganglion versus motorneuron isolation), researchers typically use heterogeneous cultures of glia. A procedure to isolate glia initially established by McCarthy DQGGH9HOOLVLQKDVEHHQPRGL¿HGE\YDULRXVJURXSVEXWKDVURXJKO\UHPDLQHG intact for over 25 years [28]. In this procedure, perinatal (postnatal day 1 to 4) rat or mouse cerebral brain cells are plated at high density (2× 105/cm2) in serumsupplemented medium after removal of the meninges and mechanical or enzymatic dissociation. At this point, glia are still dividing and, with the help of serum and HQGRJHQRXVO\SURGXFHGF\WRNLQHVZLOOFUHDWHDFRQÀXHQWOD\HURIDVWURF\WHVJOLDO precursor cells, and microglia within 6 days. The astrocytes in these cultures tend WRIRUPDFRQÀXHQWOD\HURQWRSRIWKHSRO\'O\VLQH±FRDWHGSRO\VW\UHQHZLWKWKH loosely adherent microglia resting on top of the astrocyte layer. To isolate primary cultures of microglia, researchers take advantage of these differences in adherence and shake the cultures two weeks after plating and then again three weeks after plating. Although the culture can be shaken further, the microglia from additional shakes are no longer used in order to avoid experimental error derived from clonal expansion of particularly adherent microglia. The media are collected after each shake and centrifuged, resulting in microglia that are around 95% pure and an astrocyte layer that is more than 98% pure. Shaking occurs on a standard rotary shaker at room temperature, although the times and speeds vary from lab to lab (our lab shakes for less than 3 hours at 180 RPM). The longer and faster the cells are shaken, the purer the astrocyte culture will be. However, shaking also activates the microglia, so longer shake times may result in more basal microglial activation. Astrocytes grown this way can survive for many weeks and can be subcultured several times, while microglia cannot survive for more than a few days without the supporting astrocyte layer. This method by McCarthy and de Vellis forms the basis for the primary glial cell isolation procedure, but each research group tends to make adjustments based on their own experience. Each group varies details such as the shake date, duration, speed, and frequency; the media; and the serum formulation. 4.2.2.3 Single Cell Type Primary Cultures: Primary Microglia Our laboratory routinely maintains primary enriched-microglia cultures, which are prepared from the whole brains of 1-day-old Fisher 344 rat pups or mice according to a variation of the McCarthy and de Vellis procedure [29]. After removing meninges and blood vessels, the brain tissue (minus olfactory bulbs and cerebellum) is gently triturated and seeded (5 × 107, approximately 2.5 rat pup brains or 4 mouse


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pup brains) in 150-cm3ÀDVNV7KHFXOWXUHPHGLXPFRQVLVWVRI'0(0)PHGLD supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 mM nonessential amino acids, 50 U/ml penicilOLQ DQG OJPO VWUHSWRP\FLQ &XOWXUHV DUH PDLQWDLQHG DW & LQ D KXPLGL¿HG atmosphere of 5% CO2 and 95% air. One week after seeding, the media is replaced. 7ZRZHHNVDIWHUVHHGLQJZKHQWKHFHOOVUHDFKDFRQÀXHQWPRQROD\HURIJOLDOFHOOV microglia are shaken off. Afterward, cells are resuspended in a treatment media (DMEM-F12 media supplemented with 2% FBS, 50 U/ml penicillin, and 50 ug/ml streptomycin) and replated at 1 × 105 in a 96-well plate. Cells are treated 12 to 24 hours after seeding the enriched microglia. Media can be replaced in the original 150-cm3 ÀDVNRIPL[HGJOLDDQG a week later, an additional shake of microglia can be harvested. Other than this standard culture preparation, there are other preparations that are less commonly used. Microglia that are 92 to 97% pure can be bulk isolated from older rats, as described Basu et al., who used a series of digestive incubations, nylon meshes, and centrifugation steps to isolate microglia from 8 to 12 week Sprague–Dawley rats [30]. Other cells known to participate in the glial scarring reaction, such as meningeal cells [26] and O2A precursor cells [24], can be isolated but have not yet been used widely in biocompatibility experiments. 4.2.2.4

Single Cell Type Primary Cultures: Primary Astrocytes

The astrocyte isolation procedure is the inverse of the microglia isolation procedure since microglia are shaken off and discarded while the astrocytes are maintained and subcultured. Dobbertin et al. used 2-day-old Sprague–Dawley rats, enzymatically triturated small pieces of tissue using 0.1% trypsin in HBSS for 20 minutes, DQGVHHGHGSRO\'O\VLQHFRDWHGÀDVNVZLWKIHWDOFDOIVHUXPLQ'0(0>@ The cultures were shaken between the 8th and 12th days, and the cells were subFXOWXUHGDQGWUHDWHGZLWK0RIWKHDQWLPLWRWLFF\WRVLQHȕ'DUDELQRIXUDQRVLG (Ara-C) in serum-free media to remove any remaining proliferating glial precursors. Remaining microglia were removed by treatment with 10 mM L-leucine methyl ester (LME), which is a phagocyte toxin. The more shakings that the astrocytes go through, the more microglia are removed, and a purer astrocyte culture is generated. After weekly shakings for 6 weeks, a 98% pure astrocyte culture remains. However, this method will produce a VLJQL¿FDQWQXPEHURIW\SHDVWURF\WHVVRRXUJURXSSUHIHUVWRDFTXLUHDVWURF\WHV using a primary cortical astrocyte method, as previously described [31] with a slight PRGL¿FDWLRQV >@ $JDLQ ZKROH EUDLQV IURP GD\ROG UDWV DUH LVRODWHG DQG WKH meninges are removed. However, for astrocytes, the cerebral cortices are dissected and subjected to enzymatic digestion for 15 min in DMEM/F-12 containing 2.0 mg/ ml porcine trypsin and 0.005% DNase I. The tissue is then mechanically disaggregated using a 60-µm cell dissociation kit (Sigma-Aldrich, St. Louis, Missouri) to yield a mixed glial cell suspension. The cell suspension is centrifuged for 10 min at 300 g and resuspended in fresh culture medium: DMEM/F-12 supplemented with 10% FBS, 100 µM nonessential amino acids, 100 µM sodium pyruvate, 200 µM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. The cells are plated on


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75-cm 2SRO\VW\UHQHWLVVXHFXOWXUHÀDVNVDQGPDLQWDLQHGDW&&22, and 95% DLUXQWLOFRQÀXHQF\IRUZHHNV)UHVKPHGLXPLVUHSOHQLVKHGHYHU\WRGD\V)ROORZLQJFRQÀXHQF\WKHFHOOVDUHSODFHGRQDQRUELWDOVKDNHUDWUHYROXWLRQVPLQ for 6 hrs to remove contaminating cells (mostly microglia). The cells are harvested with 0.1% trypsin/EDTA in Hank’s Balanced Salt Solution and plated in either T25 ÀDVNV RU PP 3HWUL GLVKHV DW D GHQVLW\ RI WR × 106 cells. Experimental VWXGLHVDUHSHUIRUPHGZLWKLQWRZHHNVRILQLWLDOSODWLQJ6SHFL¿FDOO\FHOOVDUH WUHDWHGZKHQWKH\DUHFRQÀXHQWDSSUR[LPDWHO\ZHHNDIWHUWKHODVWVHHGLQJ8SRQ treatment, cultures are switched to fresh medium containing 2% heat-inactivated )%6P0/JOXWDPLQHP0VRGLXPS\UXYDWH8POSHQLFLOOLQDQGȝJPO streptomycin. 4.2.2.5

Primary Cell Coculture

A step up in complexity from primary cultures of a single cell type is cocultures where two different cell types from two different sources are combined. The result is a culture that allows the study of cell–cell interactions between two cell types and is not limited by the different sensitivities of two different cell types to one isolation procedure. For example, sensitive neurons can be isolated by one procedure in an embryonic animal, while hardier astrocytes can be isolated by a different procedure in an adult animal. The two cell suspensions are seeded together, or one atop the other, and neuron–astrocyte interactions can be observed, or behaviors only observed in the presence of both cell types together can be tested. Although these cultures may be more relevant to the in vivo situation since cells are combined with other cell types that they normally interact with in vivo, relevance may be lost if different sources and isolation procedures result in behavior that is not physiologically relevant. 7RWHVWWKHHIIHFWRIGLIIHUHQWPDWHULDOVLQLQÀXHQFLQJFRUWLFDODVWURF\WHDELOLW\ to promote neuronal growth, Biran et al. seeded cerebellar granule neurons isolated from 7-day-old rats through a panning procedure atop a layer of astrocytes isolated IURP3UDWFHUHEUDOFRUWH[WKDWKDGEHHQSXUL¿HGDZD\IURPPLFURJOLDDQGRWKHU cells by shaking (as described in the previous section) [33]. Since the P-1 dissection procedure does not yield neurons, yet neurons were important in assessing the growth-promoting properties of cortical astrocytes, the coculture was necessary to answer the question of biocompatibility posed by the researchers. They were able to XVHWKHFXOWXUHWR¿QGQRGLIIHUHQFHLQDVWURF\WHJURZWKSURPRWLQJDELOLW\EHWZHHQ different materials. In one of the few attempts to look at astrogliosis in vitro, Guenard and colleagues used dorsal root ganglion (DRG) neurons prepared from E15 rats plated on top of astrocytes from E21 rat cortex or 2- to 3-month-old rat spinal cord to study the effect of mechanical axonal injury on astrocyte proliferation, GFAP expression, ECM deposition, and process orientation [34]. DRG neuron explants provide longliving neurons with large axons used to study axonal damage and regrowth after injury, whereas smaller neurons from the cortex (the astrocyte cell source) may have EHHQGLI¿FXOWWRLVRODWHRUDQDO\]H+LUVFKDQG%DKUVHHGHGUHWLQDOH[SODQWVIURP E16 rat embryos on top of adult optic nerve astrocytes or P-1 to P-3 cortical astrocyte


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OD\HUV WR ¿QG WKDW WKH UHWLQDO JDQJOLRQ FHOO D[RQV SUHIHUUHG WR JURZ RQ DVWURF\WH regions of the culture rather than the contaminating meningeal cell regions, putatively because of the different mix of inhibitory ECM expressed by the different basal layer cell types [35]. In another study, cortical neonatal astrocytes that were JURZQRQDVWUHWFKDEOHVXEVWUDWHDQGWKXVDOLJQHGLQVSHFL¿FRULHQWDWLRQVEHFDXVHRI continuous mechanical strain in one direction, were used as the base layer to culture P-1 DRG neurons [36]. The study showed that aligned astrocytes also resulted in aligned ECM deposition and aligned neuronal process growth, thus potentially laying the groundwork for engineered glial substrates for spinal cord repair. Although astrocyte-neuron cocultures are most common, neurons can also be cocultured with other cell types, such as Schwann cells [34] and meningeal cells [35]. 4.2.2.6 Multicell Primary Cultures: An In Vitro Model of Glial Scarring The glial scarring response to implanted biomaterials involves at least three interacting cell types: neurons, astrocytes, and microglia. To keep the in vitro cell culture system as relevant to the in vivo environment as possible, all three cell types should optimally be isolated in the same isolation procedure from the same animal at the VDPHWLPH8QOLNHH[SHULPHQWHUVZKRJRWRJUHDWOHQJWKVWRLVRODWHDVSHFL¿FFHOO type and remove “contaminating” cells, our approach has been to isolate and culture these interacting cell types together, thus more closely reproducing the in vivo enviURQPHQW7KHEHQH¿WLVWKDWWKHFRPSOLFDWHGJOLDOVFDUULQJUHVSRQVHZKLFKUHTXLUHV cell–cell interaction between at least three cell types, can be reproduced in vitro (Figure 4.6) [37]. We have been able to reproduce the glial scarring response to biomaterials placed in such culture, and the in vitro approach allows us to create a time course of events leading to glial scarring while potentially recording from the culture at the same time. We plan to further use the in vitro model to explore the mechanisms behind glial scar formation and further develop the model as a way to test new bioFRPSDWLELOLW\DSSURDFKHVWKDWEHFRPHDYDLODEOHLQWKHQHXURSURVWKHWLFV¿HOG The increased complexity also comes with several drawbacks as compared to simpler cell line or single cell type primary cell culture. First, the variability between culture preparations and experimenters is greater as more parameters can SRWHQWLDOO\EHYDULHG7KHFHOO±FHOOLQWHUDFWLRQVDUHPRUHGLI¿FXOWWRREVHUYHDQG analyze, as seven interactions are now possible between the three different cell types (A–A, B–B, C–C, A–B, A–C, B–C, A—B–C), whereas only one interaction is present in single cell type cultures (A–A), or three possible interactions are present in less complex cocultures cultures (A–A, B–B, A–B). Furthermore, since all the cell types have to be present in one isolation procedure, early embryonic (E14 to E17) cells are used to keep neurons alive, thus potentially reducing the culture’s relevance to adult in vivo behavior. Understanding the tradeoffs involved in using a more complex culture, we have GHFLGHGWKDWWKHEHQH¿WV²QDPHO\WKHUHFUHDWLRQRIWKHJOLDOVFDUDURXQGHOHFWURGH materials in vitro—outweigh the limitations in the system described above. The neuron–glia cell culture system contains all of the cell types relevant to glial scar formation. It is an embryonic day 14 midbrain culture that contains the physiologically relevant mix of approximately 40% neurons, 10% microglia, and 50% astrocytes


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FIGURE 4.6 $WKURXJK% 7ULSOHÀXRUHVFHQWODEHOLQJRIDPRGHOHOHFWURGHLQQHXURQ±JOLD culture with DAPI staining nuclei blue, GFAP staining green, and OX-42 staining microglia red shows the relative positions of different cells near the wire after 10 days in culture. Just as observed in vivo, there is a layer of microglia (red) adjacent to the microwire and astrocytes (green) outside of the microglial layer showing upregulated GFAP. The image in (B) shows WKHJOLDOVFDUULQJDWDKLJKHUPDJQL¿FDWLRQFOHDUO\YLVXDOL]LQJWKHSUHYDOHQFHRIPLFURJOLD around the microwire. For reference, the wire diameter is 50 µm in all images. (Adapted from Polikov, V. S., Block, M. L., Fellous, J. M., Hong, J. S., and Reichert, W. M., Biomaterials 27, 5368–5376, 2006. With permission.) (See color insert.)

>@7RJHQHUDWHWKHFXOWXUH¿UVWWKHPLGEUDLQLVGLVVHFWHGRXWDQGWKHPHQLQJHV removed. Then, the cells are mechanically triturated with various sized pipette tips and seeded at 5 × 105/well into poly-D-lysine-coated 24-well plates. Cells are maintained DW&LQDKXPLGL¿HGDWPRVSKHUHRI&22 and 95% air, in minimal essential medium (MEM) containing 10% FBS, 10% horse serum (HS), 1 g/l glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 µM nonessential amino acids, 50 U/ml penicillin, and 50 µg/ml streptomycin. Seven-day-old cultures are used for treatment after a media change to MEM containing 2% FBS, 2% HS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, and 50 µg/ml streptomycin. Treatment involves several different interventions to induce the culture to reproGXFHWKHVFDUULQJRULQÀDPPDWRU\EHKDYLRUWKDWOHDGVWRUHFRUGLQJHOHFWURGHIDLOXUH in vivo [37]. One simple treatment option is to add 10 ng/ml of LPS into the culture, ZKLFKUHVXOWVLQPLFURJOLDODFWLYDWLRQLQÀDPPDWRU\F\WRNLQHUHOHDVHDQGQHXURQDO bystander damage. To simulate physical damage and cell death characteristic of the mechanical injury sustained during device insertion, a scrape model is used (Figure $ ,QWKLVPRGHODQDUHDRIWKHFRQÀXHQWFHOOOD\HULVVFUDSHGRIIZLWK a cell scraper or a pipette tip, and cells are monitored as they repopulate the damaged region. A treatment to reproduce the foreign body response and glial scarring


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FIGURE 4.7 (A) Time course of cellular events in response to a scrape wound. The area scraped free of cell is on the left of the dotted line. Astrocytes are seen to send processes (arrows) into the wound beginning at 6 hours and continuing through 48 hours, and completely recolonize the wound by 7 days. GFAP-negative spindle-shaped precursor cells (arrowheads) that do not stain for microglial markers but stain for vimentin (not shown) migrate into and colonize the wound ahead of the GFAP-positive processes. Microglia migrate to and spread out within the wound by 24 hours, and their numbers increase over time, until by 7 days there are more microglia inside the wound than in the surrounding culture. (B) Time course of cellular events in response to the wire placement. Microglia attach to the wire as early as 6 hours and increase in number until a layer of microglia 1 to 2 cells thick is formed covering the length of the wire. This layer remains through 10 days in culture. Astrocytes show no response to the microwire until 10 days after treatment, a layer of activated astrocytes with upregulated GFAP forms around the microwire, mimicking the glial scarring seen in vivo. (Adapted from Polikov, V. S., Block, M. L., Fellous, J. M., Hong, J. S., and Reichert, W. M., Biomaterials 27, 5368–5376, 2006. With permission.) (See color insert.)


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around an implant that occurs as a result of chronic electrode implantation involves the placement of short (2 to 5 mm) pieces of microwire into the culture and observe the cell reaction to the foreign body (Figure 4.7B). We use stainless steel wire because it is inexpensive, easy to sterilize, and is used in some microwire recording arrays, although we have used other materials with little observable difference in FHOOXODUUHVSRQVH(DFKFXOWXUHLV¿[HGDWDFHUWDLQWLPHSRLQWDIWHUWUHDWPHQWDQG immunostained for different cell markers to differentiate neuronal (MAP-2, NeuN cell markers), microglial (IBA-1, OX-42), and astrocyte (GFAP) behavior. For the /36WUHDWPHQW¿[DWLRQLVXVXDOO\GRQHLQWKH¿UVWGD\VVLQFHWKHPD[LPDOF\WRkine response is at 24 hours. The scrape is looked at with a time course ranging from VHYHUDOKRXUVWRGD\VZKHQWKH³ZRXQG´LV¿OOHG DQGWKHPLFURZLUHWUHDWPHQWLV extended as long as the culture is stable, typically 10 days. 7KLV FXOWXUH V\VWHP KDV EHHQ XVHG URXWLQHO\ WR VWXG\ WKH QHXURLQÀDPPDWRU\ processes underlying progressive neurological diseases such as Parkinson’s disease and Alzheimer’s disease [2,29,38–47]. Removal of each of the different cell types destroys our ability to follow these processes, as each cell type contributes something to the overall disease process. We encounter the same logic with reproducing glial scarring behavior around biomaterials placed in culture. To recreate the microglial migration and attachment to the wire, the astrocyte upregulation of GFAP and glial scar, and the neuronal isolation from the recording surface, we need to have microglia, astrocytes, and neurons in the culture. A culture without microglia could survive for longer periods than the 3 weeks we can maintain neuronal survival, and a culture without neurons could be isolated from postnatal or even adult animals, yet critical cell types would not be present in these situations. To our NQRZOHGJHRXUVLVWKH¿UVWDQGRQO\GHPRQVWUDWLRQRIWKLVVFDUULQJEHKDYLRUWREH published in vitroGXHLQSDUWWRWKHVSHFL¿FSUHVHQFHRIDOOWKUHHFHOOW\SHVNQRZQ to participate in the brain’s response to implanted foreign materials. The system certainly has its drawbacks, whether it is the use of embryonic cells, the short-lived viability of the cells, or the noncortical cell source, yet no other system has produced in vitro behavior that so closely mimics the characteristics of the in vivo response. We have begun to look at E17-E18 cortical cultures that also contain a relevant mix of neurons, astrocytes, and microglia, and initial results suggest a similar glial scarring response is present in these cultures as well (unpublished results). Another multicell culture employed in our lab is the P-1 mixed glia culture. Primary mixed glia cultures are prepared from the whole brains of 1-day-old Fisher 344 rat pups or mice. After removing meninges and blood vessels, the brain tissue (minus olfactory bulbs and cerebellum) is gently triturated and 0.5 × 106 cells/well are seeded in a 24 well plate, or 0.5 × 105 cells/well in a 96 well plate. The culture medium consists of DMEM-F12 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 l M nonessential amino acids, 50 U/ml penicillin, and 50 lg/ml streptomycin. Cultures are maintained at 37°C in a KXPLGL¿HG DWPRVSKHUH RI &22 and 95% air. Three days after initial seeding, cultures are replenished with 0.5 ml/well fresh medium (24 well plate) or 0.1ml/well (96 well plate). At 7 days postseeding, cultures are treated. Upon treatment, cultures are switched to fresh medium containing 2% heat-inactivated FBS), 2 mM L-gluWDPLQH P0 VRGLXP S\UXYDWH 8PO SHQLFLOOLQ DQG ȝJPO VWUHSWRP\FLQ


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The P-1 mixed glia system could be a useful tool to study glial responses to foreign materials even without the presence of neurons.

4.2.3

BRAIN SLICES

4.2.3.1 Brain Slices: Complexity Approaching In Vivo Another way to model the brain environment is to use organotypic brain slice cultures. In contrast to primary cultures, these slices, as their name suggests, provide a three-dimensional representation of the cellular environment and preserve cell-tocell interactions. The preparation of these slices minimally raises the basal level of activation and thereby allows for a more sensitive model. In addition, fewer animals are required to create these cultures since many slices can be obtained from one brain and each slice can be maintained for months [48,49]. Finally, slices of mature animals, up to postnatal day 21 to 23, can be grown in culture, whereas primary cultures can only be produced from embryonic or neonatal animals [8]. The drawbacks to increasing the complexity are that the culture approaches the uncontrolled nature of the in vivo environment. Furthermore, while the slices are well suited for generating acute and chronic recordings of neurons in preserved circuits, the staining and ¿[DWLRQSURWRFROVQHFHVVDU\WRSHUIRUPLPPXQRF\WRFKHPLFDODQDO\VLVRQWKHFHOOXODUUHVSRQVHWRELRPDWHULDOVLVPRUHGLI¿FXOWWKDQLQWZRGLPHQVLRQDOFXOWXUHVRU even in vivo, where tissue sections are easily cut and stained. The thin slices attach WRJURZWKPHPEUDQHLQWLVVXHFXOWXUHDQGDUHGLI¿FXOWWR¿[VHFWLRQDQGVWDLQ The complexity of these models presents its own challenges. Preserving the cellular environment eliminates the ability to produce uniformly dissociated cultures DQGWKXVLQFUHDVHVWKHGLI¿FXOW\RIDWWULEXWLQJDQREVHUYHGUHVSRQVHWRDVSHFL¿FFHOO type. To assess the overall response of a particular agent, the selective vulnerability RIFHOOVLQGLIIHUHQWUHJLRQVRIWKHEUDLQZKLFKPD\QRWEHUHDGLO\LGHQWL¿DEOHPXVW be taken into account. Furthermore, the presence of extensive networks impedes examining and tracking of individual cells. Primary dissociated cultures, in contrast, allow for easy perfusion of agents without problems of tissue absorption or interaction from neighboring cells or tissues. Finally, many studies have shown that slice cultures have not consistently replicated in vivo responses [50]. Thus, the slice models may provide a promising alternative or addition to current primary models, but the individual challenges they pose warrant caution in their use. In a typical isolation procedure, the tissue slice is placed onto a semiporous membrane that is attached to a removable insert, and the assembly is placed into a PHGLD¿OOHGZHOO7KHPHPEUDQHSUHYHQWVGLUHFWFRQWDFWRIWKHPHGLDWRWKHWLVVXH A diagram of the basic components and an image of the interface setup are shown in Figure 4.8. Typically, slices from the brains of postnatal animals, in particular rats and mice, are used. Multiple slices of uniform thickness can be prepared at once by using a vibrating microtome or a less complex manual tissue chopper. Fresh slices of approximately 300 to 400 µm thick are cultured for 2 to 3 weeks to allow cells to stabilize before treatment with chemical or mechanical injuries. The slices can be observed for many weeks afterwards. Although few in the neuroprosthetics community have used brain slices to study biocompatibility, the literature abounds with studies to support the use of


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Membrane

FIGURE 4.8 Diagram and image (millipore) of the Stoppini method for culturing tissue slices. The tissue is placed on an insert and positioned in a media-containing well without direct contact by the tissue to the media.

slices as culture models. The use of multiple techniques to characterize the behavior and morphology of cells has provided documentation for numerous in vivo events, including immediate and long-term responses, activated and resting state transitions, and cellular reactions to mechanical, excitotoxic, and bacterial injuries. In other studies, slices have been cocultured with dissociated cells to examine the interDFWLRQVEHWZHHQVSHFL¿FFHOOW\SHV7KHYHUVDWLOLW\WKDWVOLFHFXOWXUHVRIIHULQGLFDWHV that they may be used to isolate and study a broad range of disease mechanisms. 4.2.3.2

Brain Slices: Biocompatibility Studies

Studies using this model to assess the biocompatibility of implants and prosthetLFVDUHOLPLWHG:LWKLQWKH¿HOGRIELRFRPSDWLELOLW\RQO\DKDQGIXORIVWXGLHVKDYH examined the response to neural electrodes. Koeneman and colleagues conducted RQH RI WKH ¿UVW VWXGLHV HPEHGGLQJ VLQJOH SRO\EHQ]\OF\FOREXWHQH LPSODQWV LQ UDW brain slices and studying the tissue response over a 2-week period [48]. Using cellVSHFL¿FPDUNHUVWRVLPXOWDQHRXVO\YLVXDOL]HWKHQXPEHUPRUSKRORJ\DQGORFDOL]Dtion of different cell types near the implant, they showed that glia and neurons made extensive contact with the implants and, further, that the implants did not promote cell death (Figure 4.9). These observations suggested that the electrodes were biocompatible. Although quantitative assays are needed to support their conclusions, the study showed that brain slice models could be used as an alternative to assess neural electrode biocompatibility and provided techniques in which such assessments could be performed. Using a different technique, Kristensen et al. assessed neural implant biocompatibility by growing hippocampal slices directly on microelectrode arrays and analyzing the structural interaction between the array and native cells [51]. Although they observed formation of a glial scar at the base of the electrode, though not at the tip, the histological patterns in slices grown on arrays were comparable to those grown on standard membranes. Furthermore, slices grown on arrays did not become more susceptible to excitotoxins and neurotoxins. These results, in addition to the possibility that the scar may have resulted from initial tissue injury, led them to conclude that the arrays are biocompatible with tissue slices.


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FIGURE 4.9 (Left) (OHFWURGHLPSODQWHGLQDȝPFRURQDOVOLFHEODFNDUURZ LQVHUWLRQ only, white = implant. (Right) Confocal image of the electrode after 7 days: blue = nuclei, orange = GFAP, green = neurons. (Adapted from Koeneman, B. A., Lee, K. K., Singh, A., He, J. P., Raupp, G. B., Panitch, A., and Capco, D. G., J. Neurosci. Methods 137, 257–263, 2004. With permission.) (See color insert.)

Bypassing traditional methods of slice culture that maintain neuronal elecrophysiological activity, Bjornsson et al. recently used 500-µm thick cortical slices to measure the effects of electrode insertion parameters on tissue strain [52]. In this case, the experimenters needed the three-dimensional structure and vascular features that are unavailable in cell culture but needed an ex vivo setup to conduct quantitative analysis on the tissue response to insertion. Their analysis found that a faster insertion results in lower tissue deformation, although large variability was IRXQG EHWZHHQ LQVHUWLRQV DQG WKLV YDULDELOLW\ ZDV KHDYLO\ LQÀXHQFHG E\ FRUWLFDO surface features such as vascular elements.

4.3 CONCLUSIONS In vivo studies have always been, and will continue to be, the gold standard in evaluating device–tissue interactions. However, in vivo studies are costly, time consuming, and often cannot provide the mechanistic insight necessary to understand the underlying problems causing device failure. Without the cellular and molecular mechanisms that controlled in vitro cell culture experiments can provide, the quest for a nonfouling, nonscarring electrode design may continue to progress through the slow, seemingly random path that subcutaneous devices such as implantable glucose sensors have taken. However, the variety and richness of the in vitro cell culture models available for the brain may accelerate or help guide the in vivo experiments and novel electrode designs. Simple cell line experiments that cost on the order of several hundreds of dollars can prevent costly problems of in vivo material cytotoxicity, while primary cell cultures can help to explain why one material attracts neurite growth when another repels neuronal attachment and elongation. Cell cultures involving multiple interacting cell types or even slices of brain tissue can reproduce glial scarring and electrode failure under controlled, observable conditions and potentially eliminate the need for in vivo experiments except as a validation of in vitro results. At all times with in vitro experimentation, one must


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keep in mind that cell cultures are an imperfect model of in vivo behavior and that as the in vitro system becomes more rigid and controlled, it moves away from the uncontrolled, complicated in vivo environment the model is emulating. However, by keeping the limitations of in vitro analysis in mind, and verifying in vitro¿QGLQJV in vivo, one can make great strides in understanding the tissue reaction to implanted electrodes and in the design of biocompatible devices by utilizing an appropriately chosen in vitro cell culture system.

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5

In Vivo Solute Diffusivity in Brain Tissue Surrounding Indwelling Neural Implants Michael J. Bridge and Patrick A. Tresco

CONTENTS 5.1 Introduction................................................................................................. 118 5.2 Methods....................................................................................................... 120 5.2.1 Device and Implantation Procedure ............................................... 120 5.2.1.1 Diffusion Experiments..................................................... 120 5.2.1.2 Histological Processing of Tissue Samples ..................... 122 5.2.1.3 Fluorescence Imaging of Samples ................................... 122 5.2.1.4 Quantitative Image Analysis ........................................... 123 5.2.1.5 Data Processing................................................................ 124 5.2.1.6 Gel Diffusivity Characterization Experiments ............... 125 5.2.1.7 Evaluation of In Vivo Diffusivity Properties: Central Nervous System (CNS)–Extracellular Space (ECS) Solute Diffusion Modeling......................... 126 5.3 Results......................................................................................................... 129 5.3.1 Gel Diffusivity Characterization Experiment................................ 129 4XDQWLWDWLYH)OXRUHVFHQFH3UR¿OH4)3 $QDO\VLVRI&16 Tissue Access Device (TAD) Implant Groups............................... 131 5.3.3 CNS–ECS Solute Diffusion Modeling........................................... 134 8SSHU6WULDWXP0HDQ,QWHQVLW\3UR¿OH6XEVHW'DWD....... 134 5.3.3.2 Evaluation of In Vivo+ROORZ)LEHU0HPEUDQH (HFM) Diffusivity ........................................................... 134 5.3.3.3 Simulation Analysis of Control and Experimental *URXS0LQXWH3UREH'LVWULEXWLRQV............................. 134 5.4 Discussion ................................................................................................... 138 5.5 Conclusion .................................................................................................. 144 AEEUHYLDWLRQV......................................................................................................... 144 References.............................................................................................................. 145

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5.1 INTRODUCTION ,QWKLVFKDSWHUZHGHVFULEHDQDQDO\WLFDODSSURDFKWKDWFDQEHXVHGWRVWXG\VROXWH diffusivity as a function of the tissue composition surrounding a device implanted LQQRUPDODJHGGDPDJHGRUGLVHDVHGEUDLQ7KHPHWKRGLVFDSDEOHRIUHVROYLQJ changes in solute diffusivity and cellular composition at the scale of a few microns. ,QWKLVFKDSWHUZHGHVFULEHWKHNH\IHDWXUHVRIWKHPHWKRGLQFOXGLQJDTXDQWLWDWLYH LPDJLQJDQGPRGHOLQJDSSURDFKWKDWFDQEHXVHGWRDVVHVVH[WUDFHOOXODUGLIIXVLRQ VXUURXQGLQJDPRGHOLPSODQWDQGZHLOOXVWUDWHKRZWKHDSSURDFKFDQEHXVHGWR VWXG\WKHLQÀXHQFHRIOLYLQJFHOOVRQWLVVXHUHPRGHOLQJDQGVROXWHWUDQVSRUW$YDLODEOHHYLGHQFHVXJJHVWVWKDWWKHDSSURDFKPD\EHXVHIXOLQVRUWLQJRXWWKHLQWULFDF\ RI WKH IRUHLJQ ERG\ UHVSRQVH DV ZHOO DV H[DPLQLQJ KRZ VROXEOH IDFWRUV UHOHDVHG IURPYDULRXVW\SHVRIWUDQVSODQWHGFHOOVDIIHFWEUDLQWLVVXHUHPRGHOLQJDQGUHJLRQDO regeneration in the central nervous system. 2XU FXUUHQW NQRZOHGJH RI WKH FHQWUDO QHUYRXV V\VWHP &16 UHVSRQVH WR LPSODQWVZRXOGEHQH¿WIURPXQGHUVWDQGLQJZKHWKHUVROXWHGLIIXVLRQLVDIIHFWHGE\ the tissue that develops around chronic implants irrespective of whether the implant LVDELRPHGLFDOGHYLFHRUDFHOOXODUEDVHGLPSODQW$VKDVEHHQGHVFULEHGLQRWKHU FKDSWHUVRIWKLVERRNWKLVVRFDOOHG³IRUHLJQERG\UHVSRQVH´GHYHORSVLUUHVSHFWLYH of the size or type of device or transplant [1–11] and has a characteristic phenotype WKDWLQFOXGHVLQÀDPPDWRU\PDUNHUVUHDFWLYHJOLRVLVDQGQHXURQDOFHOOORVV>±@ 7RGDWHVLJQL¿FDQWHIIRUWKDVEHHQIRFXVHGRQGHVFULELQJWKHHYHQWVWKDWDFFRPSDQ\ WKHLPSODQWDWLRQRIVXFKGHYLFHVWLVVXHVDQGFHOOVLQWREUDLQWLVVXHRYHUWLPHZLWK DSDUWLFXODUHPSKDVLVGLUHFWHGDWGHVFULELQJWKHWHPSRUDODQGVSDWLDOQDWXUHRIWKH HYHQWVWKDWWDNHSODFHDWWKHLPSODQW±WLVVXHLQWHUIDFHDQGDVVHVVLQJWKHSRWHQWLDORI the response to affect device or transplant function. By all indications, a full underVWDQGLQJRIZKDWQHHGVWREHGRQHWRFRQVLVWHQWO\LQWHUIDFHYDULRXVGHYLFHVDQGOLYing transplants with a variety of potential neural targets is still a ways off. Indeed, LWDSSHDUVWKDWWKHVFLHQWL¿FEUHDGWKDQGGHSWKKDVQRWVXI¿FLHQWO\DGYDQFHGDVKDV for example, occurred with cochlear implants, so that the challenge of these newer WKHUDSHXWLFDSSURDFKHVFDQVKLIWIURPRQHRIODFNRIVFLHQWL¿FNQRZKRZWRRQHRI engineering. 'LI¿FXOWLHVDVVRFLDWHGZLWKWKHGHOLYHU\RIDJHQWVWRWKH&16KDYHOHGWRDJURZLQJQXPEHUDQGYDULHW\RILQWHUYHQWLRQDODSSURDFKHV>@$OWKRXJKWKHIHDVLELOity of focal delivery devices [5,17–20] and various neuroprosthetic applications has EHHQHVWDEOLVKHG>±@WKHHI¿FDF\RIYDULRXVDSSURDFKHVLVRIWHQFRPSURPLVHG E\FKURQLFXVH,WKDVEHFRPHFOHDUWKDWLPSURYHPHQWRI&16LPSODQWWHFKQRORJ\ ZLOOUHTXLUHDEHWWHUXQGHUVWDQGLQJRIWKHQDWXUHRIWKHWLVVXHWKDWGHYHORSVDGMDFHQW WRLPSODQWVDVZHOODVDEHWWHUXQGHUVWDQGLQJRIKRZWRPRGXODWHWKHSURSHUWLHVRI the tissue. 7RGDWHDQXPEHURIH[SHULPHQWDOWHFKQLTXHVKDYHEHHQXWLOL]HGWRFKDUDFWHUize solute transport in the extracellular space (ECS) of the CNS. Pioneering studies ZHUHSHUIRUPHGWKURXJKPHDVXUHPHQWRIUDGLRODEHOHGFRPSRXQGVLQWLVVXHVHFWLRQV at different time points following ventricle perfusion in animals [25–27]. Derivation RI WLVVXH GLIIXVLYLW\ SURSHUWLHV ZDV DFFRPSOLVKHG E\ HPSOR\LQJ VROXWH GLIIXVLRQ PRGHOVWRWKHDQDO\VLVRISUREHGLVWULEXWLRQ0RUHUHFHQWO\DXWRUDGLRJUDSK\RIWKLQ


In Vivo Solute Diffusivity in Brain Tissue

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VHFWLRQVDORQJZLWKLPDJHDQDO\VLVKDVEHHQXVHGWRDVVHVVUDGLRODEHOHGVROXWHGLIfusion from implanted polymeric controlled release devices [28]. 7KHUHDOWLPHLRQWRSKRUHWLFWHWUDPHWK\ODPPRQLXPWHFKQLTXHDOVRUHIHUUHGWR as the tetramethyl ammonium [TMA+@PLFURHOHFWURGHPHWKRG KDVEHHQXVHGIRU in vivo assessment of CNS–ECS diffusivity and to assess changes associated with a QXPEHURISK\VLRORJLFDODQGSDWKRORJLFDOFRQGLWLRQV>±@,QWKLVPHWKRG70$+ LVLQWURGXFHGE\LRQWRSKRUHVLVIURPDQHOHFWURGHDOLJQHGSDUDOOHOWRDGRXEOHEDUreled TMA+LRQVHQVLWLYHHOHFWURGH,6( VHWDWD¿[HGGLVWDQFHDZD\IURPDVRXUFH electrode. Diffusion of released TMA+ is then assessed through real-time measurement of ion concentrations in tissues where the TMA+ ISM is located. Local point concentration versus time measurements coupled with appropriate diffusion models are then employed to evaluate CNS–ECS diffusivity parameters. A limitation of the real-time iontophoretic method is that only relatively small molecular weight (MW) charged molecules (e.g., TMA+ 'D FDQEHXVHGDVGLIIXVLRQSUREHVDQGWZR DFXWHSHQHWUDWLQJLQMXULHVDUHUHTXLUHG 7KHLQWHJUDWLYHRSWLFDOLPDJLQJ,2, WHFKQLTXHLVDQin vitroWHFKQLTXHSUHYLously utilized to characterize macromolecule diffusion in CNS tissue [32–34]. A EROXVRIÀXRUHVFHQWO\ODEHOHGVROXWHLVUHOHDVHGLQWRDWKLFNaP EUDLQVHFtion mounted under a standard upright microscope. Diffusion is then monitored DQGPHDVXUHGE\FDSWXULQJÀXRUHVFHQFHLPDJHVDWLQFUHDVLQJORQJHUWLPHLQWHUYDOV 7KHRUHWLFDOH[SUHVVLRQVDUHWKHQHPSOR\HGWRDFFRXQWIRUERWKLQIRFXVDQGRXWRI IRFXVÀXRUHVFHQFHVLJQDOV6XFKPHDVXUHPHQWV\LHOGDVHULHVRISUREHGLVWULEXWLRQ SUR¿OHVIRUWKHWLPHSRLQWs collected, from which diffusivity is evaluated using an exponential model of solute diffusion. Based on methods similar to the IOI techQLTXH PRUH UHFHQWO\ PXOWLSKRWRQ PLFURVFRS\ KDV EHHQ HPSOR\HG WR HYDOXDWH WKH GLVWULEXWLRQRIODEHOHGQHUYHJURZWKIDFWRUDQGGH[WUDQSUREHVLQEUDLQVOLFHV>@ 7KHLQYHVWLJDWRUVVXJJHVWWKDWWKHWHFKQLTXHLVDQLPSURYHPHQWVLQFHRXWRIIRFXV ÀXRUHVFHQFHGRHVQRWEOXUUHFRUGHGLPDJHVWKHUHE\VLPSOLI\LQJLQWHUSUHWDWLRQRI SUREHGLVWULEXWLRQV $QRWKHUUHFHQWO\GHYHORSHGWHFKQLTXHUHIHUUHGWRDV³GXDOSUREH´PLFURGLDO\VLV KDV EHHQ HPSOR\HG WR LQYHVWLJDWH &16±(&6 GLIIXVLYLW\ SDUDPHWHUV LQ UDW EUDLQV >@ $ PLFURGLDO\VLV SUREH LV XVHG WR LQIXVH D UDGLRWUDFHU LQWR WKH UDW EUDLQ DQG DQRWKHU PLFURGLDO\VLV SUREH SRVLWLRQHG LQ SUR[LPLW\ WR WKH ¿UVW a PP LV then utilized to sample changes in the local solute concentration over time. Fitting of local point concentration versus time using appropriate mathematical models WKHQHQDEOHVGHULYDWLRQRIEXON&16±(&6GLIIXVLYLW\SDUDPHWHUVIRUWKHLQWHUVWLWLDO UHJLRQORFDWHGEHWZHHQWKHSUREHV 5HFHQWO\ZHGHVFULEHGDQRYHOWLVVXHDFFHVVGHYLFH7$' EDVHGRQKROORZ¿EHU PHPEUDQH+)0 EUDLQWLVVXHLPSODQWVWKDWZHUHXVHGWRH[DPLQHZKHWKHUFHOOV WUDQVSODQWHGLQWRWKH7$'LQÀXHQFHGVROXWHGLIIXVLRQLQWKHEUDLQWLVVXHWKDWVXUURXQGVWKHLPSODQW>±@8QOLNHRWKHUPHWKRGV7$'VDVVHVVKRZYDULRXVWLVVXH FRPSRQHQWVDIIHFWWKHWUDQVSRUWRIVROXEOHIDFWRUVDIWHUDFKURQLFLQWHUYHQWLRQ,Q WKLVFDVHWKHGHYLFHFDQEHLPSODQWHGSULRUWRLQWURGXFLQJWKHFHOOVRUWKHVROXWH PROHFXOHVZKLFKDOORZVRQHWRGHVLJQH[SHULPHQWVWKDWDVVHVVHVWKHIRUHLJQERG\ UHVSRQVHDWGLIIHUHQWWLPHSRLQWVDQGWRH[DPLQHZKDWPLJKWEHH[SHFWHGIROORZLQJ the transplantation of cells after a long or short indwelling times. Also, since the


120


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5.2 METHODS 5.2.1

DEVICE AND IMPLANTATION PROCEDURE

A schematic of the cell encapsulation device is shown in Figure 5.1A. The implants ZHUHIDEULFDWHGDQGVWHULOL]HGDVGHVFULEHGSUHYLRXVO\>±@$SKRWRJUDSKRID representative device is shown in Figure 5.1F. A poly(acrylonitrile-vinyl chloride) 3$139& +)0ZDVIDEULFDWHGDVGHVFULEHG>@ with an asymmetric morphology ZLWKDQLQQHUGLDPHWHURIaPDQGZDOOWKLFNQHVVRIaPVHH)LJXUH( 7KHN'DGH[WUDQGLIIXVLRQFRHI¿FLHQWIRUWKHQDwYHPHPEUDQHXVHGZDVa× 10_8cm 2/sec [42]. Cell coil constructs were loaded with cells and implanted using stereotaxic PHWKRGV DV SUHYLRXVO\ GHVFULEHG>±@ &ROODJHQHQWUDSSHG PHQLQJHDO FHOOVRU collagen matrix alone were transferred into the devices prior to implantation. CelOXODUFRQVWUXFWVFDQEHSUHSDUHGXVLQJDYDULHW\RIFHOOW\SHVLQFOXGLQJDQFKRUDJH and nonanchorage-dependent cell types using a density of 3.0 × 104 entrapped cells per HFM construct. Prior to implantation loaded coil constructs are maintained in serum-free culture media so as not to induce reactivity not associated with the implanted cells. 7KHRULHQWDWLRQRIWKHGHYLFHLPSODQWHGZLWKLQWKHUDWEUDLQLVGHSLFWHGLQ)LJXUH%$SKRWRJUDSKRID7$'LPSODQWHGLQWKHUDWEUDLQLVVKRZQLQ)LJXUH* 7\SLFDOO\RUDQLPDOVDUHXWLOL]HGIRUERWKWKHFRQWUROJURXSLHFROODJHQ only) DQGH[SHULPHQWDOJURXSVLHFROODJHQHQWUDSSHGPHQLQJHDO¿EUREODVWV 2QFHWKH device is implanted and anchored to the cranium, the cell coil element is inserted LQWRWKH+)0FKDPEHUFigure 5.1C), the access cap is snapped in place, and the overlying dermis is closed with sutures. Alternatively, the cells or a sustainedUHOHDVHSRO\PHUFDQEHSODFHGLQWKHLPSODQWHGGHYLFHDWDODWHUWLPHSRLQWWRVWXG\ WKHLQÀXHQFHRIWKHFHOOVRUGUXJVRQWLVVXHUHPRGHOLQJ 5.2.1.1 Diffusion Experiments Following a recovery period animals are reanesthetized, the dermis overlying the implanted TAD is opened, the access cap is removed, and the cell coil construct is ZLWKGUDZQIURPWKHGHYLFH:LWKWKHDLGRIDORZÀRZPLFURV\ULQJHSXPSDO\VLQH



121

FIGURE 5.1 6LPSOL¿HGVFKHPDWLFVRI$ &16WLVVXHDFFHVVGHYLFH% LPSODQWHGGHYLFH RULHQWDWLRQLQUDWEUDLQ& LQVHUWLRQRIFHOOFRLOFRQVWUXFWDQG' ORDGLQJDQGH[WUDFWLRQ RISUREHVROXWLRQLQWRDQGIURPWKHLQWUDOXPHQDOFKDPEHURIWKHGHYLFHIRU&16GLIIXVLYLW\ H[SHULPHQW UXQV ( 6FDQQLQJ HOHFWURQ PLFURJUDSK RI 3$139& KROORZ ¿EHU PHPEUDQH HPSOR\HG LQ DVVHPEO\ RI 7$' 7KH DUURZ SRLQWV WR WKH LQWUDOXPHQDO SHUPVHOHFWLYH VNLQ layer of the HFM. (F) Photograph of actual TAD employed in the study. (G) Photograph of 7$'LPSODQWHGLQUDWEUDLQZLWKDFFHVVFDSUHPRYHG

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Histological Processing of Tissue Samples

$YLEUDWRPH7HFKQLFDO3URGXFWV,QWHUQational, St. Louis, Missouri) is utilized to PDNHKRUL]RQWDOWLVVXHVHFWLRQVDFURVVWKHUHWULHYHGEUDLQVDPSOHV6OLFHVDWWR PDUHVHULDOO\FROOHFWHGDQGVWRUHGLQZHOOSODWHVFRQWDLQLQJDî3%6VROXWLRQ ZLWK1D$DW&7KURXJKRXWVHFWLRQLQJDQGVXEVHTXHQWSURFHVVLQJRIVHFWLRQV FDUHLVWDNHQWRPLQLPL]HOLJKWH[SRVXUHRI75GH[WUDQODGHQWLVVXHVHFWLRQV %DWFKSURFHVVLQJRIVHFWLRQVLVXWLOL]HGIRUDOOVXEVHTXHQWVWDLQLQJSURFHGXUHV WROLPLWVWDLQLQJYDULDELOLW\DQGWRHQVXUHFRQVLVWHQWWUHDWPHQWRIDOOVDPSOHV,QFXEDWLRQRIVHFWLRQVZLWKFHOOVSHFL¿FPDUNHUVDOORZVUHJLRQDOLGHQWL¿FDWLRQRISDUWLFXODUFHOOW\SHVDVZLOOEHLOOXVWUDWHGKHUHXVLQJPDUNHUVIRUUHDFWLYHDVWURF\WHV DQGPDFURSKDJHVDQGUHDFWLYHPLFURJOLD$¿UVWVHWRIWLVVXHVHFWLRQVFRQVLVWLQJRI HYHU\IRXUWKVHULDOVHFWLRQPVSDFLQJ LVVWDLQHGIRUJOLDO¿EULOODU\DFLGLFSURWHLQ*)$3 DQLQWHUPHGLDWH¿ODPHQWSURWHLQNQRZQWREHXSUHJXODWHGE\UHDFWLYH DVWURF\WHVFRPSULVLQJJOLDOVFDUV>@6HFWLRQVDUHH[SRVHGWRDJRDWVHUXPLQ 1×3%6ZLWKWULWRQWRSHUPHDEOL]HFHOOPHPEUDQHVIRU+DWURRPWHPSHUDWXUH a& 3ULPDU\ DQWLERG\ FRQVLVWLQJ RI DSSURSULDWH DQWLVHUD IRU LQVWDQFH SRO\FORQDOUDEELWDQWL*)$3GLOXWLRQPRXVH,J*'DNR LVDSSOLHGRYHUQLJKWDW &$QDSSURSULDWHVHFRQGDU\DQWLERG\VXFKDV$OH[D±ODEHOHGJRDWDQWLUDEELW IgG (H+L) (1:220 dilution, Molecular PUREHV LVthen applied for a minimum of 1 +DWa&$GGLWLRQDOVHFWLRQVVHUYHDVSULPDU\FRQWUROVWKURXJKLQFXEDWLRQZLWK RQO\WKHDSSOLHGVHFRQGDU\DQWLERG\ $ VHFRQG VHW RI WLVVXH VHFWLRQV ZLWK P VSDFLQJ HYHU\ HLJKWK VHFWLRQ LV VWDLQHG IRU DQRWKHU PDUNHU IRU LQVWDQFH (' DQ DQWLJHQ PDUNHU FRPPRQO\ HPSOR\HGWRGLVFULPLQDWHEHWZHHQDFWLYDWHGPLFURJOLDDQGPDFURSKDJHVLQWKH&16 >@:LWKWKHH[FHSWLRQRIH[SRVXUHWRWULWRQGXULQJWKHEORFNLQJVWHSDVLPLODU LPPXQRVWDLQLQJSURWRFROLVXVHGIRU('GHWHFWLRQ7KHSULPDU\DQWLERG\FRQVLVWV of mouse anti-ED1 (1:1000 dilution, rat IgG1, Serotec), and the secondary is Alexa ±ODEHOHGJRDWDQWLPRXVH,J*GLOXWLRQ0ROHFXODU3UREHV ('VWDLQLQJ primary control sections are also prepared similar to the previous protocol. All sections are then counterstained with the nuclear dye 4’,6-diamidino-2-pheQ\OLQGROHGLK\GURFKORULGH'$3,0ROHFXODU3UREHV PRXQWHGRQVWDQGDUGPLFURVFRSHVOLGHVXVLQJ)OXRURPRXQW*0ROHFXODU3UREHV DQGVWRUHGLQWKHGDUNDW& $GGLWLRQDOVHFWLRQVWREHXWLOL]HGWRDVVHVVWKHUHODWLYHFRQFHQWUDWLRQVRI75'H[WUDQSUREHDFURVVWKHWUDQVPHPEUDQHUHJLRQDUHDOVRPRXQWHGRQVOLGHV 5.2.1.3

Fluorescence Imaging of Samples

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(5.1)

where Iexp and IÀDWUHSUHVHQWWKHH[SHULPHQWDODQGÀDW¿HOGFDOLEUDWLRQLPDJHVDQGS UHSUHVHQWVDSL[HOLQWHQVLW\VFDOHIDFWRUGHULYHGIURPSHDNLQWHQVLW\YDOXHVDSSHDULQJ LQFDOLEUDWLRQLPDJHV,PDJHVDUHWKHQVWRUHGDVWDJJHGLPDJH¿OHIRUPDWV7,))V which maintain intensity data as discrete channel information. 5.2.1.4 Quantitative Image Analysis 7R HQVXUH QRQELDVHG ÀXRUHVFHQFH SL[HO LQWHQVLW\ VDPSOLQJ RI FROOHFWHG LPDJHV D /DEYLHZ1DWLRQDO,QVWUXPHQWV&R$XVWLQ7H[DV ±EDVHGLPDJHDQDO\VLVSURJUDP ZDVFUHDWHGDQGTXDOL¿HGLQSUHOLPLQDU\VWXGLHVVSHFL¿FDOO\IRUWKLVH[SHULPHQWDO application (introduced in [40]). In the analysis program created (see Figure 5.2) three points were utilized to delineate a reference arc at the outer wall of the HFM. 7KHDQJXODUORFDWLRQUDGLXVDQGVL]HRIWKHUHIHUHQFHDUFLVDGMXVWDEOHHQDEOLQJ FRRUGLQDWLRQRIWKHUDGLDOD[LVEHWZHHQVHFWLRQVDVZHOODVH[FOXVLRQRISURFHVVLQJ GHIHFWVWKDWPD\EHSUHVHQWLQLPDJHVVXFKDVDLUEXEEOHV2QFHDUHIHUHQFHDUFLV GHOLQHDWHGTXDQWLWDWLYHÀXRUHVFHQFHLQWHQVLW\VDPSOLQJSUR¿OHV4)3V DUHJHQHUDWHGH[WHQGLQJQRUPDOWRWKHDUFDWDGHQVLW\RIaGHJUHHSUR¿OHDQGRQDYHUDJH IRUWKHSUHVHQWVWXG\\LHOGHGaVDPSOLQJSUR¿OHVSHULPDJHDQDO\]HG7KH4)3V JHQHUDWHGDUHUDGLDOO\LQGH[HGLQaPLQFUHPHQWVWKDWRULJLQDWHPEDFN IURPWKHGHOLQHDWHGDUFWRHQDEOHDQDO\VLVRIWKHWUDQVPHPEUDQHUHJLRQVDQGUDQJH IURP WR P LQ WRWDO OHQJWK $W HDFK UDGLDO LQFUHPHQW DQ DQWLDOLDV SL[HO H[WUDFWLRQ DOJRULWKP LV DSSOLHG WR GHULYH DQ LQWHQVLW\ YDOXH EDVHG RQ D ZHLJKWHG averaging of pixels in nearest proximity (as depicted in inset of Figure 5.2). 7KHFRPSRVLWH4)3IRUWKHLPDJHLVWKHQFDOFXODWHGE\DYHUDJLQJDOOFRLQFLGLQJ LQFUHPHQWVRIWKHVDPSOLQJSUR¿OHVJHQHUDWHGDQGLVSHUIRUPHGVLPXOWDQHRXVO\IRU DOOFRORUFKDQQHOVSUHVHQWLQLPDJHV:KHQSUR¿OHLQWHQVLW\DYHUDJLQJLVSHUIRUPHG RQVROXWHGLIIXVLRQSUREHGLVWULEXWLRQVYDOXHVIDOOLQJRXWVLGHRIWZRVWDQGDUGGHYLDWLRQVDUHGLVFDUGHGDQGWKHDYHUDJHUHFDOFXODWHG7KLV¿OWHULVQRWDSSOLHGWRKLVWRlogical stains since punctate patterns are expected, given that the populations of cell W\SHVRILQWHUHVWDUHGLVFUHWHUHJLRQDOHQWLWLHVOLNHO\WRYDU\LQWKHFLUFXPIHUHQWLDO


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FIGURE 5.2 'HPRQVWUDWLRQ RI WKH TXDQWLWDWLYH ÀXRUHVFHQFH SUR¿OLQJ VDPSOLQJ PHWKRG employed in study. Three points (A, B, and C) are used to delineate a reference arc at the RXWHUVXUIDFHRIWKH+)05DGLDOO\GH¿QHGVDPSOLQJDUUD\VDUHWKHQJHQHUDWHGDWUHJXODUO\ VSDFHGLQWHUYDOV$VGHSLFWHGLQWKHLQVHW¿JXUHPXOWLSOHSL[HOVLQWKHLPPHGLDWHYLFLQLW\RI each sampling array point are used in the evaluation of its intensity value.

orientation of samples. The presence of tissue section processing irregularities, such DVSDUWLDOWHDUVLQWKHWLVVXHGLVSODFHPHQWRIWLVVXHIURPWKHPHPEUDQHLQWHUIDFHRU DLUEXEEOHHQWUDSPHQWZLWKLQWKHFDSWXUHUHJLRQUHVXOWVLQWKHUHMHFWLRQRIDOO4)3V generated for that image. However, only rarely do all four orientations captured for DVSHFL¿FWLVVXHVHFWLRQKDYHWREHH[FOXGHGIURPDQDO\VHV 5.2.1.5

Data Processing

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125

calculation dextran curves are normalized through division with the intensity value DWWKHUDGLXVUHSUHVHQWLQJWKHPHPEUDQH±WLVVXHP±W LQWHUIDFH

5.2.1.6

Gel Diffusivity Characterization Experiments

Gel medium utilized for gel diffusivity characterization experiments consisted of D UDWLR RI SRUFLQH VNLQ JHODWLQ WR KLJKWHPSHUDWXUHPHOW DJDURVH DW D weight-to-volume (w/v) content. Gel solutions were prepared through addition of gelatin and agarose components to a 1×3%6VROXWLRQZLWK1D$$ZHOOSODWH VHUYHGDVWKHPROGIRUWKHHPEHGGLQJSURFHVVZLWK7$'VPDLQWDLQHGUDGLDOO\FHQWHUHGDQGQRUPDOWRWKHERWWRPVXUIDFHRIZHOOVGXULQJVROLGL¿FDWLRQRIWKHKHDWHG JHOVROXWLRQ:HOOVZHUH¿OOHGZLWKWKHJHOVROXWLRQXSWRWKHPLGSRLQWRIWKHDFFHVV SRUWFRPSRQHQWRIWKH7$'*HOHPEHGGHG7$'VDPSOHVZHUHWKHQUHPRYHGIURP molds and placed in 6 well plates containing 1×3%6 VROXWLRQ ZLWK 1D$ WR ensure their complete hydration prior to diffusion runs. The protocol used for in vitroJHOGLIIXVLRQH[SHULPHQWVFORVHO\PLPLFNHGWKDW used for in vivoH[SHULPHQWV7KHOHYHORI3%6VROXWLRQLQZHOOVZDVEURXJKWHYHQ WRWKDWRIWKHWRSVXUIDFHRIJHOV7KH3%6VROXWLRQLQWKHLQWUDOXPHQDOFKDPEHURI WKH7$'ZDVWKHQH[FKDQJHGZLWKDQ$OH[D±ODEHOHGVWUHSWDYLGLQVROXWHSUREH 0:aN'D0ROHFXODU3UREHV LQ3%6DWDFRQFHQWUDWLRQRIaPJPO7KH ODEHOHGSURWHLQZDVWKHQDOORZHGWRGLIIXVHLQWRWKHVXUURunding gel for 10- (n = 2), 20- (n = 2), 30- (n = 1), 60- (n = 1), 120- (n = 1), or 180-minute (n = 1) periods. At the HQGRIHDFKGLIIXVLRQUXQWKHFKDPEHUZDVÀXVKHGZLWK3%6DQGORDGHGZLWK JOXWDUDOGHK\GH3%6 ¿[DWLYH VROXWLRQ 7KH VXUURXQGLQJ EXIIHU VROXWLRQ ZDV DOVR UHSODFHGZLWKWKH¿[DWLYHVROXWLRQ6DPSOHVZHUHWKHQVWRUHGDW&IRUDPLQLPXP of 12 h prior to sectioning. )ROORZLQJ¿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îPDJQL¿FDWLRQZDVUHTXLUHGJLYHQWKHH[WHQWRISUREHGLIIXVLRQ)RXULPDJHVUHSUHVHQWLQJXSSHUDQGORZHUOHIWDQGULJKWTXDGUDQWVZHUH FDSWXUHG SHU VHFWLRQ 4)3 DQDO\VLV RI LPDJHV ZDV WKHQ SHUIRUPHG DV GHVFULEHG DERYH0LFURIUDFWXUHVDQGLUUHJXODULWLHVSUHVHQWDWWKHJHO±PHPEUDQHLQWHUIDFHWKDW UHVXOWHG IURP 7$' H[WUDFWLRQ DQG VHFWLRQLQJ SURFHVVHV UHTXLUHG GLVFDUGLQJ RI D ODUJHQXPEHUaWR RIWKHFRPSRVLWH4)3VSULRUWRHYDOXDWLRQRIPHDQLQWHQVLW\SUR¿OHV


126


5.2.1.7 Evaluation of In Vivo Diffusivity Properties: Central Nervous System (CNS)–Extracellular Space (ECS) Solute Diffusion Modeling $SSUR[LPDWLRQ RI &16 WLVVXH GLIIXVLYLW\ SURSHUWLHV ZDV DFFRPSOLVKHG E\ ¿WWLQJ WKH REVHUYHG 75ODEHOHG N'D GH[WUDQ SUREH PHDQ LQWHQVLW\ SUR¿OHV ZLWK WKDW GHULYHG IURP VLPXODWLRQV HPSOR\LQJ D WKHRUHWLFDO PRGHO GHVFULELQJ VROXWH GLIIXVLRQLQWKH&16±(&6*LYHQWKDWSUR¿OHVUHSUHVHQWWUDQVLHQWVWDWHGLVWULEXWLRQVDQG given the expected variations in the diffusivity characteristics of the HFM intraluPHQDOVSDFHPHPEUDQHZDOOVSDFHUHDFWLYHPLFURJOLD±PDFURSKDJHOD\HUJOLDOVFDU OD\HUDQGWKHXQDIIHFWHGQDwYH &16WLVVXHUHJLRQVZHHOHFWHGWRHPSOR\DUDGLDO ¿QLWHHOHPHQWGLIIHUHQFHPRGHOWKDWHQDEOHVRQHWRDFFRXQWIRUGLIIHUHQFHVLQWKHLU GLIIXVLYLW\SURSHUWLHV)LWWLQJRIH[SHULPHQWDOGDWDE\VLPXODWHGUHVXOWVZDVDFFRPSOLVKHGWKURXJKWULDODQGHUURUDGMXVWPHQWVRISDUDPHWHUVUHOHYDQWWRWKH&16±(&6 solute diffusion model. A similar approach was utilized to estimate an effective GLIIXVLYLW\FRHI¿FLHQWIRULPSODQWHG3$139&+)0VDVZHOODVIRUWKHVWUHSWDYLGLQ SUREHLQWKHJHOPHGLXPHPSOR\HG The mathematical model employed for CNS–ECS diffusion simulations was EDVHG RQ )LFN¶V VHFRQG ODZ DSSOLHG WR D V\VWHP LQ ZKLFK VROXWHGLIIXVLRQ RFFXUV symmetrically in a radially outward direction:

wC (r , t ) wt

§ w 2C ( r , t ) · D¨ ¸ 2 © wr ¹

(5.2)

where C(r,t) is the solute concentration at a distance r from the origin at time t, and D LV WKH VROXWH GLIIXVLRQ FRHI¿FLHQW (TXDWLRQ RQO\ SURYLGHV D PDFURVFRSLF description of solute diffusion in a homogeneous system (i.e., continuum diffusion RUIUHHVROXWLRQPHGLXP VXFKDVZDWHU)RUWKLVUHDVRQ)LFN¶VVHFRQGODZLVFRPPRQO\HPSOR\HGWRHYDOXDWHWKHGLIIXVLRQFRHI¿FLHQWRIDSDUWLFXODUVROXWHLQSXUH water (Dw ,WLVDFRQYHQLHQWEHQFKPDUNIRUVROXWHGLIIXVLRQLQKHWHURJHQHRXVV\Vtems such as tissue tREHFRPSDred. However, on a microscopic scale solute diffusion in the CNS is restricted to the complex geometric structures that comprise the ECS pathways. The presence of solute in tissue is therefore discontinuous in nature DQGKDVKLVWRULFDOO\EHHQLQWURGXFHGLQWLVVXHGLIIXVLRQPRGHOVWKURXJKLQFRUSRUDtion of a term referred to as the void volume fraction (B). In regard to CNS–ECS solute diffusion modeling, the void volume fraction simply represents the ratio of ECS volume to overall tissue volume. ,QWKHOLWHUDWXUHUHODWHGWRWKLVVXEMHFWZKHQDQHTXDWLRQRIFRQWLQXXPGLIIXVLRQVXFKDV(TXDWLRQ LVXVHGLQWKHHYDOXDWLRQRIWLVVXHGLIIXVLYLW\SURSHUWLHV it is common to refer to resultant values of DDVDSSDUHQWGLIIXVLRQFRHI¿FLHQWVDa). In addition, investigators of CNS–ECS refer to the degree to which solute diffusion in tissue is reduced (i.e., hindered diffusion) compared to diffusion in pure water as ³WRUWXRVLW\´>@.QRZOHGJHRIDaVXEVHTXHQWO\FDQEHXVHGWRFDOFXODWHWKH ECS diffusivity parameter tortuosity (M GH¿QHGDV

O © 2008 by Taylor & Francis Group, LLC

Dw / Da

(5.3)


127

The geometrical symmetry in the two-dimensional radial model employed for WKLV DQDO\VLV HQDEOHV VLPSOL¿FDWLRQ WR D RQHGLPHQVLRQDO V\VWHP IRU &16±(&6 VROXWH GLIIXVLRQ VLPXODWLRQV 7KH ¿QLWH HOHPHQW V\VWHP LV WKHUHIRUH FRPSRVHG RI DOLQHDUQRGDODUUD\RIHOHPHQWVWKDWRULJLQDWHVLQWKHFHQWHURIWKH+)0,Q¿QLWH GLIIHUHQFHIRUP(TXDWLRQ PD\EHDSSUR[LPDWHG

Ci ,n 1 Ci ,n 't

Di Ci 1,n 2Ci,n Ci 1,n , 'r 2

(5.4)

where %rUHSUHVHQWVWKHGLVWDQFHEHWZHHQQRGHVDQG%t represents the incremental WLPHLQWHUYDOXVHGIRUWKHVLPXODWLRQUXQ1RWHWKDWWKHULJKWKDQGVLGHRI(TXDWLRQ PXOWLSOLHGE\%t yields the incremental change in solute concentration (%Ci) for nodal element i7KHRWKHUWHUPVSUHVHQWLQ(TXDWLRQ DUHGH¿QHGDV

Ci ,n ri

C (ri , tn )

('r )i, for 0 d i d inorm

tn

('t )n, for 0 n d nmax

Di

w Dint ra ° D ° memb ° a ® Dmicro ° a ° Dscar a °¯ Dnorm

where nmax

0 d i d iintra iintra i d imemb for imemb i d imicro imicro i d iscar iscar i d inorm

tend / 't and iintra imemb

(5.5)

Rintra / 'r Rmemb / 'r

where imicro Rmicro / 'r iscar Rscar / 'r inorm Rnorm / 'r

where Rintra, Rmemb, Rmicro, Rscar, and Rnorm, respectively, delineate the radial distances IURPWKHRULJLQWRWKHLQWUDOXPHQDODQGH[WUDOXPHQDOPHPEUDQHVXUIDFHVDQGWKH RXWHUWLVVXHERXQGDULHVRIWKHUHDFWLYHPLFURJOLDJOLDOVFDUDQGQDwYH&16WLVVXH a LVGH¿QHGEDVHGRQDWWDLQLQJ regions. The apparent diffusivity of scar tissue ( Dscar DUHDVRQDEOH¿WWRWKHREVHUYHGUHVXOWVZKLFKLQWKHFDVHRIRXUSUHOLPLQDU\VWXG\ a \LHOGHGWKHEHVW¿WVZKHQGH¿QHGZLWKDGLVWDQFHGHSHQGHQF\LH Dscar f (ri )). Diffusion of solute occurs radially outward in the experimental two-dimensional V\VWHPWKDWWKHOLQHDU¿QLWHHOHPHQWPRGHOLVLQWHQGHGWRVLPXODWH7KHUHIRUHQRGDO elements (ri) in our linear model need to represent two-dimensional annular regions (Ai) of the system. Since the annular regions grow in size as elements move outward from the origin (i.e., Ai > Ai-1), it is necessary to incorporate this aspect into the mathHPDWLFDOGHVFLSWLRQRIWKHOLQHDUV\VWHPSUHVHQWHGLQ(TXDWLRQ 7KLVZDVDFFRPSOLVKHGE\UHIRUPXODWLQJ(TXDWLRQ WRVROYHIRUHOHPHQWDOFKDQJHVLQPDVV%mi) at each incremental time period (%t ZLWKWKHHOHPHQWDOFRQFHQWUDWLRQVQRZGH¿QHG

Ci

mi / Ai ,

,QVHUWLQJ(TXDWLRQ LQWR(TXDWLRQ DQGVROYLQJIRU%mi yields © 2008 by Taylor & Francis Group, LLC

(5.6)

128


'mi

Di (Ci,n Ci 1,n ) Ai 1 (Ci,n Ci 1,n ) Ai 't 'r 2

mi ,n 1 mi ,n

(5.7)

where

Ai

D i (ri 1 ) 2 (ri ) 2 S

(5.8)

7KH¿UVWWHUPRQWKHULJKWVLGHRI(TXDWLRQ B i) accounts for the void volume fraction of the ECS diffusivity parameter in our diffusion model, with B micro, B scar, and B normGH¿QHGLQDPDQQHUVLPLODUWRDiLQ(TXDWLRQ ZLWKWKHH[FHSWLRQWKDW B i = 1 for i imemb. The elemental mass at time t = n + 1 is then calculated as

mi,n 1

mi,n 'mi,n 1

(5.9)

The CNS–ECS solute diffusivity simulation therefore consisted of solving of (TXDWLRQ LQFRQMXQFWLRQZLWK(TXDWLRQ ZLWKDQHZQRGDOPDVVDUUD\>m0, m1, … mnorm@DQGWKHVXEVHTXHQWFRQFHQWUDWLRQQRGDODUUD\XVLQJ(TXDWLRQ FDOFXODWHGDWHDFKLQFUHPHQWDOWLPHLQWHUYDO5HSRUWHGVLPXODWHGSUREHGLVWULEXWLRQ SUR¿OHVUHSUHVHQWWKHQRGDOFRQFHQWUDWLRQDUUD\YDOXHVSUHVHQWDWWKHHQGRIDVLPXODWLRQUXQV7RHQVXUHWKHVWDELOLW\RIWKHVLPXODWLRQ%tZDVFKRVHQE\

't

'r 2 2 Dimin

(5.10)

where Dimin represents the lowest value of Di applied in the simulation. $WWKHRQVHWRIVWXGLHVLWZDVXQFOHDUZKHWKHUWKHPDVVRISUREHDYDLODEOHLQ WKHFKDPEHUVKRXOGEHPRGHOHGDV¿QLWHRULQ¿QLWHJLYHQWKHH[WUDSUREHVROXWLRQ placed in the access port region of TADs during diffusion runs. For this reason VLPXODWLRQVZHUHFRQGXFWHGXVLQJWZRLQLWLDOFRQGLWLRQVUHSUHVHQWDWLYHRI¿QLWHDQG LQ¿QLWHVROXWHSUREHUHVHUYRLUV7KHLQLWLDOFRQGLWLRQVHPSOR\HGIRUD¿QLWHVROXWH PDVV LQ WKH LQWUDOXPHQDO VSDFH ZLWK PHPEUDQH DQG WLVVXH UHJLRQV YRLG RI VROXWH was

Ci (ri ,t0 )

100 ® ¯ 0

for

0 d i d iintra iintra i d inorm

(5.11)

)RU LQ¿QLWH UHVHUYRLU VLPXODWLRQ UXQV VROXWH PDVV LQ WKH LQWUDOXPHQDO VSDFH ZDV VSHFL¿HG

Ci (ri ,tn ) 100 for 0 d i d iintra

and 0 d n d nmax

(5.12)

7KH LQ¿QLWH VLQN ERXQGDU\ FRQGLWLRQ LPSRVHG RQ WKH ODVW HOHPHQW RI WKH QRGDO array was



Ci (ri , tn ) 0 for i imax

129

and 0 d n d nmax

(5.13)

The value of imaxXVHGIRUVLPXODWLRQVODUJHHQRXJKWKDWQRSUREHZDVSUHVHQWDWWKH preceding node at the end of simulation runs. $OO &16 VROXWH GLIIXVLRQ VLPXODWLRQV XWLOL]HG D SXUH ZDWHU SUREH GLIIXVLYLW\ w value Dintra 3.72 u107 cm 2V7KLVIUHHPHGLXPGLIIXVLYLW\YDOXHIRUDN'DGH[tran solute, also used in the calculation of tortuosity values cited in this study, was DQHVWLPDWHEDVHGRQWKHHPSLULFDOFRUUHODWLRQ>@ D = 7.667 × 10 –5(MW) –0.47752, where MWLVWKHPROHFXODUZHLJKWRIWKHGH[WUDQPROHFXOH7KHPHPEUDQHGLIIXsivity value (Dmemb XVHGZDVEDVHGXSRQWKHUHVXOWVRIVHSDUDWHVLPXODWLRQDQDO\VHVXVLQJSDUDPHWHUVIRUDOOWLVVXHUHJLRQVEDVHGRQWKRVHRIQDwYH&16WLVVXH$Q a = 7.5 × 10 -8 cm2/s, derived from apparent diffusivity value of normal tissue, Dnorm the literature [32], and a void fraction value of normal tissue, B norm = 0.21, also a a , Dscar , B miderived from the literature [48], were used for initial simulations. Dmicro , and B ZHUHGHWHUPLQHGWKURXJK¿WWLQJRIH[SHULPHQWDOO\REWDLQHGSUR¿OHV scar cro The values %r PDQG%t = 0.2s were used for in vivo solute diffusion simulation analyses of control and experimental group data. )RUJHOGLIIXVLRQVLPXODWLRQVWKHUHJLRQRXWVLGHRIWKHPHPEUDQHZDVDVVXPHG to have a constant diffusivity value and void fraction value of 1. In addition, a pure w ZDWHUGLIIXVLYLW\YDOXHIRUWKHODEHOHGVWUHSWDYLGLQaN'D RI Dintra 7.54 u 107 2 cm VZDVXVHGEDVHGXSRQWKHWKHRUHWLFDOHTXDWLRQRIIUHHPHGLXPSURWHLQGLIIXsion [49]: D = A/(MW)m, where A = 2.85 × 10 –5 cm 2s_1g1/3mol_1/3 and m = 1/3. A value of Dmemb = 1.8 × 10 –7 cm 2VZDVDOVRXVHGDQGZDVHVWLPDWHGEDVHGXSRQSUHYLRXV FKDUDFWHUL]DWLRQVRIWKH+)0SHUIRUPHGXVLQJDUDQJHRIGH[WUDQVROXWHSUREHV7KH value of %rXVHGWRVLPXODWHVROXWHGLIIXVLRQLQJHOVZDVP

5.3 RESULTS 5.3.1

GEL DIFFUSIVITY CHARACTERIZATION EXPERIMENT

Fluorescent microscopy images representing each of the time periods utilized for test runs appear in Figure 5.3. Circular voids located near the corners of images GHOLQHDWHWKHUHJLRQLQLWLDOO\RFFXSLHGE\+)0SULRUWRVHFWLRQLQJRI¿[HGVDPSOH gels. Characteristic of solute diffusion phenomenon, increasing the length of time of WHVWUXQVUHVXOWHGLQVHULDOO\JUHDWHUSUREHGLVWULEXWLRQVZLWKLQVDPSOHV 7KH UHVXOWV RI 4)3 DQDO\VLV RI FDSWXUHG ÀXRUHVFHQW LPDJHV LV VKRZQ LQ )LJXUHZKHUHWKHQRUPDOL]HGPHDQLQWHQVLW\SUR¿OHVIRUWKHYDULRXVWLPHJURXSV are plotted. TKHFRQFHQWUDWLRQVRISUREHREVHUYHGIRUWKHDQGPLQXWHJURXSV appear to drop off exponentially, while the longer runs appear sigmoidal. The sigPRLGDOVKDSHRIWKHODWWHUSUR¿OHVLVLQGLFDWLYHRIGLIIXVLRQLQDV\VWHPGULYHQE\D VRXUFHFRQWDLQLQJD¿QLWHPDVVRIVROXWH $¿QLWHHOHPHQWPRGHORIVROXWHGLIIXVLRQVLPXODWLRQWKDWLQFRUSRUDWHGHOHPHQWV VSDWLDOO\UHSUHVHQWDWLYHRIWKH7$'FKDPEHU+)0ZDOODQGDGMDFHQWJHOPHGLXP ZDVWKHQXWLOL]HGWRHVWLPDWHWKHHIIHFWLYHGLIIXVLYLW\FRHI¿FLHQWRIWKHVWUHSWDYLGLQ SUREH E\ ¿WWLQJ RI WKH DQG PLQXWH UHVXOWV %DVHG XSRQ WKH UHVXOWV RI simulation analyses, a free medium effective diffusivity value of Deff = 6.78 × 10 –7 © 2008 by Taylor & Francis Group, LLC

130


FIGURE 5.3 Representative 10× ÀXRUHVFHQFH PLFURVFRS\ LPDJHV RI $OH[D ±ODEHOHG VWUHSWDYLGLQLQZYJHODWLQ±DJDURVHJHOIRUH[SHULPHQWDOUXQWLPHVDVLQGLFDWHG LQLPDJHV7KHFLUFXODUYRLGUHJLRQVSUHVHQWLQLPDJHVZHUHRULJLQDOO\RFFXSLHGE\WKH+)0 WKDWKDGWREHH[WUDFWHGIURPJHOVSULRUWRWKHLUVHFWLRQLQJ

FIGURE 5.4 1RUPDOL]HG PHDQ LQWHQVLW\ SUR¿OHV RI $OH[D ±ODEHOHG VWUHSWDYLGLQ LQ ZYJHODWLQ±DJDURVHJHOIRUH[SHULPHQWDOUXQWLPHVDVLQGLFDWHGLQWKHJUDSK 7KH³QÝDOXHVGLVSOD\HGDGMDFHQWWRWKHSUR¿OHODEHOVUHSUHVHQWWKHQXPEHURIFRPSRVLWH 4)3SUR¿OHVXWLOL]HGLQWKHHYDOXDWLRQRIPHDQLQWHQVLW\SUR¿OHV7KHSUR¿OHVZHUHQRUPDOL]HGEDVHGXSRQWKHPHDQLQWHQVLW\YDOXHDWWKHVXUIDFHRIWKHJHOLQLWLDOO\DGMDFHQWWRWKH 7$'PHPEUDQHPLQWKHFKDUW



131

FIGURE 5.5 $OH[D ±ODEHOHG VWUHSWDYLGLQ QRUPDOL]HG PHDQ LQWHQVLW\ SUR¿OHV IRU and 20-minute experimental run time sample groups along with 10- and 20-minute simuODWHGSUREHGLVWULEXWLRQSUR¿OHVDWWDLQHGIURPVLPXODWLRQUXQVWKDWXWLOL]HGDQHIIHFWLYHJHO GLIIXVLRQFRHI¿FLHQWRI× 10_7 cm 2VWRDWWDLQWKHGLVSOD\HG¿WVRIREVHUYHGH[SHULPHQWDO SUREHGLVWULEXWLRQSUR¿OHV7KHVLPXODWHGSUREHGLVWULEXWLRQSUR¿OHVZHUHQRUPDOL]HGEDVHG RQ WKH YDOXH SUHVHQW DW WKH PHPEUDQHJHO r P DW WKH FRQFOXVLRQ RI WKH PLQXWH simulation run.

cm 2/s was derived. Figure 5.5 displays the results of 10- and 20-minute simulated SUREHGLVWULEXWLRQSUR¿OHVWKDWEHVW¿WWKHH[SHULPHQWDOPHDQSUREHLQWHQVLW\SUR¿OHVDOVRLQFOXGHGLQ¿JXUH

5.3.2

QUANTITATIVE FLUORESCENCE PROFILE (QFP) ANALYSIS OF CNS TISSUE ACCESS DEVICE (TAD) IMPLANT GROUPS

Included in Figure DUHÀXRUHVFHQWPLFURJUDSKVRIVHFWLRQVFROOHFWHGIURPWKH upper striatum region that are representative of the totality of those captured for the control (A to C) and experimental (D to F) groups. GFAP staining (Figure 5.6A,D) revealed that often the glial scar found in the cell-transplanted group was displaced DGLVWDQFHDZD\IURPWKH+)0ZKHUHDVWKDWRIFRQWUROVQRFHOOV DSSHDUHGDGMDFHQWWRWKHPHPEUDQHZLWKDVWURF\WHSURFHVVHVVRPHWLPHVSURMHFWHGLQWRWKHPDFrovoid spaces of the wall. ED1 staining (Figure 5.6B,E) revealed that the region EHWZHHQWKHPHPEUDQHDQGJOLDOVFDULQWKHFHOOWUDQVSODQWHGJURXSVHFWLRQVZDV GHQVHO\SRSXODWHGE\UHDFWLYHPLFURJOLDDQGPDFURSKDJHFHOOV7KLVLVLQFRQWUDVWWR the no-cell-transplant or control group sections, where ED1 staining was typically REVHUYHGRQO\LQQHDUSUR[LPLW\WRWKHP±WLQWHUIDFH 7KHGHJUHHRIFHOOXODULW\EDVHGRQ'$3,QXFOHDUVWDLQLQJ)LJXUH$%'( IRXQGLQWKHPHPEUDQHZDOOUHJLRQZDVDOVRVLJQL¿FDQWO\GLIIHUHQWEHWZHHQJURXSV with a greater population of cells found residing in experimental group secWLRQV$QRWKHUGLIIHUHQFHREVHUYHGEHWZHHQWKHJURXSVUHODWHGWRWKHLQWHQVLW\RI


132


FIGURE 5.6 5HSUHVHQWDWLYH ÀXRUHVFHQW PLFURJUDSKV RI XSSHU VWULDWXP LPDJHV FDSWXUHG IURPFRQWURO$WR& DQGH[SHULPHQWDO'WR) JURXSEUDLQVHFWLRQV7KHPLFURJUDSKVDUH FRPSRVHGRI$DQG' '$3,EOXH DQG*)$3JUHHQ &DQG( '$3,DQG('JUHHQ DQG & DQG ) N'D 75GH[WUDQ UHG SUREH GLVWULEXWLRQV (See color insert following page 110.)

75GH[WUDQSUREHFigure &) IRXQGLQWKHWLVVXHUHJLRQLPPHGLDWHO\DGMDFHQW WRWKH+)0,QJHQHUDOWKHSUREHGLVWULEXWLRQLQH[SHULPHQWDO VHFWLRQVDSSHDUHG more diffuse than that found in control sections. 0HDQLQWHQVLW\SUR¿OHVHYDOXDWHGEDVHGRQWKHHQWLUHVHWRIFRPSRVLWH4)3SUR¿OHVFROOHFWHGIRUWKHXSSHUVWULDWXPUHJLRQDSSHDULQFigure 5.7, where results for the no-cell control (A) and cell-transplanted or experimental (B) groups are displayed in VHSDUDWHJUDSKV7KHPSRVLWLRQRQWKH[D[LVLQWKHVHJUDSKVUHSUHVHQWVWKHP±W LQWHUIDFHZLWKQHJDWLYHYDOXHVUHÀHFWLQJGDWDZLWKLQWKHPDFURYRLGUHJLRQVRIWKH +)0ZDOO:KHQ*)$3DQG('FRPSRVLWHSUR¿OHVLQGDWDVHWV$DQG%ZHUHQRUPDOL]HGWRWKHLUUHVSHFWLYHSHDNLQWHQVLW\YDOXHVWKHLUPHDQSUR¿OHSHDNVIHOOEHORZ WKHYDOXHRIXQLW\7KLVLVLQGLFDWLYHRIYDULDWLRQLQWKHVFDUULQJUHVSRQVHEHWZHHQ and around the circumference of sections. In contrast to the no-cells control group,



133

FIGURE 5.7 N'D75GH[WUDQ*)$3DQG('PHDQLQWHQVLW\SUR¿OHVDVVHVVHGIURP WKHHQWLUHGDWDVHWRIFRPSRVLWH4)3SUR¿OHVFROOHFWHGIRUWKHXSSHUVWULDWXPUHJLRQLQFRQWURO$ DQGH[SHULPHQWDO% VDPSOHJURXSV³QÝDOXHVGLVSOD\HGLQOHJHQGVUHSUHVHQWWKH QXPEHURIFRPSRVLWH4)3SUR¿OHVXWLOL]HGLQWKHFDOFXODWLRQRIPHDQLQWHQVLW\SUR¿OHV7KH 75GH[WUDQ SUR¿OH IRU WKH H[SHULPHQWDO JURXS ZDV DGGLWLRQDOO\ QRUPDOL]HG EDVHG RQ WKH SURSRUWLRQ RI SUREH SUHVHQW DW WKH P±W LQWHUIDFH UHODWLYH WR WKDW DVVHVVHG IRU WKH FRQWURO group.

variations in the scarring response were especially pronounced in the cell-transplant experimental group samples )LJXUH% ZKHUH FOHDUO\ GLVFHUQDEOH SHDNV DUHODFNLQJLQERWKPDUNHUSUR¿OHV,QVRPHH[SHULPHQWDOJURXSVHFWLRQVWKHVFDU FRPSRVLWLRQUHÀHFWHGWKDWSUHVHQWLQFRQWUROVDPSOHVZKLOHLQRWKHUVWKH('+ reacWLYHPLFURJOLDDQGPDFURSKDJHOD\HUKDGVLJQL¿FDQWO\LQ¿OWUDWHGPDFURYRLGUHJLRQV RIWKHPHPEUDQHDQGH[WHQGHGWHQVRIPLFURQVDZD\IURPWKHPHPEUDQH7KHRYHUODSRI*)$3DQG('SUR¿OHVZDVDOVRLQGLFDWLYHRIUDGLDOGH¿QHGVSDWLDOYDULDWLRQV LQWKHVFDUULQJUHVSRQVHREVHUYHGZLWKLQJURXSV$VVKRZQLQ)LJXUHWKHVKDSH RIWKH75GH[WUDQSUR¿OHVZDVFOHDUO\GLVWLQFWEHWZHHQWKHJURXSV7KHSUR¿OHIRU


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the experimental group was normali]HGEDVHGXSRQWKHSURSRUWLRQRISUREHSUHVHQW at the m–t interface relative to that assessed for the control. The applied normalL]DWLRQ WKHUHIRUH LQGLFDWHV WKDW WKH FRQFHQWUDWLRQ RI 75GH[WUDQ SUREH DW WKH P±W LQWHUIDFHIRUPHQLQJHDO¿EUREODVWLPSODQWVLHWKHH[SHULPHQWDOJURXS ZDVa that of the control group.

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DQDYHUDJHDSSDUHQWGLIIXVLRQFRHI¿FLHQWWRGHOLQHDWHVROXWHWUDQVSRUWWKURXJKWKH affected tissue region (i.e., reactive microglia and macrophage and astrocyte layHUV ,WVKRXOGEHQRWHGWKDWDOOVLPXODWHGGDWDSUR¿OHVSUHVHQWHGKHUHUHVXOWHGIURP VLPXODWLRQUXQVZKHUHWKHSUREHFRQFHQWUDWLRQLQWKHLQWUDOXPHQDOFKDPEHUZHUH DVVXPHGWREHFRQVWDQWLHLQ¿QLWHUHVHUYRLUPRGHO 7KH³EHVW¿W´75GH[WUDQSUR¿OHVIRUWKHH[WHQWVRIUHDFWLYHPLFURJOLDDQGPDFURSKDJHOD\HUVZHUHREWDLQHGIRUPDQGPIRUWKHFRQWURODQGH[SHULPHQWDO groups, respectively. Recalling that decreases in tortuosity represent increases in tissue GLIIXVLYLW\VHH(TXDWLRQ H[DPLQDWLRQVRIWKHWLVVXHWRUWXRVLW\YDOXHVREVHUYHG LQWKHUHDFWLYHPLFURJOLDDQGPDFURSKDJHOD\HUIRUERWKVWXG\JURXSVM = 1.76 versus IRUQDwYHWLVVXH VXJJHVWWKLVUHJLRQLVOHVVUHVLVWDQWWRN'DGH[WUDQGLIIXVLRQ 8 a a E\DIDFWRURI Dmicro 1.2 u107 cm2/s than Dnorm 7.5 u10 cm2V 8QOLNHWKH VLJQL¿FDQWFKDQJHLQWRUWXRVLW\LGHQWL¿HGLQWKLVUHJLRQWKHYRLGIUDFWLRQYDOXHVXVHG WRSURGXFHWKHVH¿WVGLGQRWGLIIHUIURPWKDWRIQDwYHWLVVXHZLWKB micro = B norm = 0.21. 7KHUDGLDOH[WHQWVRIWKHJOLDOVFDUOD\HUVGHOLQHDWHGLQEHVW¿WVLPXODWLRQUXQV ZHUHPDQGPIRUWKHFRQWURODQGH[SHULPHQWDOJURXSVUHVSHFWLYHO\7KH a values of Dscar for the control group ranged from 3.00 × 10 –8 to 7.32 × 10 –8 cm 2/s, the HTXLYDOHQWRIȜUDQJHGIURPaWRaDQGZHUHVSHFL¿HGE\OLQHDUO\LQFUHDVa LQJWKHDSSDUHQWGLIIXVLYLW\E\ × 10 –8 cm2/s per node. The values of Dscar for © 2008 by Taylor & Francis Group, LLC

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FIGURE 5.9 5HVXOWVDWWDLQHGIURP¿QLWHHOHPHQW&16±(&6VROXWHGLIIXVLRQVLPXODWLRQV $ N'D 75GH[WUDQ QRUPDOL]HG PHDQ LQWHQVLW\ SUR¿OH IRU WKH XSSHU VWULDWXP FRQWURO JURXS VXEVHW DORQJ ZLWK D EHVW ¿W PLQXWH VLPXODWHG SUREH GLVWULEXWLRQ SUR¿OH DWWDLQHG IURP VLPXODWLRQ DQDO\VLV 7KH VLPXODWHG SUREH GLVWULEXWLRQ SUR¿OH ZDV QRUPDOL]HG EDVHG on the value at the m–t interface (r P DWWKHFRQFOXVLRQRIWKHPLQXWHVLPXODWLRQ UXQ % *)$3 DQG (' PHDQ LQWHQVLW\ SUR¿OHV IRU WKH FRQWURO JURXS VXEVHW DORQJ ZLWK &16±(&6WRUWXRVLW\SUR¿OHVHYDOXDWHGEDVHGRQDSSDUHQWGLIIXVLRQFRHI¿FLHQWVXWLOL]HGLQ WKHVLPXODWLRQUXQWKDWSURGXFHGWKHSUR¿OHWKDWEHVW¿WWKHREVHUYHGGH[WUDQSUREHGLVWULEXWLRQ³QÝDOXHVGLVSOD\HGLQOHJHQGVUHSUHVHQWWKHQXPEHURIFRPSRVLWH4)3SUR¿OHVXWLOL]HG LQWKHFDOFXODWLRQRIPHDQLQWHQVLW\SUR¿OHV



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WKH H[SHULPHQWDO JURXS ZHUH VSHFL¿HG LQ D VLPLODUO\ OLQHDUO\ LQFUHDVLQJ PDQQHU and ranged from 3.50 × 10–8 to 7.21 × 10 –8 cm 2VWKHHTXLYDOHQWRIOUDQJLQJIURP aWRaYDULHGE\a× 10 –8 cm 2/s per node. These results suggest that in FRQWURODQGH[SHULPHQWDOJURXSVWKHOHDGLQJERUGHURIWKHJOLDOVFDULVPRUHUHVLVWDQWWRN'DGH[WUDQGLIIXVLRQE\IDFWRUVRIDQGUHVSHFWLYHO\)RUERWK VWXG\JURXSVDYRLGIUDFWLRQYDOXHRIĮ scar ZDVXVHGWRSURGXFHEHVW¿WSUREH GLVWULEXWLRQSUR¿OHV 7KHUHVXOWVRIEHVW¿WVLPXODWLRQVDWWDLQHGIRUERWKVWXG\JURXSVDOVRLQGLFDWHDQ a DSSDUHQWGLIIXVLYLW\YDOXHIRUWKHXQDIIHFWHGEUDLQWLVVXHUHJLRQRI Dnorm 7.5 u 108 2 cm VDQGDYRLGIUDFWLRQYDOXHRIĮ norm 7KHVHYDOXHVDUHHTXDOWRWKRVHLQLWLDOO\ XWLOL]HG IRU VLPXODWLRQ DQDO\VHV VHOHFWHG EDVHG RQ SUHYLRXVO\ FLWHG VWXGLHV )RUWKLVUHDVRQDVHULHVRIVHYHQVLPXODWLRQVIRUERWKVWXG\JURXSVZHUHSHUIRUPHG a ranged from 7.2 × 10 –8 to 7.8 × 10 –8 cm 2/s. In a similar where the value of Dnorm PDQQHUDVHULHVRIVHYHQVLPXODWLRQVZHUHSHUIRUPHGLQZKLFKWKHYDOXHRIĮ norm ranged from 0.19 to 0.23. None of the results attained from these sets of simulations SURGXFHGDQLPSURYHG¿WWRWKHH[SHULPHQWDOO\GHULYHG75GH[WUDQPHDQLQWHQVLW\ SUR¿OHVGDWDQRWVKRZQ 7KHEODFNER[¿WUHVXOWVVKRZQIRUWKHH[SHULPHQWDOJURup (Figure 5.10) treats WKH DIIHFWHG WLVVXH UHJLRQ DV D VLQJOH HQWLW\ ³PLFUR VFDU´ ZLWK WRUWXRVLW\ DQG YRLG IUDFWLRQ YDOXHV FRQVLGHUHG WR EH FRQVWDQW 7KH VLPXODWHG SUREH GLVWULEXWLRQ a SUR¿OHVKRZQIRUWKLV¿WZDVDWWDLQHGXVLQJDUDGLDOH[WHQWRIPZLWK Dmicro scar –8 2 7.08 × 10 cm VWKHHTXLYDOHQWRIȜ§DQGĮ micro+scar = 0.21. Based upon this result, the affected tissue region for the experimental group, overall, is slightly more UHVLVWLYHWR WKH GLIIXVLRQ RI N'D GH[WUDQ WKDQ XQDIIHFWHG WLVVXH E\ D IDFWRU RI 1.06.

5.4

DISCUSSION

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139

ORZFRQFHQWUDWLRQVLWLVDEOHWRSURYLGHDUHODWLYHO\¿UPVXEVWUDWHDWURRPWHPSHUDWXUH+RZHYHUHPSOR\LQJWKHWHFKQLTXHUHTXLUHVSUREHLPPRELOL]DWLRQDWWKHFRQFOXVLRQRIWHVWUXQV6LQFHDJDURVHLVQRWDSURWHLQDFHRXVVXEVWUDWHLWZDVQHFHVVDU\ WRLQFRUSRUDWHJHODWLQLQWRWKHJHOPL[WXUHWRHQDEOHFRQMXJDWLRQRIWKHSUREHWRD VWUXFWXUDOFRPSRQHQWRIWKHPHGLXP)RUDVLPLODUUHDVRQWKH$OH[DODEHOHGVWUHSWDYLGLQZDVXVHGLQOLHXRIWKHO\VLQH¿[DEOHGH[WUDQWRPD[LPL]HWKHSUREHPHGLXP LPPRELOL]DWLRQSRWHQWLDORIWKHV\VWHP The use of in vitro gel diffusion characterization experiments to validate in vivo LQYHVWLJDWLRQLVZHOOHVWDEOLVKHGLQWKLVDUHDRIUHVHDUFK/HYLQHWDO>@XWLOL]HG UDGLRDFWLYLW\PHDVXUHPHQWVRIVHULDODJDUVHFWLRQVWRYDOLGDWHWKHGLIIXVLRQFRHI¿FLHQWVRIVROXWHVLQEUDLQWLVVXHIURPWKHFRQFHQWUDWLRQVRISUREHHYDOXDWHGDWWKHHQG RIGLIIXVLRQH[SHULPHQWV%ODVEHUJHWDOXVHGWKHGLIIXVLRQRIUDGLRODEHOHGSUREHV LQDJDUJHOPHGLXPWRHVWLPDWHIUHHPHGLXPLHSXUHZDWHU GLIIXVLRQFRHI¿FLHQWV for calculation of ECS tortuosity values [27]. Nicholson and Phillips [29] used the TMA+PLFURHOHFWURGH PHWKRG LQ DJDU JHOV WR YDOLGDWH WKH WHFKQLTXH DQG PRGHOV employed to investigate solute diffusion in the CNS–ECS. Agarose diffusion experLPHQWVKDYHDOVREHHQHPSOR\HGWRYDOLGDWHWKH,2,H[SHULPHQWDOPHWKRGXVHGWR LQYHVWLJDWHN'DGH[WUDQ>@DQGDOEXPLQ>@GLIIXVLYLW\LQWKH&16±(&6/DVW WKH1LFKROVRQJURXSHPSOR\HGDJDUJHOPHGLXPWRYDOLGDWHWKHLUTXDQWLWDWLYHGXDO SUREHPLFURGLDO\VLVWHFKQLTXH>@ The gel studiHVFRQ¿UPHGDQXPEHURIFULWLFDOH[SHULPHQWDODVVXPSWLRQV)LUVW LWZDVQHFHVVDU\WRHVWDEOLVKWKDW+)0EDVHGGHOLYHU\RIVROXWHSUREHWRDQDGMDFHQW PHGLXP ZDV DWWULEXWDEOH WR GLIIXVLYH DQG QRW FRQYHFWLYHGULYHQ WUDQVSRUW SURFHVVHV 6HFRQG ZH YDOLGDWHG WKDW ÀXRUHVFHQW PLFURVFRS\ LPDJLQJ RI VDPSOH VHFWLRQVSURYLGHGDQDGHTXDWHIRXQGDWLRQIRUH[WUDFWLQJVROXWHSUREHGLVWULEXWLRQ SUR¿OHVIRU4)3DQDO\VLV7KLUGOLJKW¿HOGFRUUHFWLRQDQGEDFNJURXQGVXEWUDFWLRQ SURFHVVLQJRIUDZLQWHQVLW\SL[HOLPDJHVIRUGHWHUPLQLQJUHODWLYHÀXRUHVFHQWLQWHQVLW\KDGWREHDVVHVVHG)RXUWKDOLQHDUFRUUHODWLRQEHWZHHQUHODWLYHÀXRUHVFHQFH LQWHQVLW\DQGVROXWHSUREHFRQFHQWUDWLRQKDGWREHHVWDEOLVKHGVRWKDW4)3DQDO\VLV FRXOGEHHPSOR\HGIRUDVVHVVLQJUDGLDOSUREHGLVWULEXWLRQVIURPFDSWXUHGLPDJHV /DVW ZH KDG WR GHPRQVWUDWH WKDW WKH ¿QLWH HOHPHQW PRGHO HPSOR\HG KHUH ZRXOG UHOLDEO\LQWHUSUHWVROXWHGLIIXVLYLW\SURSHUWLHVEDVHGRQUHSOLFDWLRQRIH[WUDFWHGVDPSOHSUREHGLVWULEXWLRQSUR¿OHV ,WLVRXUEHOLHIWKDWWKHUHVXOWVSUHVHQWHGKHUHIRUWKHJHOGLIIXVLYLW\VWXG\YDOLGDWH WKHUREXVWQHVVRIWKHPHWKRGRORJ\HPSOR\HGLQSUHYLRXVO\GHVFULEHGVWXGLHV>@ 7KHUDWLRQDOHIRUWKLVFRQFOXVLRQVWHPVIURPH[DPLQDWLRQRIWKHTXDOLW\RIWKHFXUYH ¿WVEHWZHHQDFWXDODQGVLPXODWHGDQGPLQXWHSUREHGLVWULEXWLRQSUR¿OHVDQG WKHHVWLPDWHGHIIHFWLYHGLIIXVLRQFRHI¿FLHQW\LHOGHGWKURXJKWKHPHWKRGVDSSOLHGLQ the example cited here (see Figure 5HFDOOWKDWWKHWKHRUHWLFDOHTXDWLRQIRUSURtein diffusion yields a value of Dw = 7.54 × 10 –7 cm 2/s as a rough approximation of the diffusivity of streptavidin in free medium. Based on the results of our example, ZHHVWLPDWHGDQHIIHFWLYHIUHHGLIIXVLRQFRHI¿FLHQWRIDeff = 6.78 × 10 –7 cm 2/s for VWUHSWDYLGLQLQRXUJHOV\VWHP7KLVZRXOGVHHPWREHUHDVRQDEOHVLQFHRXUYDOXH RQO\GLIIHUVIURPWKHWKHRUHWLFDODSSUR[LPDWLRQE\a7KHIDFWWKDWERWKDQG PLQXWHVLPXODWLRQVSURGXFHWKHVDPHHVWLPDWHGYDOXHLPSOLHVWKHHI¿FDF\RIWKH


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DGHTXDWHO\LPPRELOL]LQJN'DGH[WUDQSUREHVLQWKH(&6VRIEUDLQWLVVXH>@ DVZHOODVQRQ&16WLVVXHV>@7KHGHFLVLRQWRXWLOL]HO\VLQH¿[DEOHN'D75 dextrans was, in part, made to further ensure prevention of postmortem diffusion of WKHGH[WUDQVROXWHSUREH)UHH]LQJWKHPRELOLW\RIWKHVROXWHLVHVSHFLDOO\FULWLFDOIRU WKLVLQYHVWLJDWLRQJLYHQWKHOHYHORIVSDWLDOUHVROXWLRQUHTXLUHGWRGLVWLQJXLVKPLQXWHFKDQJHVLQWKHGLIIXVLYLW\FKDUDFWHULVWLFVDFURVVDWKLQEDQGRIWLVVXH$VHFRQG UHDVRQIRUVHOHFWLQJDO\VLQH¿[DEOHSUREHZDVWRHQDEOHHYDOXDWLRQRIUHODWLYHSUREH FRQFHQWUDWLRQVLQWKHPHPEUDQHUHJLRQ%\XVLQJDO\VLQH¿[DEOHGH[WUDQLPPRELOL]DWLRQRQ3$139&PHPEUDQHVXUIDFHVLVDFFRPPRGDWHGWKURXJKFRQMXJDWLRQ ZLWKDGVRUEHGSURWHLQRQWKHPHPEUDQHVXUIDFH In the current method, a high degree of consistency is maintained in the meaVXUHPHQWRIWLVVXHVROXWHFRQFHQWUDWLRQGLVWULEXWLRQV$PRQJWKHZD\VZHDGGUHVVHG this issue was through implementing a strict set of guidelines regarding the capWXULQJRILPDJHVVXFKDVFRQVWDQWFDSWXUHWLPHVXWLOL]DWLRQRIWKHEOXHVSHFWUXP '$3, IRUDOLJQPHQWRIVHFWLRQVRQWKHPLFURVFRSHDQGE\OLPLWLQJH[SRVXUHVWR DYRLGEOHDFKLQJSKHQRPHQRQ,QDGGLWLRQQHZOLJKW¿HOGFDOLEUDWLRQLPDJHVZHUH DFTXLUHGDWWKHRQVHWRIHDFKPLFURVFRS\VHVVLRQ7KH4)3LPDJHDQDO\VLVVRIWZDUH SURJUDPXVHGWRHYDOXDWHVROXWHGLVWULEXWLRQSUR¿OHVZDVDOVRVSHFL¿FDOO\GHYHORSHG for the current study application. The software facilitates the referencing of origins of individual radial sampling arrays at the m–t interface, which is the most readily GLVWLQJXLVKDEOHIHDWXUHFRPPRQLQDOOVHFWLRQVDQDO\]HG7KHFLUFXPIHUHQWLDOVDPSOLQJGHQVLW\RIGHJUHHVSUR¿OHHIIHFWLYHO\SURYLGHVFRPSOHWHFDQYDVVLQJRIWKH DQDO\VLVUHJLRQJLYHQWKDWKLJKHUSUR¿OLQJGHQVLWLHVSURGXFHLQVLJQL¿FDQWFKDQJHV in composite point intensity standard deviation values. Our decision to apply diffusivity model analyses to the upper striatum region LV SULPDULO\ EDVHG RQ WKH UHDVRQDELOLW\ RI WKH DVVXPSWLRQ WKDW SUREH GLIfusion in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¿QGLQJV UHJDUGLQJ ORQJWHUP UHVROXWLRQ UHVSRQVHV WR LPSODQWHG GHYLFHV DQGPDWHULDOV>±±@7KHN'D75GH[WUDQSUR¿OHDWWDLQHG IRUWKHH[SHULPHQWDOJURXSVXEVHWFRQWDLQVDYDOOH\LQWKH('+ reactive microglia DQGPDFURSKDJHUHJLRQWKDWFDQQRWEHWKHSURGXFWRIVLPSOHVROXWHGLIIXVLRQDQG PD\UHSUHVHQWXSWDNHRIWKHVROXWHE\PDFURSKDJHV,WLVOLNHO\WKDWRQO\DSVHXGR matrix is present in this region since reactive microglia and macrophage cells are QRUPDOO\UHVSRQVLEOHIRUWKHUHPRYDORIFHOOXODUGHEULVDSURFHVVWKDWUHTXLUHVWKHVH FHOOV WR EUHDN GRZQ H[LVWLQJ FRPSRQHQWV RI WKH H[WUDFHOOXODU PDWUL[ WR IDFLOLWDWH WKHLUPLJUDWLRQWKURXJKDIIHFWHGWLVVXHV>@(YHQWKRXJKDOGHK\GH¿[DWLRQ ZDVDSSOLHGLWLVSRVVLEOHWKDWWLVVXHLQWKLVUHJLRQZDVORRVHO\DWWDFKHGSHUKDSV


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FRQWULEXWLQJWRWKHREVHUYHGIUHTXHQFLHVRIWLVVXHGHWDFKPHQWIURPWKHP±WLQWHUIDFH 8QGHU WKLV FLUFXPVWDQFH GXULQJ VHFWLRQLQJ RI EUDLQ VDPSOHV RU VWDLQLQJ RI VHFWLRQVLQGLVFHUQLEOH\HWVLJQL¿FDQWIUDJPHQWVRIWKHWLVVXHPD\KDYHGHWDFKHG DORQJZLWKERXQGGH[WUDQSUREH,IWKLVSKHQRPHQRQGLGRFFXUWKHQWKHLQWHQVLW\ RIWKH('SUR¿OHZRXOGDOVREHGHSUHVVHGLQWKLVUHJLRQ7KHDSSURDFKWDNHQWR PRGHOWKLVUHJLRQZDVEDVHGRQWKHDVVXPSWLRQWKDWWKH75GH[WUDQSUREHLQWHQVLWLHVIRXQGDWWKHP±WLQWHUIDFHDQGWKHSHDNWKDWOLHVDGMDFHQWWRWKHYDOOH\aP UHSUHVHQWHGUHOLDEOHYDOXHVIRUWKHFRQFHQWUDWLRQVRISUREHSUHVHQWLQWKHVHORFDWLRQV at the end of 30-minute diffusion runs. Based upon this two-point treatment of the data, it was only appropriate to apply a constant apparent diffusivity value for this region in simulation analyses. $VLPLODUWZRSRLQWWUHDWPHQWZDVDSSOLHGLQWKHGHWHUPLQDWLRQRIVROXWHSUREH HIIHFWLYH GLIIXVLYLW\ WKURXJK WKH PHPEUDQH ZKHUH LQWHQVLW\ YDOXHV SUHVHQW DW WKH LQWUDOXPHQDOVXUIDFHDQGP±WLQWHUIDFHZHUHXVHGWRDVVHVVWKH¿WRIVLPXODWHGGDWD This is clearly apparent in Figure 5.9 since it is at these radial positions (0 and 90 P WKDWWKHVLPXODWHGFXUYHLQWHUVHFWVWKHDFWXDO75GH[WUDQPHDQLQWHQVLW\SUR¿OH HYDOXDWHG IURP 4)3 DQDO\VHV 7KH UHVXOWV RI VLPXODWLRQV EDVHG RQ WKLV DSSURDFK VXJJHVWWKDWWKHGLIIXVLYLW\RIWKHPHPEUDQHWRZDUGVWKHN'DGH[WUDQGHFUHDVHG WRDSSUR[LPDWHO\RILWVQDwYHLHSUHLPSODQWDWLRQ GLIIXVLYLW\Dmemb = 0.9 × 10 –8 cm 2/s versus 6.0 × 10 –8 cm2V 7KLVVLJQL¿FDQWGHFOLQHREVHUYHGLQWKHSHUPHDELOLW\RIWKHPHPEUDQHLVOLNHO\SULPDULO\WKHUHVXOWRISURWHLQIRXOLQJRIWKHSRUHV FRPSULVLQJ WKH SHUPVHOHFWLYH VNLQ OD\HU DQG SHUKDSV WR D VPDOO GHJUHH RZLQJ WR FHOOXODULQ¿OWUDWLRQRIWKHPDFURYRLGUHJLRQVRIWKHPHPEUDQH 6HYHUDODVSHFWVRIWKHWLVVXHGLIIXVLYLW\UHVXOWVOHQGFUHGLELOLW\WRWKHWHFKQLTXHV that were employed. As depicted in the results presented in Figures 5.9A and 5.10A, the overall VLPLODULW\ RI DFWXDO DQG VLPXODWHG SUREH GLVWULEXWLRQ SUR¿OHV DWWDLQHG IRUERWKVWXG\JURXSVVXJJHVWWKHUREXVWQHVVRIWKH¿QLWHHOHPHQWPRGHOHPSOR\HG in this study. Additionally, the apparent diffusivity values derived from simulation analyses for the unaffected CNS tissue regions (rPDQGrPIRUWKH a control and experimental groups, respectively) were Dnorm = 7.5 × 10 –8 cm 2/s for ERWKVWXG\JURXSV$SSDUHQWGLIIXVLYLW\YDOXHVDWWDLQHGIRUWKHUHDFWLYHPLFURJOLD and macrophage and glial scar regions are also in agreement regarding the trends REVHUYHGLQWKHUHVXOWVIRUWKHVHUHJLRQV7KHUHVXOWVIRUWKLVUHJLRQZHUHLQDJUHHPHQWZLWKSUHYLRXVO\SXEOLVKHG¿QGLQJV>@DQGWKHVLPLODULW\RIWKH¿QGLQJVIRU WKHDIIHFWHGWLVVXHUHJLRQVWRJHWKHUVXJJHVWWKHUHOLDELOLW\RIWKHWLVVXHGLIIXVLYLW\ characterization methodology employed in the current study. Tortuosity (M), as related to CNS–ECS solute diffusion, is generally considHUHGE\LQYHVWLJDWRUVDVFRPSRVHGRI³JHRPHWULF´DQG³YLVFRVLW\´FRPSRQHQWV>@ :KLOHWKHJHRPHWULFFRPSRQHQWUHÀHFWVWKHLQFUHDVHGGLVWDQFHDVROXWHWUDYHOVDVLW circumnavigates nonlinear extracellular pathways, the viscosity component origiQDWHVIURPIULFWLRQDOIRUFHVLPSRVHGRQVROXWHVE\WKHSUHVHQFHRIODUJHPDWUL[SURWHLQV DQG RWKHU FHOOPHPEUDQHDVVRFLDWHG PRLHWLHV ,2,EDVHG VWXGLHV HPSOR\LQJ GH[WUDQVROXWHGLIIXVLRQSUREHVKDYHDOVRUHYHDOHGWKDW(&6WRUWXRVLW\LVGHSHQGHQW on solute molecular weight, with M values increasing from 1.77 to 2.25 as the MW LQFUHDVHV IURP WR N'D >@ ,I WKH JHRPHWULF FRPSRQHQW UHPDLQV UHODWLYHO\ FRQVLVWHQWIRUHDFKRIVROXWH0:VFKDUDFWHUL]HGDUHDVRQDEOHDVVXPSWLRQVLQFHWKH



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WLVVXHFHOOXODUFRPSRVLWLRQVDQGPRUSKRORJLHVGLGQRWYDU\EHWZHHQSUREHVWKHQ the results of the IOI study may suggest that the viscosity component is the primary determinant of overall tortuosity for larger MW solutes. Characterization of reactive gliosis from the perspective of macromolecule GLIIXVLYLW\ SURSHUWLHV KDV RQO\ UHFHQWO\ EHHQ H[DPLQHG >@ 7KLV VWXG\ DQG WKH UHVXOWVSUHVHQWHGKHUHVXJJHVWWKDWWKHSHUPHDELOLW\RIDJOLDOVFDULVPRVWUHVWULFWLYH at its leading edge (MYDOXHVH[FHHGLQJDQGRIQRUPDO DQGJUDGXDOO\ increases in the transition to unaffected tissue. The presence of a denser extracellular matrix architecture in this region, which would increase the viscosity component RIWRUWXRVLW\LVDFKDUDFWHULVWLFWKDWKDVSUHYLRXVO\EHHQDVVRFLDWHGZLWKDVWURJOLRVLV >@:HDOVRREVHUYHGDGHFUHDVHGB value (0.17 versus 0.21) in the glial scar UHJLRQIRUERWKJURXSV7KLVPD\EHWKHUHVXOWRIWKHPRUSKRORJ\RIFHOOVSUHVHQWLQ WKHJOLDOVFDUUHJLRQZKLFKLVJHQHUDOO\GHVFULEHGE\GHQVHO\SDFNHGUHDFWLYHDVWURcytes with hypertrophied and tightly interdigitating processes [43]. Important to the GHYHORSPHQWRI&16LPSODQWDWLRQGHYLFHVLVWKHHOXFLGDWLRQRIWKHVLJQL¿FDQFHRID persistent reactive microglia and macrophage region on the diffusivity properties. ,QWKLVUHVSHFWLWLVFOHDUEDVHGRQRXUUHVXOWVWKDWWKHH[WHQWRIWKHJOLDOVFDUGLVWLQJXLVKHGVROHO\RQWKHEDVLVRIGLIIXVLYLW\ZDVODUJHUZKHQWKLVSHUVLVWHQWUHDFWLYH OD\HUZDVSUHVHQWYHUVXVP 7R RXU NQRZOHGJH WKH GLIIXVLYLW\ SURSHUWLHV RI ('+-reactive microglia and PDFURSKDJHWLVVXHDUHFRPSOHWHO\XQNQRZQ2XUUHVXOWVVXJJHVWWKDWGLIIXVLYLW\RI WKHN'DGH[WUDQSUREHLQWKLVUHJLRQZDVVLJQL¿FDQWO\KLJKHUZLWKDQHVWLPDWHG a a WRUWXRVLW\RIaRIQRUPDO Dmicro = 1.2 × 10 –7 cm2/s versus Dnorm = 7.5 × 10 –8 2 cm V 6XUSULVLQJO\WKHYDOXHRIWKHYRLGYROXPHIUDFWLRQREVHUYHGLQWKLVUHJLRQ was similar to that of normal tissue (B 7KLV¿QGLQJVXJJHVWVDGHFUHDVHLQ the viscosity component of tortuosity that may indicate the presence of a less dense, loosely integrated extracellular matrix architecture in this region. It is also potenWLDOO\DWWULEXWDEOHWRGHFUHDVHVLQWKHJHRPHWULFFRPSRQHQWRIWRUWXRVLW\VLQFHWKH morphology of microglia and macrophage cells, and therefore extracellular spacial characteristics, vary from that of normal CNS tissue. 7KH PHWKRGRORJ\ GHVFULEHG KHUH LV XQLTXH FRPSDUHG WR PHWKRGV SUHYLRXVO\ used to examine tissue diffusivity properties. The TMA+-microelectrode and dualSUREH PLFURGLDO\VLV WHFKQLTXHV UHO\ XSRQ PHDVXUHPHQWV RI ORFDO SRLQW FRQFHQWUDtions versus time and are therefore inherently limited to the evaluation of average DSSDUHQWGLIIXVLYLWLHVDFURVVWKHEXONUHJLRQRYHUZKLFKWKHSUREHVVSDQ7KH,2,DQG PXOWLSKRWRQPLFURVFRS\WHFKQLTXHVDVVHVVFKDQJHVLQSUREHGLVWULEXWLRQVRYHUWLPH 7KHUHVROXWLRQRISUREHSUR¿OHVLQWKHVHPHWKRGVZDVVDFUL¿FHGLQOLHXRIHYDOXDWLQJ DWLPHVHULHVRIG\QDPLFGLVWULEXWLRQVIURPZKLFKRQO\DQDYHUDJHDSSDUHQWGLIIXVLYLW\RIDUHJLRQLVHVWLPDWHG:KLOHVLPLODUWRRXUWHFKQLTXHLQWKDWWKHGLVWULEXWLRQRI SUREHoccurs at a sinJOHWLPHSRLQWHIIRUWVWRHPSOR\WKHUDGLRODEHO±DXWRUDGLRJUDSK\WHFKQLTXHVWRDWWDLQSUR¿OHVRIVLPLODUUHVROXWLRQWRWKRVHDFKLHYHGLQWKLVVWXG\ KDYH\HWWREHUHSRUWHG%\XVLQJ4)3DQDO\VLVLQFRQMXQFWLRQZLWK¿QLWHHOHPHQW VLPXODWLRQVZHZHUHDEOHWRSURGXFHDWLVVXHGLIIXVLYLW\PDSSLQJIRUWKHUHJLRQRI interest. )LQDOO\ LW LV LQWHUHVWLQJ WR QRWH WKDW WKH EODFN ER[ ¿W VHH Figure 5.7), representative of an average apparent tissue diffusivity value evaluated for the entire


144


DIIHFWHGEUDLQWLVVXHUHJLRQVXJJHVWVWUDQVSRUWSURSHUWLHVFRPSDUDEOHWRXQDIIHFWHG a –8 cm 2/s versus D a –8 cm 2/s (in tissue regions with Dmicro scar = 7.08 × 10 norm = 7.5 × 10 WHUPVRIWRUWXRVLW\Ȝ§YHUVXV ,PSOLFLWLQWKLVUHVXOWLVWKDWFDUHVKRXOG EH WDNHQ LQ WKH LQWHUSUHWDWLRQ RI UHVXOWV REVHUYHG IURP GLIIXVLYLW\ PHDVXUHPHQWV GHULYHG IURP WHFKQLTXHV EDVHG RQ WKH WZRSRLQW PHWKRGRORJ\ 7KH FXUUHQW VWXG\ revealed opposite directions in tissue diffusivity properties of the compositionally GLVWLQFWEXWDGMDFHQWOD\HUVRIWLVVXHDFURVVDUHODWLYHO\VPDOOUHJLRQVSDQQLQJRQO\ aP

5.5

CONCLUSION

7KHUHVXOWVRIERWKin vitro gel diffusivity and in vivoEUDLQWLVVXHGLIIXVLYLW\VWXGies suggest the validity of the methodology employed to examine solute diffusion SURSHUWLHVLQWKH(&6RI&16WLVVXHV6ROXWHSUREHGLVWULEXWLRQVSUHVHQWLQWLVVXH DQGJHOVHFWLRQVFRXOGEHH[WUDFWHGWKURXJKTXDQWLWDWLYHÀXRUHVFHQFHSUR¿OLQJDQDO\VLV 7KH TXDOLW\ RI WKH VLPXODWHG ¿WV DWWDLQHG LQ FRPSDULVRQ WR H[WUDFWHG SUREH GLVWULEXWLRQSUR¿OHVDOVRVXSSRUWVWKHXWLOLW\RIWKH¿QLWHHOHPHQWPRGHOHPSOR\HG to derive tissue diffusivity properties. Based on the methodology employed, for DN'DGH[WUDQVROXWHWKHGLIIXVLYLW\RIWKHUHDFWLYHPLFURJOLDDQGPDFURSKDJH OD\HULVVLJQL¿FDQWO\KLJKHUWKDQQRUPDOWLVVXHZKLOHWKDWRIWKHJOLDOVFDUUHJLRQLV VLJQL¿FDQWO\ORZHU 7RGDWHDQXPEHURIFOLQLFDOVWXGLHVKDYHGLVFXVVHGWKHDGYDQWDJHVRIDXWRORJRXV ¿EUREODVWV IRU VLWHVSHFL¿F DQG VXVWDLQHG GHOLYHU\ ZKLFK FDQ EH HDVLO\ FROOHFWHG IURP WKH DIIHFWHG SDWLHQW¶V RZQ VNLQ RYHU RWKHU W\SHV RI FHOOV LQ WHUPV RI delivering therapeutic molecules into target CNS tissue. These results suggest that HYHQWKRXJKWKHDPRXQWVRIWKHUDSHXWLFPROHFXOHVPD\EHUHGXFHGE\DQHQKDQFHG IRUHLJQERG\UHVSRQVHWR¿EUREODVWVLPSODQWHGLQEUDLQWLVVXHWKHUDSHXWLFPROHFXOHV EHORZN'DFDQVWLOOGLIIXVHLQWRDGMDFHQWWDUJHW&16WLVVXH:LWKWKHDGYDQFHVLQ JHQHWLFHQJLQHHULQJZHPD\EHDEOHWRVXSSUHVVWKHUHOHDVHRIQDWXUDOO\SURGXFHG SUR¿EURJHQLFF\WRNLQHVWKXVUHGXFLQJWKHOHYHORIWKHIRUHLJQERG\UHVSRQVHWRWKH WUDQVSODQWHGFHOOVDQGLPSURYHWKHHI¿FDF\RIWKHUDSHXWLFPROHFXOHGHOLYHU\XVLQJ VXFKWUDQVSODQWHGFHOOW\SHV$OWHUQDWLYHO\LWPD\EHZLVHWRLGHQWLI\FHOOW\SHVWKDW produce less of an encapsulation response for use as sustained-release vehicles in DGXOWEUDLQWLVVXH

ABBREVIATIONS M CNS Dw Da DAPI ECS (' *)$3 +)0

Tortuosity Parameter Central nervous system 'LIIXVLRQFRHI¿FLHQWLQZDWHU $SSDUHQWGLIIXVLRQFRHI¿FLHQW Nuclear dye 4’,6-diamidino-2-phenylindole, dihydrochloride Extracellular space $QWLJHQLFPDUNHUWKDWGLVFULPLQDWHVPDFURSKDJHVIURPPLFURJOLD *OLDO¿EULOODU\DFLGLFSURWHLQ +ROORZ¿EHUPHPEUDQH



,60 PAN-PVC 3%6 4)3 TAD TMA+ TR

145

,RQVHOHFWLYHPHPEUDQH Poly(acrylonitrile-vinyl chloride) 3KRVSKDWHEXIIHUHGVDOLQH 4XDQWLWDWLYHÀXRUHVFHQFHSUR¿OH Tissue access device Tetramethyl ammonium Texas Red

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