Ocular Periphery and Disorders

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OCULAR PERIPHERY AND DISORDERS

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OCULAR PERIPHERY AND DISORDERS EDITORS

DARLENE A. DARTT Harold F. Johnson Research Scholar Schepens Eye Research Institute Harvard Medical School Boston, MA USA

REZA DANA Massachusetts Eye and Ear Infirmary Schepens Eye Research Institute Harvard Medical School Boston, MA USA

PETER BEX Schepens Eye Research Institute Harvard Medical School Boston, MA USA

LINDA K. MCLOON Department of Ophthalmology University of Minnesota Minneapolis, Minnesota USA

PATRICIA D’AMORE Schepens Eye Research Institute Harvard Medical School Boston, MA USA

JERRY Y. NIEDERKORN UT Southwestern Medical Center at Dallas Dallas, Texas USA

Amsterdam • Boston • Heidelberg • London • New York • Oxford Paris • San Diego • San Francisco • Singapore • Sydney • Tokyo Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK Copyright # 2011 Elsevier Ltd. All rights reserved Material in the text originally appeared in the Encyclopedia of the Eye, edited by Darlene A. Dartt, Joseph C. Beshare and Reza Dana (Elsevier Ltd. 2010) No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at (http://elsevier.com/locate/permissions), and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-382042-6 For information on all Academic Press publications visit our website at www.elsevierdirect.com Printed and bound in Spain 11 12 13

10 9 8 7 6 5 4 3 2 1

CONTENTS

Contributors Introduction

I.

ix–xii xiii–xiv

EXTRAOCULAR AND EYELID MUSCLES: STRUCTURE, FUNCTION, AND PATHOPHYSIOLOGY

Eyelid Anatomy and the Pathophysiology of Blinking

C Evinger

Differentiation and Morphogenesis of Extraocular Muscles Extraocular Muscles: Extraocular Muscle Anatomy

3

D M Noden

L K McLoon and S P Christiansen

Extraocular Muscles: Extraocular Muscle Metabolism Extraocular Muscles: Proprioception and Proprioceptors

9 17

F H Andrade

27

R Blumer

33

Abnormal Eye Movements due to Disease of the Extraocular Muscles and Their Innervation A Serra and R J Leigh

39

Extraocular Muscles: Functional Assessment in the Clinic

45

II.

S P Christiansen and L K McLoon

STRUCTURE AND FUNCTION OF THE TEAR FILM, OCULAR ADNEXA, CORNEA AND CONJUNCTIVA IN HEALTH AND PATHOGENESIS IN DISEASE

Tear Film

J P Craig, A Tomlinson, and L McCann

Meibomian Glands and Lipid Layer Lacrimal Gland Overview

51

T J Millar, P Mudgil, and S Khanal

60

M C Edman, R R Marchelletta, and S F Hamm-Alvarez

68

Lacrimal Gland Hormone Regulation Lacrimal Gland Signaling: Neural

A K Mircheff, D W Warren, and J E Schechter D Zoukhri

83

Lids: Anatomy, Pathophysiology, Mucocutaneous Junction

T Wojno

Overview of Electrolyte and Fluid Transport Across the Conjunctiva Conjunctival Goblet Cells

74

R R Hodges and D A Dartt

O A Candia and L J Alvarez

91 99 108

Ocular Mucins

M Berry

116

Tear Drainage

F P Paulsen and L Braüer

126

P Asbell and D Brocks

133

Cornea Overview

Corneal Epithelium: Cell Biology and Basic Science

M A Stepp

143

v

vi

Contents

Corneal Nerves: Anatomy Corneal Nerves: Function

C F Marfurt C Belmonte

150 158

Corneal Epithelium: Wound Healing Junctions, Attachment to Stroma Receptors, Matrix Metalloproteinases, Intracellular Communications G M Gordon and M E Fini

164

Corneal Epithelium: Transport and Permeability

171

Stem Cells of the Ocular Surface

P S Reinach, F Zhang, and J E Capo´-Aponte

Y Du and J L Funderburgh

178

The Surgical Treatment for Corneal Epithelial Stem Cell Deficiency, Corneal Epithelial Defect, and Peripheral Corneal Ulcer N Koizumi and S Kinoshita

185

Refractive Surgery

193

S Marcos, L Llorente, C Dorronsoro, and J Merayo-Lloves

Refractive Surgery and Inlays Contact Lenses

R M M A Nuijts, M Doors, N G Tahzib, and L P J Cruysberg

N Carnt, Y Wu, and F Stapleton

Imaging of the Cornea

201 207

S C Kaufman, M Fung, D Raja, and N Kramarevsky

213

The Corneal Stroma

J L Funderburgh

219

Corneal Dystrophies

B H Feldman and N A Afshari

226

Corneal Imaging: Clinical Corneal Scars

S Garg and R F Steinert

D G Dawson

Corneal Endothelium: Overview

256 D R Whikehart

Regulation of Corneal Endothelial Function

272

J A Bonanno and S P Srinivas

Regulation of Corneal Endothelial Cell Proliferation Artificial Cornea

234

283

Q Lu, T A Fuchsluger, and U V Jurkunas

M A Rafat, J M Hackett, P Fagerholm, and M Griffith

290 296

Drug Delivery to Cornea and Conjunctiva: Esterase- and Protease-Directed Prodrug Design R S Talluri, S Hariharan, P K Karla, and A K Mitra

303

Knock-Out Mice Models: Cornea, Conjunctiva, Eyelids and Lacrimal Gland C-Y Liu, and H Liu

315

Gene Therapy for the Cornea, Conjunctiva, and Lacrimal Gland C Siddappa, and R R Mohan

III.

W W-Y Kao,

A Sharma, A Ghosh, 327

IMMUNE REGULATION OF THE CORNEA AND CONJUNCTIVA AND ITS DYSREGULATION IN DISEASE

Adaptive Immune System and the Eye: Mucosal Immunity

A K Mircheff

Adaptive Immune System and the Eye: T Cell-Mediated Immunity R D Vicetti Miguel Innate Immune System and the Eye

339

K C McKenna and 347

M S Gregory

354

Dynamic Immunoregulatory Processes that Sustain Immune Privilege in the Eye

J Y Niederkorn

361

Immunosuppressive and Anti-Inflammatory Molecules that Maintain Immune Privilege of the Eye A W Taylor

367

Antigen-Presenting Cells in the Eye and Ocular Surface

373

Dry Eye: An Immune-Based Inflammation Penetrating Keratoplasty

P Hamrah and R Dana

M E Stern and S C Pflugfelder

T H Flynn and D F P Larkin

Immunopathogenesis of HSV Keratitis

390

K Buela, G Frank, J Knicklebein, and R Hendricks

Immunopathogenesis of Onchocerciasis (River Blindness)

381

E Pearlman and K Gentil

396 401

Contents

Immunopathogenesis of Pseudomonas Keratitis L D Hazlett Immunobiology of Acanthamoeba Keratitis J Y Niederkorn Molecular and Cellular Mechanisms in Allergic Conjunctivitis

vii

406 413 V L Calder

419

Pathogenesis of Fungal Keratitis E Pearlman, S Leal, A Tarabishy, Y Sun, L Szczotka-Flynn, Y Imamura, P Mukherjee, J Chandra, M Momany, S Hastings-Cowden, and M Ghannoum

426

Conjunctiva Immune Surveillance

431

E Knop and N Knop

Defense Mechanisms of Tears and Ocular Surface Corneal Epithelium: Response to Infection

A M McDermott

Elizabeth A Szliter-Berger and L D Hazlett

444 452

Inflammation of the Conjunctiva

T Nishida

459

Concept of Angiogenic Privilege

B Regenfuss and C Cursiefen

465

Corneal Angiogenesis

M S Cortina and D T Azar

Avascularity of the Cornea

IV.

R J C Albuquerque and J Ambati

470 478

VISUAL ACUITY RELATED TO THE CORNEA AND ITS DISORDERS

Pupil

P D R Gamlin and D H McDougal

Acuity

M D Crossland

Contrast Sensitivity Astigmatism Myopia

P Bex

487 494 500

M J Cox

506

F A Vera-Diaz

517

Amblyopia

D M Levi

525

Hyperopia

E Harb

529

Index

535


CONTRIBUTORS

N A Afshari Duke University Medical Center, Durham, NC, USA

V L Calder UCL Institute of Ophthalmology, London, UK

R J C Albuquerque University of Kentucky, Lexington, KY, USA

O A Candia Mount Sinai School of Medicine, New York, NY, USA

L J Alvarez Mount Sinai School of Medicine, New York, NY, USA

J E Capo´-Aponte U.S. Army Aeromedical Research Laboratory (USAARL), Fort Rucker, AL, USA

J Ambati University of Kentucky, Lexington, KY, USA

N Carnt Institute for Eye Research, Sydney, NSW, Australia

F H Andrade University of Kentucky Medical Center, Lexington, KY, USA

J Chandra Case Western Reserve University, Cleveland, OH, USA

P Asbell Mount Sinai Hospital, Department of Ophthalmology, New York, NY, USA D T Azar University of Illinois at Chicago, Chicago, IL, USA C Belmonte Instituto de Neurociencias de Alicante, Universidad Miguel Hernańdez-Consejo Superior de Investigaciones Cientı´ficas, San Juan de Alicante, Spain M Berry Bristol Eye Hospital, Bristol, UK P Bex Schepens Eye Research Institute, Boston, MA, USA R Blumer Medical University of Vienna, Vienna, Austria J A Bonanno Indiana University, Bloomington, IN, USA D Brocks Mount Sinai Hospital, Department of Ophthalmology, New York, NY, USA L Braüer Martin Luther University Halle-Wittenberg, Halle, Germany K Buela University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

S P Christiansen Boston University School of Medicine, Boston, MA, USA M S Cortina University of Illinois at Chicago, Chicago, IL, USA M J Cox University of Bradford, Bradford, UK J P Craig University of Auckland, Auckland, New Zealand M D Crossland UCL Institute of Ophthalmology/Moorfields Eye Hospital, London, UK L P J Cruysberg University Hospital Maastricht, Maastricht, The Netherlands C Cursiefen Friedrich-Alexander University Erlangen-Nuernberg, Erlangen, Germany R Dana Harvard Medical School, Boston, MA, USA D A Dartt Schepens Eye Research Institute, Boston, MA, USA D G Dawson Emory University School of Medicine, Atlanta, GA, USA M Doors University Hospital Maastricht, Maastricht, The Netherlands

ix

x

Contributors

C Dorronsoro Consejo Superior de Investigaciones Cientı´ficas, Madrid, Spain

S F Hamm-Alvarez University of Southern California School of Pharmacy, Los Angeles, CA, USA

Y Du University of Pittsburgh, Pittsburgh, PA, USA

P Hamrah Harvard Medical School, Boston, MA, USA

M C Edman University of Southern California School of Pharmacy, Los Angeles, CA, USA

E Harb New England College of Optometry, Boston, MA, USA

C Evinger SUNY Stony Brook, Stony Brook, NY, USA

S Hariharan University of Missouri–Kansas City, Kansas City, MO, USA

P Fagerholm Linko¨ping University Hospital, Linko¨ping, Sweden B H Feldman Philadelphia Eye Associates, Philadelphia, PA, USA M E Fini University of Southern California, Los Angeles, CA, USA

S Hastings-Cowden University of Athens, Athens, GA, USA L D Hazlett Wayne State University School of Medicine, Detroit, MI, USA

T H Flynn Moorfields Eye Hospital, London, UK

R Hendricks University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

G Frank University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

R R Hodges Schepens Eye Research Institute, Boston, MA, USA

T A Fuchsluger Schepens Eye Research Institute, Boston, MA, USA J L Funderburgh University of Pittsburgh, Pittsburgh, PA, USA

Y Imamura Case Western Reserve University, Cleveland, OH, USA U V Jurkunas Schepens Eye Research Institute, Boston, MA, USA

M Fung University of Minnesota, Minneapolis, MN, USA

W W-Y Kao University of Cincinnati, Cincinnati, OH, USA

P D R Gamlin University of Alabama at Birmingham, Birmingham, AL, USA

P K Karla University of Missouri–Kansas City, Kansas City, MO, USA

S Garg University of California, Irvine, Irvine, CA, USA

S C Kaufman University of Minnesota, Minneapolis, MN, USA

K Gentil University of Bonn, Bonn, Germany

S Khanal University of Western Sydney, NSW, Australia

M Ghannoum Case Western Reserve University, Cleveland, OH, USA

S Kinoshita Kyoto Prefectural University of Medicine, Kyoto, Japan

A Ghosh University of Missouri–Columbia, Columbia, MO, USA G M Gordon University of Southern California, Los Angeles, CA, USA M S Gregory Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA

J Knicklebein University of Pittsburgh School of Medicine, Pittsburgh, PA, USA E Knop Charite´ – Universita¨tsmedizin Berlin, Berlin, Germany N Knop Hannover Medical School, Hannover, Germany

M Griffith University of Ottawa Eye Institute, Ottawa, ON, Canada

N Koizumi Kyoto Prefectural University of Medicine, Kyoto, Japan

J M Hackett University of Ottawa, Ottawa, ON, Canada

N Kramarevsky University of Minnesota, Minneapolis, MN, USA

Contributors D F P Larkin Moorfields Eye Hospital, London, UK

R R Mohan University of Missouri–Columbia, Columbia, MO, USA

S Leal Case Western Reserve University, Cleveland, OH, USA

M Momany University of Athens, Athens, GA, USA

R J Leigh Case Western University, Cleveland, OH, USA D M Levi University of California, Berkeley, Berkeley, CA, USA C-Y Liu University of Cincinnati, Cincinnati, OH, USA H Liu University of Cincinnati, Cincinnati, OH, USA L Llorente Consejo Superior de Investigaciones Cientı´ficas, Madrid, Spain Q Lu Schepens Eye Research Institute, Boston, MA, USA R R Marchelletta University of Southern California School of Pharmacy, Los Angeles, CA, USA S Marcos Consejo Superior de Investigaciones Cientı´ficas, Madrid, Spain C F Marfurt Indiana University School of Medicine – Northwest, Gary, IN, USA L McCann Glasgow Caledonian University, Glasgow, UK A M McDermott University of Houston, Houston, TX, USA D H McDougal University of Alabama at Birmingham, Birmingham, AL, USA K C McKenna University of Pittsburgh, Pittsburgh, PA, USA L K McLoon University of Minnesota, Minneapolis, MN, USA J Merayo-Lloves Consejo Superior de Investigaciones Cientı´ficas, Madrid, Spain T J Millar University of Western Sydney, NSW, Australia A K Mircheff University of Southern California, Los Angeles, CA, USA A K Mitra University of Missouri–Kansas City, Kansas City, MO, USA

xi

P Mudgil University of Western Sydney, NSW, Australia P Mukherjee Case Western Reserve University, Cleveland, OH, USA J Y Niederkorn University of Texas Southwestern Medical Center, Dallas, TX, USA T Nishida Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan D M Noden Cornell University, Ithaca, NY, USA R M M A Nuijts University Hospital Maastricht, Maastricht, The Netherlands F P Paulsen Martin Luther University Halle-Wittenberg, Halle, Germany E Pearlman Case Western Reserve University, Cleveland, OH, USA S C Pflugfelder Baylor College of Medicine, Houston, TX, USA M A Rafat University of Ottawa Eye Institute, Ottawa, ON, Canada D Raja University of Minnesota, Minneapolis, MN, USA B Regenfuss Friedrich-Alexander University Erlangen-Nuernberg, Erlangen, Germany P S Reinach The State University of New York, New York, NY, USA J E Schechter University of Southern California, Los Angeles, CA, USA A Serra University of Sassari, Sassari, Italy A Sharma University of Missouri–Columbia, Columbia, MO, USA C Siddappa University of Missouri–Columbia, Columbia, MO, USA S P Srinivas Indiana University, Bloomington, IN, USA F Stapleton University of New South Wales, Sydney, NSW, Australia

xii

Contributors

R F Steinert University of California, Irvine, Irvine, CA, USA

A W Taylor Schepens Eye Research Institute, Boston, MA, USA

M A Stepp The George Washington University Medical Center, Washington, DC, USA

A Tomlinson Glasgow Caledonian University, Glasgow, UK

M E Stern Allergan Inc, Irvine, CA, USA

F A Vera-Diaz Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA

Y Sun Case Western Reserve University, Cleveland, OH, USA

R D Vicetti Miguel University of Pittsburgh, Pittsburgh, PA, USA

L Szczotka-Flynn Case Western Reserve University, Cleveland, OH, USA

D W Warren University of Southern California, Los Angeles, CA, USA

Elizabeth A Szliter-Berger Wayne State University School of Medicine, Detroit, MI, USA

D R Whikehart The University of Alabama at Birmingham, Birmingham, AL, USA

N G Tahzib University Hospital Maastricht, Maastricht, The Netherlands

T Wojno The Emory Clinic, Atlanta, GA, USA

R S Talluri University of Missouri–Kansas City, Kansas City, MO, USA A Tarabishy Case Western Reserve University, Cleveland, OH, USA

Y Wu Institute for Eye Research, Sydney, NSW, Australia F Zhang The State University of New York, New York, NY, USA D Zoukhri Tufts University, Boston, MA, USA

INTRODUCTION

Protection of the entire eye from the external environment and maintenance of a clear optical pathway through the aqueous humor, lens, and vitreous to the retina are the functions of the ocular periphery. The outermost portion of the periphery is the eyelids that protect the eye through blinking and preserve visual acuity through the movement of the eye by the exceedingly specialized extraocular muscles. The next layer of protection is the tear film, secreted by the ocular adnexa, and the epithelia of the ocular surface, composed of the cornea and conjunctiva. The tears and ocular surface epithelia protect the eye through numerous coordinating layers of structural and functional mechanisms. The tears and cornea also must retain their transparency and maintain a smooth optical surface. Dysfunction and dysregulation of the ocular periphery in disease can compromise the entire visual system and lead to loss of visual acuity, inflammation, and infection, thus jeopardizing the function of the entire eye and, in severe cases, cause loss of vision. This book focuses on both the normal functioning of the tissues of the ocular periphery and their pathophysiology in disease. This volume provides a unique collection of chapters on the multiple, diverse tissues that comprise the ocular periphery and function to protect vision. The goal of this book is to provide a comprehensive and contemporary review of the structure and function of the ocular periphery in health and disease. The book is organized into four sections including I. Extraocular and Eyelid Muscles: Structure, Function, and Pathophysiology, II. Structure and Function of the Tear Film, Ocular Adnexa, Cornea, and Conjunctiva in Health and Pathogenesis in Disease, III. Immune Regulation of the Cornea and Conjunctiva and Its Dysregulation in Disease, and IV. Visual Acuity Related to the Cornea and Its Disorders. Section I is devoted to the extraocular muscles and the eye lids, the muscles that move the eye and the lids. Chapters include several chapters on the anatomy, function, metabolism, and pathophysiology of the specialized extraocular muscles, along with a chapter on eyelid function and pathophysiology. Final chapters discuss diseases of the extraocular muscles and the clinical diagnosis of the dysfunction of these muscles. The authors have particularly highlighted the special features of the extraorbital muscles that allow them to function without fatiguing, unlike other skeletal muscles as well as specific diseases that preferentially spare or involve these muscles critical for binocular vision. Section II focuses on two major areas: first, the tear film and the tissues that secrete it and second, the epithelia (cornea and conjunctiva) that form the anterior surface of the eye. Initial chapters in this section focus on the multiple components of the complex tear film and the mechanisms by which they are secreted by the meibomian gland, lacrimal gland, and conjunctiva. The next chapters discuss the structure and function of the conjunctiva and the three layers of the cornea, each of these layers with its specialized functions to maintain the clarity of the cornea while protecting the eye from mechanical, thermal, chemical, and pathogenic challenges of the external environment. Additional chapters in this section focus on corneal disease as well as new modalities for understanding ocular surface dysfunction and repairing this dysfunction. Section III highlights the unique mechanisms that the cornea uses to respond to immune and infectious challenges. Multiple chapters focus on different aspects of the immune and angiogenic privilege of the cornea that is unique to this tissue, and compare it with the more ‘‘normal’’ response of the conjunctiva. The many facets of the complex immune response of the cornea are presented. In addition, the multiple mechanisms that are responsible for the avascularity of the cornea are discussed. Another theme of this section is inflammation and its involvement with dry eye disease and infectious diseases of the ocular surface. All together, sections I, II, and III present the multiple layers of structure and diverse overlapping mechanisms that are in place to prevent breach of the interior of the eye by mechanical, thermal, chemical, and pathogenic threats from the external environment and the diseases that result when these defenses are overwhelmed. Section IV is devoted to the cornea, but in this section, the visual acuity of the cornea is highlighted. Chapters discuss normal visual optics and the conditions that result from changes in corneal shape that disrupt the visual axis and lead to decreased vision.

xiii

xiv

Introduction

Each chapter contains text readable to a scientist outside the field of the article, multiple multimedia and color figures to illustrate the most important points of the chapter, and a list of references to provide more in-depth information. Chapters are primarily directed at scientists looking for an entry point into a field tangential to their specialty as well as at graduate students and postdoctoral fellows in eye research. The chapters will be especially useful to scientists designing introductory or generalized courses that cover diverse fields of eye research. Scientists writing review articles or chapters will also find the book’s chapters especially useful as a starting point. The many introductory chapters are written at the level to be understood by undergraduates at university and public libraries, but include enough information to satisfy the more advanced needs of graduate students and postdoctoral fellows. The in-depth chapters on more focused research areas are ideal for postdoctoral fellows and experienced scientists. The plentiful illustrations will be especially helpful in understanding the more complicated points as well as illustrating basic processes and anatomy. Finally, I thank the other editors of this book who chose the chapters to be included in this book as well as the chapter authors who devoted considerable time to proof-reading these articles. I also thank all the authors for their excellent contributions and Robin R. Hodges for her excellent managerial assistance. Darlene A. Dartt, Ph.D. Boston, MA USA October 24, 2010

I. EXTRAOCULAR AND EYELID MUSCLES: STRUCTURE, FUNCTION, AND PATHOPHYSIOLOGY


Eyelid Anatomy and the Pathophysiology of Blinking C Evinger, SUNY Stony Brook, Stony Brook, NY, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Blepharospasm – A dystonic muscle contraction disorder characterized by forceful, bilateral, and uncontrolled closure of the eyelids. Hemifacial spasm – A muscle contraction disorder characterized by forceful uncontrolled contraction of the facial muscles on one side of the face. Levator palpebrae superioris muscle – A skeletal muscle innervated by cranial motor nerve 3 whose primary function is elevation of the upper eyelid. Mu¨ller’s muscle – A smooth muscle that runs from the inferior surface of the tendon of the levator palpebrae superioris to insert into the tarsal plate. It receives sympathetic nervous system innervation. Omnipause neurons – Part of the brainstem neural circuit that controls saccades. These neurons fire during fixation and cease firing before and during saccadic movements. Stimulation of omnipause neurons interrupts saccades. Orbicularis oculi muscle – A circumferential muscle of facial expression innervated by the facial nerve that lies just deep to the skin within both eyelids as well as the surrounding bones of the orbital margin. Its main function is closure of the eyelid. Paresis – Partial paralysis of a skeletal muscle resulting in muscle weakness. Retractor bulbi muscle – Skeletal muscles innervated by cranial nerve 6 whose function is to retract the eyeball into the orbit causing movement of the nictitating membrane over the surface of the eye. Saccades – Rapid eye movements that redirect the line of sight so that the image of interest falls on the fovea of the retina. Vergence – The coordinated movements of both eyes in opposite directions in order to maintain binocular vision.

Organization of the Eyelid System To understand the neural control of eyelids, the basis of neurological diseases affecting eyelid control, and how the

eyelids protect the eye, think about the evolutionary origins of eyelids. For fish, a class of vertebrates without eyelids, eye protection primarily requires avoiding objects hitting and damaging the cornea. To avoid objects hitting the eyeball, fish retract their eyes into the orbit by co-contracting their extraocular muscles. Thus, cornea protection was initially linked to neural circuits whose primary goal was to move the eye. When vertebrates moved onto the land, the development of eyelids was a critical step in reducing the dehydrating effects of air on the cornea. Although goblet cells, lacrimal and meibomian glands produce the fluids to coat the corneal surface, it is blinking of the eyelid that spreads the tears to restore the tear film, which maintains corneal hydration. In addition, blinking removes small objects from the surface of the eye and provides some protection from objects getting into the eye. Although blinking is essential for maintaining the cornea, lid closure has the undesirable side effect of blocking vision. Thus, an eyelid control system must generate blinks that minimally disrupt vision while adequately protecting the cornea. The nervous system deals with this constraint by developing fast eyelid closure and opening without carefully controlling absolute eyelid position. The other restriction on the nervous system’s management of the eyelids is that they must move synchronously with vertical eye movements to avoid blocking vision. Overcoming this problem requires the nervous system to control eyelid position accurately. The eyelid control system accomplishes this feat by linking itself to the eye movement system. The melding of the eyelid system with the eye movement system reveals itself first in the anatomical organization of the eyelids. Only four forces act on the upper eyelid (Figures 1(a) and 2). (1) The phasically active orbicularis oculi (OO) muscle actively closes the eyelid. The ipsilateral facial (VII) nucleus innervates the OO muscle. (2) The tonically active levator palpebrae superioris (LP) muscle actively elevates the upper eyelid. LP innervation arises from the oculomotor (III) nucleus. (3) Raising the eyelid stretches the superior transverse (Whitnall’s) ligament and the lateral and medial canthal tendons to create a passive downward force. Thus, the lowest energy state for the eyelid is closed. (4) Mu¨ller’s smooth muscle (Figure 2), which bridges the belly and the tendon of the LP, raises the eyelid approximately 1.5 mm with sympathetic activation. Post-ganglionic nerves from the superior cervical ganglion innervate Mu¨ller’s muscle. The interaction between the first three forces (OO, LP, and passive downward forces) enables

3

4

Extraocular and Eyelid Muscles: Structure, Function, and Pathophysiology

Human Pos

Closed

Open

LP

WL 10 deg

APO CT

100 ms

OOemg

(a) (c) SO Guinea pig (Pos)

Pos

R1

2 deg

LP

100 ms R2 OOemg (b)

OOemg (d) SO

Figure 1 Forces acting on the eyelid and mammalian blinking. (a) Opening the eye stretches the levator palpebrae (LP) aponeurosis (APO), the superior transverse ligament (Whitnall’s ligament, WL), and the medial and lateral canthal tendons (CT) to create passive downward forces. The lowest energy state for the eyelid system is closed. (b) Lid (Pos) lowering during a blink results from a transient relaxation of the LP followed by a phasic activation of the orbicularis oculi (OO) muscle. Raising the eyelid occurs as the LP resumes its tonic activity following the completion of OO activity. Gray indicates LP activity and black indicates OO activity. (c, d) Individual examples of a reflex blink evoked by stimulation of the supraorbital branch of the trigeminal nerve (SO ▲) for a human (c) and a guinea pig (d). R1 is the short latency response and R2 is the long latency response seen after nerve stimulation. Abbreviations: OOemg, orbicularis oculi EMG; Pos, upper eyelid position.

the eyelids to blink rapidly yet accurately match the vertical position of the eyeball. The characteristic rapid eyelid closure of a blink followed by lid opening at approximately half the speed of lid closure follows directly from the anatomical organization of the eyelid (Figures 1(b)–1(d)). A blink begins with relaxation of the tonically active LP muscle. LP relaxation releases the passive downward forces to initiate lid closure. The phasically active OO muscle discharge combines with the passive downward forces to lower the eyelid rapidly. As the OO muscle relaxes, the LP muscle slowly resumes its tonic discharge. This LP contraction raises the upper eyelid. Eyelid elevation is slower than lid closure because the LP muscle must work against the passive downward forces. The point at which the tonically active LP force matches the passive downward force created by tendon and ligament stretching determines final lid position. This anatomical organization is conserved so that the pattern of blinking is similar among mammals (Figures 1(c) and 1(d)).

In contrast to the interactions between the OO and LP muscles and passive downward forces with blinking, the coordination of eyelid motion with vertical eye movement arises from the antagonistic interactions between the LP muscle and the passive downward forces. The linkage with eye movements occurs because the LP behaves like the superior rectus muscle, which rotates the eye upward. Embryologically, the LP muscle arises from the superior rectus muscle, and LP motor neurons are always adjacent to superior rectus motor neurons in the oculomotor nucleus. LP and superior rectus motor neurons exhibit similar patterns of activity except during a blink. The tonic firing frequency of superior rectus and LP motor neurons correlates with vertical eye position. With an upward saccadic eye movement, superior rectus and LP motor neurons generate a burst of action potentials followed by an increased tonic firing frequency that holds the eye in the new elevated position. A downward saccade results from a cessation of tonic activity followed by a lower frequency tonic discharge to hold the eye in the


5

Epithelium

Fornix Facial nerve

Preseptal region of orbicularis oculi muscle

Müller’s muscle Pretarsal region of orbicularis oculi muscle

Tarsal glands Conjunctiva Figure 2 Montage of a sagittal section of the eyelid from an adult rabbit stained with hematoxylin and eosin. Courtesy of Dr. Linda K. McLoon.

depressed position. When the LP motor neurons transiently cease discharging during a downward saccadic eye movement, unopposed passive downward forces lower the eyelid. When the LP motor neuron resumes its activity at a lower tonic firing frequency, the new balance point between passive downward forces and active upward LP muscle force establishes the final eyelid position. With an upward eye movement, the increased LP motor neuron firing frequency pulls the eyelid upward until the LP muscle and passive downward forces match. Although it seems counter-intuitive that passive downward forces rather than the OO muscle act as the antagonist to the LP muscle with eye movements, it is clear that the OO does not participate in eyelid movement with vertical saccades. For example, individuals with OO denervation created by seventh nerve palsy exhibit nearly normal saccadic lid movements with vertical saccadic eye movements, but abnormally slow blinks. Further evidence of the evolutionary linkage of blinking with the oculomotor system is that blinks frequently occur with saccadic eye movements. These gaze-evoked blinks most commonly accompany large saccades to visual targets that do not have a strong behavioral significance. The advantage of combining blinks with saccades is that visual suppression occurs during both blinks and saccades. A gaze-evoked blink refreshes the tear film,

but does not produce more loss of vision than the saccadic eye movement. The evolutionary linkage also appears in the eye movements associated with blinking, blink-evoked eye movements. When looking straight ahead, there is an adducting and downward rotation of the eye with each blink. Nevertheless, the state of the eyelid system determines the trajectory of blink-evoked eye movements. These movements exhibit an upward trajectory in both eyes and are smaller than normal in individuals with a unilateral seventh nerve palsy. Eyeball retraction is also a component of these blink-evoked eye movements. For mammals, the eyeball retraction with blinking results from extraocular muscle co-contraction and contraction of the retractor bulbi muscle. This accessory extraocular muscle is innervated by motor neurons in the accessory abducens nucleus that send their axons to the orbit as part of the VIth nerve. Extraocular muscle co-contraction with blinking in mammals appears to reflect the evolutionary origins of the eyelid control system from eye retraction of fish. Despite blink-evoked eye movements accompanying all blinks, neither gaze-evoked nor reflex blinks occurring with a saccade prevent the eye from achieving its desired endpoint. With a simultaneous blink and saccade, the eyes follow a complex trajectory instead of the nearly straight path of a saccadic eye movement alone. This complex

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trajectory results in a significantly slower saccade than when the saccade occurs alone. The linkage between blinking and saccadic eye movements indicates that blinking interacts with the brainstem circuits that generate saccadic eye movements. This interaction is apparent when reflex blinks increase the speed of saccadic eye movements in individuals with abnormally slow saccades, initiate saccade oscillations, and alter the speed of vergence eye movements where the eyes are moving in opposite directions. The neurophysiological mechanism that may underlie these effects is the activity of omnipause neurons, which control fixation, and are associated with reflex blinks. Tonically active omnipause neurons gate saccadic eye movements. Omnipause neurons cease discharging immediately before and during all saccadic eye movements and reflex blinks. Microstimulation of omnipause neurons blocks reflex blinks as well as saccadic eye movements. The evolutionary origins of eyelid control functionally intertwine blinking and saccadic eye movements so that blinks frequently occur with saccadic eye movements, blinking modifies the trajectory of eye movements, and blinking and saccadic eye movements utilize some of the same brainstem neuronal circuits.

Modifiability of the Blink System In order to respond to challenges to the eyelid’s protective function, the neural control of blinking exhibits significant adaptive plasticity. The most common challenge to the protective function of blinking is the development of dry eye. Cornea irritation created by tear film inadequacy rapidly initiates several changes. First, the trigeminal system becomes more excitable, so that a given blinkevoking stimulus elicits a bigger blink than before cornea irritation. In addition to increased blink excitability, the trigeminal reflex blink circuit begins to ‘oscillate’ in response to a blink-evoking stimulus (Figure 3). In this condition, a single stimulus evokes a reflex blink followed

by blink oscillations, one or more additional large blinks. Blink oscillations occur at a constant interblink interval. These modifications compensate for dry eye in at least two ways. The reduced threshold for evoking a blink produces more frequent blinking so that blinks can occur before significant disruptions of the tear film. The larger blinks increase the amount of meibum released over the tear film. Increasing the thickness of the oily meibum layer superficial to the aqueous layer reduces aqueous evaporation. Disruptions of these normally adaptive mechanisms may underlie neurological disorders involving the eyelids, benign essential blepharospasm (BEB) and hemifacial spasm (HFS). We have an outline of the neural basis for these adaptive modifications of the blink reflex initiated by eye irritation. The adaptive changes occur in trigeminal blink circuits, but not at OO motor neurons or reticular neurons receiving bilateral trigeminal inputs. If just one eye experiences irritation, stimulating the ophthalmic branch of the trigeminal nerve on the irritated side elicits adaptive blink modifications in both eyelids. Stimulating the contralateral trigeminal nerve associated with the unaffected eye, however, elicits normal, unadapted blinks in both eyes. Consistent with this result, spinal trigeminal neurons in the corneal-evoked blink circuit discharge before blink oscillations, and this discharge correlates with the pattern of OO activity producing the blink oscillation. Although the adaptive processes clearly involve the trigeminal system, the cerebellum is also essential for blink adaptation. Blink-related neurons in the cerebellum are active with blink adaptation, and lesioning the cerebellum blocks this form of motor learning. A hypothesis for the function of this brainstem–cerebellum circuit is that the cerebellum recognizes an error signal produced by eye irritation and then initiates modifications in spinal trigeminal blink circuits to compensate for the eye irritation. These adaptive modifications in the trigeminal blink circuits may involve long-term potentiation (LTP)- and long-term depression (LTD)-like processes.

Reflex blink

SO

15 deg 250 (ms)

Blink oscillations

Figure 3 Stimulating the supraorbital branch of the trigeminal nerve (SO ▲) evokes a reflex blink followed by additional blinks with a constant interblink interval, blink oscillations. Each upper eyelid position trace is a record from a single trial from an individual with dry eye.


As discussed in the next section, the cerebellum appears to be an important element in creating the symptoms of dystonic movement disorders such as BEB.

Benign Essential Blepharospasm and Hemifacial Spasm BEB is a focal dystonia characterized by involuntary bilateral spasms of lid closure, trigeminal reflex blink hyperexcitablity, and photophobia (i.e., excessive sensitivity to light). BEB frequently begins with a complaint of ocular discomfort. The available evidence indicates that BEB arises from the confluence of a predisposing and a precipitating factor. Although the predisposing factor has not been identified, the genetic basis for other forms of dystonia suggests that the predisposing factor for BEB is genetic. The precipitating factor appears to be ocular irritation. Consistent with eye irritation as the precipitating factor is that BEB characteristics appear to be an exaggeration of the normally adaptive response to dry eye. For most individuals, the spasms of lid closure in BEB are rapid, repetitive contractions of the OO muscle. This spasm pattern is equivalent to shortening the interblink interval of the blink oscillations developed in response to dry eye (Figure 3). BEB patients exhibit trigeminal reflex blink hyperexcitability. One adaptive response to dry eye is to elevate trigeminal reflex blink excitability, although this increase accompanying dry eye is not as profound as occurs with BEB. The trigeminal hyperexcitability with BEB is sufficient to explain the photophobia. Consistent with the exaggeration of dry eye hypothesis, photophobia is present with dry eye although the light sensitivity is not as debilitating as with BEB. Thus, the exaggerated dry eye hypothesis proposes that BEB begins with the onset of dry eye or significant eye irritation. The nervous system initiates blink modifications to compensate for this irritation, but the genetic predisposition prevents the nervous system from recognizing that the adaptive changes corrected the ocular irritation. The nervous system responds by further increasing these adaptive modifications until the normally compensatory mechanisms becomes so maladaptive as to create the BEB syndrome. Although genetic modifications probably create the predisposing environment for BEB, the genetics underlying this focal dystonia are not yet clear. Most investigators argue that there is an autosomal dominant transmission with reduced penetrance in BEB. One challenge to linking genetics to specific types of dystonia, however, is that individuals with the same genetic mutation may exhibit very different forms of dystonia. For example, individuals with the DYT1 mutation responsible for the most common form of generalized dystonia may exhibit generalized dystonia, focal dystonia, or may be asymptomatic. Individuals with generalized dystonia, focal dystonia, and

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asymptomatic DYT1 carriers all exhibit a similar pattern of abnormal brain activity. Positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) scans reveal hyperactivity in the basal ganglia, primary motor cortex, supplementary motor area, putamen, thalamus, and cerebellum. In patients with BEB, these areas are also active, but the focus of abnormal activity is in the brainstem and cerebellum. Abnormal cerebellar activity with BEB supports the hypothesis that abnormal blink adaptation involving the cerebellum is critical in the development of BEB. Consistent with the hypothesis that a genetic predisposing factor affects adaptation or motor learning, individuals with BEB exhibit exaggerated LTPlike plasticity of blink circuits relative to normal subjects. There are two clear examples in which exaggeration of the adaptive processes initiated by eye irritation or dry eye leads to BEB. A small number of individuals with Bell’s palsy develop blepharospasm. The ocular irritation produced by incomplete eye closure, the precipitating factor, initiates unabated modifications that produce the BEB syndrome. As predicted by the exaggerated dry eye hypothesis, implanting gold weights in the paretic (weakened) eyelid, enabling nearly complete closure of the paretic eyelid, reduces eye irritation and allows a resolution of BEB signs. Combining predisposing and precipitating factors can also create a BEB-like syndrome in rats. In this model, a chemical lesion reducing approximately 30% of the dopamine neurons in the substantia nigra pars compacta elevates the excitability of the trigeminal blink reflex circuits. This increased trigeminal excitability acts as the predisposing factor. Transient eye irritation produced by crushing a facial nerve branch providing a portion of the OO innervation acts as the precipitating factor. The reduced eyelid mobility created by this procedure causes eye irritation that initiates adaptive blink modifications. Although the eye irritation resolves following nerve regrowth, rats continue to exhibit spasms of lid closure caused by repetitive bursts of OO activity and hyperexcitable trigeminal reflex blinks. Thus, the evidence indicates that BEB occurs in individuals genetically predisposed to the disorder who experience a precipitating condition, ocular irritation. The ocular irritation initiates a series of normally adaptive modifications. In the presence of the predisposing condition which creates an abnormal environment for motor learning, these modifications become exaggerated to create the signs of BEB. HFS begins as spontaneous, unilateral spasms of eyelid closure. Over a period of weeks to months, the spasms expand to include the rest of the facial muscles on that side of the face. Another characteristic of HFS is synkinesis in which there is an involuntary activation of multiple muscles that normally do not act together. An example of synkinesis is that stimulating the supraorbital branch of the trigeminal nerve would strongly activate the mentalis muscle, as well as the OO, in HFS patients. Unlike BEB,

8


HFS is unilateral and involves most of the ipsilateral facial muscles in addition to the OO. There is strong evidence, however, that HFS also arises from a combination of predisposing and precipitating factors that disrupt normal motor learning. The accepted precipitating factor for HFS is arterial compression at the root entry zone of the facial nerve. The most common blood vessels affecting the facial nerve in HFS are the anterior inferior cerebellar artery, the posterior inferior cerebellar artery, or the vertebral artery. Microsurgical decompression of the facial nerve typically reduces or eliminates the spasms and synkinesis of HFS so that spasms disappear in 64% and synkinesis in 53% of HFS patients within the first week following surgery. After 2–8 months, 90% of patients are spasm or synkinesis free. If arterial compression of the facial nerve alone is responsible for these signs of HFS, however, then decompression surgery should eliminate the spasms and synkinesis in less than 2–8 months. To understand the basis for this delay, it is important to consider what neural modifications might occur in response to pulsatile arterial compression of the facial nerve. A strong argument that facial nerve compression alone is insufficient to cause HFS is that 15–25% of the population exhibits arterial compression of the facial nerve, but do not develop HFS. This observation shows that HFS requires a predisposing factor as well as the precipitating factor, arterial compression of the facial nerve. Pulsatile arterial compression of the facial nerve can generate many modifications in brainstem neural circuits. Compression injury to motor neuron axons effectively weakens facial muscles, which initiates adaptive modifications. Repetitive antidromic activation of facial motor neurons by pulsatile compression of axons can alter facial motor neuron excitability or motor neuron excitability may increase because of facial motor neuron axotomy, severing the facial nerve fibers. Repetitive orthodromic activation of facial muscles by pulsatile compression of motor neuron axons can lead to a reorganization of sensory trigeminal circuits. Simultaneous orthodromic (electrical impulses traveling in the normal direction) activation of muscles that normally do not act together, for example, mentalis and OO, causes trigeminal primary afferent sensory signals from mentalis and OO muscle contraction to reach second-order trigeminal neurons synchronously. This abnormal pairing of sensory inputs can restructure trigeminal receptive fields so that the second-order trigeminal neurons respond strongly to inputs from both mentalis and OO activity instead of weakly to one and strongly to the other. Pulsatile activation of the facial nerve can also augment reflex circuit excitability because of synchronous activation of circuit elements. Second-order trigeminal neurons receiving synchronous afferent inputs innervate facial motor neurons that are already depolarized by antidromic activation.

This activity pattern can strengthen trigeminal inputs onto facial motor neurons in a spike timing-dependent plasticity-like manner. It is clear that pulsatile activation of the facial nerve can produce blink modifications, but these changes should not cause HFS by themselves. The eyelid system normally modifies itself so as to perform appropriately in the face of changes in the motor system or sensory inputs. For example, creating unexpectedly large blinks by adding weights to the upper eyelids initiates a rapid reduction in the trigeminal drive onto OO motor neurons. Similarly, chronic, repetitive facial nerve stimulation, such as occurs with pulsatile facial nerve compression, reduces blink amplitude. Thus, pulsatile facial nerve compression is insufficient to cause spasms of lid closure because the blink system will modify itself to prevent spasms of lid closure. The significant number of humans not experiencing HFS, but exhibiting arterial compression of the facial nerve, further supports this interpretation. These observations indicate that HFS, like BEB, requires a predisposing factor to develop spasms. Like BEB, an autosomal-dominant genetic mutation with low penetrance may provide the predisposing condition, which disrupts normally adaptive processes to create a pathological condition.

Further Reading Bour, L. J., Aramideh, M., and de Visser, B. W. (2000). Neurophysiological aspects of eye and eyelid movements during blinking in humans. Journal of Neurophysiology 83: 166–176. Evinger, C., Manning, K. A., and Sibony, P. A. (1991). Eyelid movements. Mechanisms and normal data. Investigative Ophthalmology and Visual Science 32: 387–400. Fukuda, H., Ishikawa, M., and Okumura, R. (2003). Demonstration of neurovascular compression in trigeminal neuralgia and hemifacial spasm with magnetic resonance imaging: Comparison with surgical findings in 60 consecutive cases. Surgical Neurology 59: 93–99; discussion 99–100. Manning, K. A. and Evinger, C. (1986). Different forms of blinks and their two-stage control. Experimental Brain Research 64: 579–588. Nielsen, V. K. and Jannetta, P. J. (1984). Pathophysiology of hemifacial spasm: III. Effects of facial nerve decompression. Neurology 34: 891–897. Rambold, H., Sprenger, A., and Helmchen, C. (2002). Effects of voluntary blinks on saccades, vergence eye movements, and saccade–vergence interactions in humans. Journal of Neurophysiology 88: 1220–1233. Sibony, P. A. and Evinger, C. (1998). Normal and abnormal eyelid function. In: Miller, N. R. and Newman, N. J. (eds.) Walsh and Hoyt’s Clinical Neuro-Ophthalmology, vol. 1, pp. 1509–1594. Baltimore, MD: Williams and Wilkins. Sparks, D. L. (2002). The brainstem control of saccadic eye movements. Nature Reviews Neuroscience 3: 952–964. VanderWerf, F., Brassinga, P., Reits, D., Aramideh, M., and Ongerboer de Visser, B. (2003). Eyelid movements: Behavioral studies of blinking in humans under different stimulus conditions. Journal of Neurophysiology 89: 2784–2796. VanderWerf, F., Reits, D., Smit, A. E., and Metselaar, M. (2007). Blink recovery in patients with Bell’s palsy: A neurophysiological and behavioral longitudinal study. Investigative Ophthalmology and Visual Science 48: 203–213.

Differentiation and Morphogenesis of Extraocular Muscles D M Noden, Cornell University, Ithaca, NY, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Mesenchyme – Embryonic cells with a fibroblast-like appearance, surrounded by extracellular matrix, lacking tight junctions with their neighbors, and often capable of undergoing extensive migratory movements. These can be of several different embryonic origins, and include cells that will contribute to many lineages. Morphogenesis – It includes those processes that establish the correct locations and three-dimensional organization of tissues and organs. This includes the proper positioning of extraocular muscles around the globe and their attachments to the sclera and orbital skeleton. Myoblasts – Mitotically active cells committed to the skeletal muscle lineage but not yet expressing muscle-specific proteins such as desmin and myosins, which generally are not evident until after myoblasts fuse to form multinucleated myofibers. Myotome – The several regions of each somite that contain progenitors of skeletal muscle progenitors. Neural crest – Mesenchymal cells that are derived from neural fold tissues and that move peripherally along well-delineated pathways to form neurons and glia of the peripheral nervous system and, in the head region, the connective tissues of the midface and branchial regions. Paraxial mesoderm – Early embryonic cells that are located beside and beneath the developing brain and spinal cord, and include the precursors of most skeletal muscles.

Introduction Muscles that move and stabilize the eye have been highly conserved during vertebrate evolution. While a few remarkable adaptations have occurred, such as co-opting of the dorsal (superior) oblique muscle to generate protective heating for the brain in some fishes, these muscles have retained an anatomical organization linking axes of the body and the eye that arose hundreds of millions of years ago. Considering their ancient status, it is logical to assume that the early development of extraocular muscles would similarly be well conserved among different species, and

therefore amenable to comparative analyses that supplant the absence of direct examination in mammals, including humans. However, compared to trunk and limb muscles, our understanding of the origins of ocular muscles and the mechanisms that initiate then maintain their development is at best fragmentary. Myogenesis of skeletal muscles is a lengthy process, with several parameters continuing to be function-dependent throughout the life of an animal. Primary myogenesis spans the period during which populations of myoblasts – committed, mitotically active muscle progenitors – arise, emigrate to their sites of differentiation, fuse to form multinucleated innervated myofibers, and establish intimate connections with connective tissues. This population forms a scaffolding, including the delineation of global and orbital domains, within which secondary myogenesis occurs. During secondary myogenesis stages, previously sequestered latent myoblasts are activated to proliferate and differentiate, forming more than 90% of the myofibers present in mature muscles and generating region-specific specialized fiber types that in most species are present before or soon after birth.

Origins of Extraocular Muscles Striated (skeletal) muscles throughout the body arise within paraxial mesoderm, which is located in close apposition to the embryonic brain and spinal cord. Exceptions to this are the avian striated ciliary muscle that is of neural crest origin, and possibly the striated muscles of the esophageal wall; however, both of these are involuntary. Among voluntary muscles, some of the more ventrally located branchial muscles arise from lateral mesoderm that is contiguous with paraxial mesoderm. Many early accounts of head myogenesis placed the embryonic origin of some eye muscles, especially the lateral rectus, in the same category as branchial (pharyngeal) arch muscles, and ascribed both to a lateral mesoderm origin. These claims were based on the sites at which muscle condensations are first grossly evident in the embryo. However, with the advent of mapping methods and assays for early muscle-specific gene expression patterns, separate and distinct sites of origin for all eye muscles within preotic (i.e., located rostral, in front of the developing inner ear) paraxial mesoderm was confirmed (Figure 1). The sites of origin of extraocular muscles parallel the sites of emergence of the three cranial motor nerves that

9

10


Prosencephalon

Extraocular Medial rectus Ventral rectus Dorsal rectus Ventral oblique

Paraxial mesoderm Lateral mesoderm 1st arch Mandibular adductors Intermandibular

3rd arch Pharyngeals Stylopharyngeus Hypaxial Laryngeal Glossal Rectus capitis

Mesencephalon

Dorsal oblique

n. V

n. IV

Retractor bulbi Lateral rectus

Metencephalon

Pterygoideus 2nd arch Stapedial Digastricus, facials

n. III

n. VI

n. VII DO n. IX

Myelencephalon n. XII

1st som.

n. X

Epaxial Biventer, splenius complexus (a)

2nd som. 3rd som. (b)

Figure 1 Sites of origin of craniofacial striated muscles in avian embryos. Panel (a) shows, in dorsal view, the locations of each muscle primordia within paraxial and lateral mesoderm. Panel (b) illustrates one mapping method used in avian embryos, wherein a small bolus of replication-incompetent retrovirus injected into early mesoderm and the embryo harvested 2 weeks later and stained for the appropriate reporter gene, in this case a bacterial galactosidase. In this embryo, in which the eye has been removed to reveal underlying tissues, only the dorsal (superior) oblique muscle was labeled.

innervate them. However, in contrast to axial and branchial muscles, sites of myogenesis are not congruent with locations of motor nerve emergence. Indeed, each of these cranial nerves needs to elongate considerably through peripheral tissues before initial contacts with target muscles are established. Some axons, such as those of the abducens nerve, must extend longitudinally beneath the brain to reach their target lateral rectus muscles, while others, such as the oculomotor nerve fibers, project perpendicular to the floor of the brain before decussating to approach their several target muscles. In extant vertebrates, head paraxial mesoderm constitutes a sparse population of mesenchymal cells (Figure 2). This contrasts with the situation in the neck and trunk regions, where paraxial mesoderm first forms somites, which are segmentally arranged, cuboidal aggregates of epithelial cells. As each somite matures, it becomes delineated into distinct myogenic (myotome) and connective

tissue-forming (sclerotome) regions. The most cranial somite is located beside the hindbrain, immediately caudal to the otic vesicle, and paraxial mesoderm rostral to this site fails to form epithelial tissues and lacks overt evidence of segmentation. Head paraxial mesoderm is present adjacent to the prospective eye-forming regions of the rostral neural plate, but is largely displaced caudally as the optic vesicles emerge and expand lateral to the diencephalon. In the midline just in front of the notochord, this population of loose paraxial mesoderm cells is contiguous with a sparse and species-variable population of prechordal mesenchymal cells. Mapping experiments in avian embryos have shown that prechordal mesoderm contributes to the genesis of extraocular muscles innervated by the oculomotor nerve (Figure 3), but it is not known whether this contribution is exclusive of or supplementary to that of paraxial mesoderm. It is not known if the same occurs in mammalian embryos.

Differentiation and Morphogenesis of Extraocular Muscles

11

Neural crest

Optic vesicle

Mesencephalon

Pharynx Lateral mesoderm

Heart tube Neural crest

(a)

Paraxial mesoderm

(b)

Figure 2 Colorized scanning electron micrographs showing the early relations of neural crest (blue) to mesodermal (red) populations in dorsal (a) and transverse (b) views. Small arrows indicate the direction of movement of the neural crest cells.

(a)

(b)

(c)

Figure 3 Contributions of prechordal mesoderm to developing extraocular muscles is shown by labeling their precursors at stage 4–5 (early gastrulation) with DiI, a fluorescent membrane-binding tag (site ‘o’ in (a)), and fixing the embryos over a day later((b), stage 12, ventral view). Labeled cells in (c) are within the eye muscle-forming region of paraxial mesoderm.

Determination of Eye Muscle Precursors Head paraxial mesoderm contains progenitors for many tissues in addition to skeletal muscle. These include cartilages and bones associated with the braincase, loose connective tissues such as meninges and adipocytes, and endothelial cells. In contrast to somites, wherein these progenitor populations are largely segregated, it appears based on mapping studies that these diverse precursors are either intermingled or contiguous in head mesoderm. The significance of this lies in the problem of generating diverse lineages. Somite cells are held in fixed positions relative to the dorsal and ventral parts of the adjacent neural tube (hindbrain and spinal cord) and overlying surface epithelium, all of which provide combinations of positive and negative regulators of early myogenesis and skeletogenesis.

A further complication – and one essential for the development of all craniofacial musculoskeletal systems – is the presence of a large, later-arising population of mesenchymal cells called the neural crest (Figure 2). Derived from neural folds either during (mammals) or shortly following (birds) closure of the cephalic neural tube (brain), these cells acquire a mesenchymal phenotype and quickly move peripherally, mostly atop underlying paraxial mesoderm. Neural crest cells from the rostral midbrain level move rostrally and caudally around the optic stalk and posterior part of the optic vesicle, then spread outwardly as the vesicle is transformed into the optic cup. After delineation of the lens from the lens placode, crest cells secondarily invade the space created anterior to the lens and establish the posterior epithelium (endothelium) and stromal populations of the cornea.

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In the trunk, members of the wnt family of growth factors are secreted by surface ectoderm and provide essential positive stimuli for muscle differentiation. The same are released by head surface epithelium, but here their effects are to retard myogenesis of branchial muscles. Arriving neural crest cells separates branchial muscle progenitors from the source of these negative effects and, augmented by the release of additional myogenesispromoting factors, allows myogenesis to progress. The extent to which eye muscle progenitors follow a similar scenario is unclear. Some extraocular precursors, particularly the lateral rectus progenitors, are embedded deep within paraxial mesoderm and are therefore quite distant from both surface ectoderm and, at early stages, neural crest cells. This deep location places the lateral rectus precursors close to the neural epithelium at the level of the future metencephalon (pons). Several experiments have established that this location provides essential cues for lateral rectus formation. When newly formed trunk paraxial mesoderm cells were excised, before they had formed somites, then grafted into the head, in place of prospective lateral rectus mesoderm cells, the transplanted cells formed a muscle that expressed molecular markers unique to the lateral rectus and established proper anatomical connections with the braincase and sclera. Small changes in the location of the implants resulted in grafted cells contributing to the dorsal oblique or branchial arch musculature. Thus, the sites within head paraxial mesoderm at which each muscle primordium forms and is specified as to its identity are highly localized. Placing a barrier between the brain and paraxial mesoderm at this region does not prevent myogenesis, but the developing muscle cells lack molecular features that define their specific identity. Together, these experiments suggest that a rich tableau of local signals is necessary for early eye muscle differentiation, with both general myogenic and individual eye muscle-specific components.

Molecular Signatures and Muscle Differentiation All skeletal myoblasts use members of a closely related set of muscle-specific transcription factors to promote and coordinate their differentiation. Two of these, myf 5 and myoD, are cross-activating regulators that are among the earliest muscle-specific genes expressed. The upstream regulatory components of these genes are body regionand muscle group-specific, and serve to integrate the diverse micro-environments surrounding each muscle group with a highly shared set of outcomes, for example, activation of genes for desmin, myosins, muscle-specific actins, and junctional receptors.

Expression of myf 5 then myoD genes in eye muscle precursors generally is slightly later than expression in trunk axial muscles, but is simultaneous with that of branchial muscles (Figure 4). Expression of these regulatory genes coincides with the onset of aggregation of most muscle precursors (Figure 5), although it is not known whether these aggregates represent the totality of muscle precursors or only a subset of primary myoblasts. By these criteria, extraocular muscles appear similar to other head and also to trunk and appendicular muscles. However, as additional features of trunk and head myogenic regulatory networks have been identified, the number of differences has exceeded the similarities, and a heretofore underlying complexity has been revealed (Table 1). This area of investigation is rapidly expanding, and rather than detail each gene currently described, a few examples of categories of differences among muscle groups will be presented. Pax3 is a regulatory gene expressed in axial and appendicular muscle precursors, and null mutations of this transcription factor (e.g., Splotch mutation) result in severe depletion of trunk muscles. However, it is not expressed in head muscle precursors, and null mutations have no discernable effect on branchial or extraocular muscles. Another pronounced difference is in the hepatocyte growth factor (HGF) – cMet growth factor-receptor complex, which is functionally required for the correct dissemination and differentiation of appendicular and tongue muscle precursors. Again, this pathway has no known role in eye and branchial myogenesis, even though HGF is expressed in and around the precursors of branchial arch, lateral rectus, and both oblique muscles. The latter example further illustrates the considerable heterogeneity among extraocular muscles. The lateral rectus is particularly enigmatic, being the only head muscle that expresses the ubiquitous trunk paraxial mesoderm marker paraxis, and together with the dorsal oblique, the transcription factor lbx1, which is present in trunk hypaxial (thoracic and abdominal wall) muscle precursors. A further complexity arises from the often-changing patterns of gene expression during the early stages of head paraxial mesoderm development. The transcription factor pitx2, which is a key mid-level participant in the integrated formation of left-right asymmetry for the heart and mid-gut, is initially expressed symmetrically and ubiquitously throughout head paraxial mesoderm (Figure 6). However, a day later, during early myogenesis stages, its expression becomes more restricted but includes the first branchial arch, lateral rectus, and both oblique muscles in addition to periocular neural crest cells. Another regulatory gene, Tbx1, which is located in the region of chromosome 22 wherein deletions cause the DiGeorge syndrome, is similarly expressed over a wide domain of mesoderm (and pharyngeal endoderm) before becoming restricted to branchial arch and the lateral rectus muscles.


2 day

3 day

4 day

5 day

Move

13

Muscle group Dorsal (superior) rectus

Move

Inferior (ventral) oblique Inferior (ventral) rectus

Move

Medial rectus Move

Superior (dorsal) oblique

Move

Lateral rectus

Move

Branchial arch

Migr.

Tongue, laryngeal Epaxial (neck) Migr.

Wing Onset of myoD transcription

Aggregation of myoblasts move migr.

Key

Onset of myf5 transcription

Myoblast movements or migrations

Myosin proteins present

Figure 4 Timetable of gene activation in extraocular and other head and trunk muscles. The most consistent difference between trunk and head muscles is that the latter show a prolonged delay between the onset of myoD expression and the synthesis of muscle-specific proteins.

2.5 day

LR BA2 BA1 BA3

DO

VO LR DR

HGC

MyoD

6 day

4 day

BA2 BA3

LR DO

BA3 BA2

DO

EP

BA1 VO

VR MR

VR HYP

HGC

Myf5

DR

BA1

VO

Myosin

Figure 5 Early differentiation and morphogenesis of head muscles in chick embryos. BA1, 2, 3, branchial arches; DO, dorsal oblique; DR, dorsal rectus; Ep, epaxial muscle precursors; Hyp, hypaxial precursors, HGC, hypoglossal cord that forms tongue muscles; LR, MR, VR, lateral, medial and ventral rectus muscles; VO, ventral oblique.

At present the significance of these spatially and temporally dynamic expression patterns is unknown. It is possible that early expressions presage the later focal appearance of certain muscles, but it is equally plausible that each of these genes has multiple and distinct functions associated with each stage. As extraocular muscles mature, they exhibit a progression of fiber types, evidenced by changes in the myosin

isoforms and related contractile and energy metabolism proteins expressed. Emergence of these complex patterns requires a series of interactions among developing myofibers, surrounding connective tissues, and innervation. In the avian embryo, the primary myofibers of most extraocular muscles express embryonic slow myosin isoforms. However, one muscle, the quadratus nictitans, which is homologous to retractor bulbi muscles and is innervated

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Table 1

Summary of myogenic regulatory networks of head and trunk

Expression sites

Genes

Trunk

Limb and tongue

Branchial

EOMs

All muscle Trunk only

myf5, myoD noggin, ptc1 pax3, c-met barx2 lbx1 paraxis pitx2 tbx1 en2 pod1 hgf myoR

þ þ þ þ þ þ þ

þ þ þ þ þ þ (þ)

þ þ þ BA1 þ BA1 þ

þ þ LR, DO LR þ LR þ DO, VO DO, VO, L1

Trunk and Head

Head only

LR BA1 BA2

DO

Allantois Left (a)

Right (b)

Figure 6 At stage 8 ((a), dorsal view) Pitx2 is expressed symmetrically throughout head paraxial (and lateral) mesoderm populations, but only on the left within trunk mesoderm. By stage 21 ((b), 4 days) it is restricted to a subset of eye muscles, the first and second branchial arch muscle masses, and periocular mesenchyme.

by the accessory abducens nerve, the myofibers are either completely fast myosin expressing, as in the quail embryo, or mixed, as in the chick embryo (Figure 7). To explore the basis for these distinct, species-specific patterns, periocular neural crest cells of the chick were replaced with comparable populations from a quail embryo. The quadratus muscle in these chimeric embryos exhibits the quail donor phenotype (Figure 7), indicating that initial differentiation of fiber types requires interactions among myofibers and encompassing connective tissues.

Muscle Morphogenesis Except for muscles that remain closely associated with the vertebral column and skull, all muscle progenitors leave their sites of origin in paraxial mesoderm and disperse into peripheral tissues. Body wall muscles do so together with sclerotome-derived connective tissue precursors, and maintaining these close spatial relations is essential

for the morphogenesis of thoracic and abdominal muscles. For appendicular myogenesis, lateral myotome-derived cells move in sequential waves of primary and secondary myoblasts into nearby limb buds, where they form longitudinal bands of future dorsal and ventral groups before segregating into individual muscles. In the hindlimbs, some of these undergo secondary dispersal to form muscles of the perineal region. Branchial muscles are comparable to body wall muscles in that they initially exhibit a constant juxtaposition with the precursors of their connective tissues, which in the head are all derived from neural crest cells, and also with the motor nerves that innervate them. This maintained registration permits continuous exchange of signals among all three components during all stages of muscle differentiation and morphogenesis. This constant contiguity has most dramatically been demonstrated for the trapezius muscle, whose precursors arise among caudal branchial arch mesodermal populations and secondarily shift caudally to attach to the scapula. Mapping studies in both bird and mouse embryos revealed that the neural crest-derived connective tissues move with, and perhaps somewhat in advance of, these myoblasts and indeed contribute to the scapula. This recapitulates an ancestral condition in which the forelimb girdle articulated with the back of the skull, as is still present in fish. Again, however, the extraocular muscles exhibit a developmental scenario unlike any other muscles. As illustrated in Figures 5 and 8, these muscle precursors move towards, then around, the equatorial region of the developing eye to assume their definitive locations. During this process, each muscle leaves the company of surrounding mesoderm cells and becomes fully encompassed by neural crest cells, which secondarily penetrate the muscle mass and form internal (e.g., endomysium) as well as surrounding (perimysium, fascia, and tendon) connective tissues. These periocular crest cells need not have originated at the same axial level as the muscles. For example, the lateral rectus muscle, the neural crest cells that will form


D.R

15

D.R

Qd. N. Qd. N.

(a)

(c)

D.R

Qd. N. Qd. N.

(b)

(d)

Figure 7 Fiber-type determination in the quadratus nictitans (Qd. N.) muscle. (a, b) Sections through this muscle in chick and quail embryos processed with antibodies to slow myosin isoforms. The quail Qd. N. lacks slow fibers, whereas in the chick both fast and slow fibers are present. (c) A control embryo in which chick neural crest cells were transplanted into a chick host, and the Qd. N. developed normally. However, when quail crest cells were grafted into a chick host embryo (d), the exclusive fast donor phenotype resulted.

Mesencephalon

Isthmus Me cep tenhalo n LR n VR Myelencephalo MR

DR

DO

VO 1.0 mm

Telencephalon

Figure 8 The movements of the dorsal (yellow arrow) and ventral (green arrow) oblique muscles from their sites of origin to their terminal locations along the equatorial zone of the globe.

its connective tissues, and the abducens nerve that innervates it originate at three separate axial levels. Indeed, these primordia do not become intimately associated until each has independently approached the periocular environment. This negates the possibility of prolonged

interactions among contiguous progenitor populations, as occurs for branchial musculoskeletal systems. The mechanisms by which aggregates of extraocular muscle primordia change both absolute and relative positions remain enigmatic. There is no precedence elsewhere in the embryo for condensations of cells moving actively through surrounding tissues. However, several lines of evidence support a model based on passive displacement of eye muscle primordia. As was shown in Figure 2, the interface between neural crest and myogenic paraxial mesoderm is initially a flat plane. Changes in the relative positions of the eye due to flexures and differential growth of the brain and expansion of the optic cup introduce distortions in this plane, but the extent to which this might affect individual eye muscles has been difficult to define. In screening for a wide range of gene expression patterns, several were found that coincided with the patterns of movements taken by some extraocular muscles (Figure 9). These reveal a set of localized distortions of the neural crest-mesoderm interface. Finger-like projections of paraxial mesoderm expand dorsally and caudally around the optic cup, becoming interdigitated with periocular neural crest populations and passively bringing the dorsal and ventral oblique muscle primordia to their definitive

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globe, where they become surrounded then infused by connective-tissue forming neural crest cells, which largely direct both the gross and microscopic differentiation of these muscles. BA1

See also: Extraocular Muscles: Extraocular Muscle Anatomy.

Further Reading Figure 9 Lateral view of a 3.5-day chick embryo showing the sites of expression of the MyoR gene. Note the finger-like projections (arrows) extending dorsal and caudal to the optic vesicle, along the sites at which the dorsal and ventral oblique are expanding.

locations. Subsequently, crest cells close behind each of these muscle primordia, creating the appearance of an island of myoblasts/myofibers amid a sea of crest cells. How these focal distortions are established is unknown. The cell surface adhesion molecule semaphorin 3A is expressed by mesodermal cells in these projections, but its role in not known.

Summary The early stages of extraocular muscle formation are well described but poorly understood mechanistically. They arise at discrete sites within unsegmented head paraxial mesoderm then launch into developmental programs that share some features with trunk and branchial muscles but are largely and surprisingly unique. Passive distortions of the mesoderm-neural crest interface bring these muscle primordia to their definitive locations around the ocular

Borue, X. and Noden, D. M. (2004). Normal and aberrant craniofacial myogenesis by grafted trunk somitic and segmental plate mesoderm. Development 131: 3967–3980. Bryson-Richardson, R. J. and Currie, P. D. (2008). The genetics of vertebrate myogenesis. Nature Reviews Genetics 9: 632–646. Evans, D. J. R. and Noden, D. M. (2006). Spatial relations between avian craniofacial neural crest and paraxial mesoderm cells. Developmental Dynamics 235: 1310–1325. Grenier, J., Teillet, M. A., Grifone, R., Kelly, R. G., and Duprez, D. (2009). Relationship between neural crest cells and cranial mesoderm during head muscle development. PLoS ONE 4: e4381. Marcucio, R. M. and Noden, D. M. (1999). Myotube heterogeneity in developing chick craniofacial skeletal muscles. Developmental Dynamics 214: 178–194. Noden, D. M. and Francis-West, P. (2006). The differentiation and morphogenesis of craniofacial muscles. Developmental Dynamics 235: 1194–1218. Noden, D. M., Marcucio, R. M., Borycki, A-G., and Emerson, C. P., Jr. (1999). Differentiation of avian craniofacial muscles. I. Patterns of early regulatory gene expression and myosin heavy chain synthesis. Developmental Dynamics 216: 96–112. Noden, D. M. and Schneider, R. A. (2006). Neural crest cells and the community of plan for craniofacial development: Historical debates and current perspectives. In: Saint-Jeannet, J. P. (ed.) Neural Crest Induction and Differentiation, pp. 1–23. Boston, MA: Landes Bioscience and Springer Science Media. Tzahor, E. (2009). Heart and craniofacial muscle development: A new developmental theme of distinct myogenic fields. Developmental Biology 327: 273–276. Yoshida, T., Vivatbutsiri, P., Morriss-Kay, G., Saga, Y., and Iseki, S. (2008). Cell lineage in mammalian craniofacial mesenchyme. Mechanisms of Development 125: 797–808.

Extraocular Muscles: Extraocular Muscle Anatomy L K McLoon, University of Minnesota, Minneapolis, MN, USA S P Christiansen, Boston University, Boston, MA, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Abducens nerve (CNVI) – The cranial motor nerve which controls contraction of the lateral rectus muscle. Aponeurosis – A large flat and dense connective tissue layer which anchors a muscle to its origin or insertion. Excyclotorsion – An outward rotation of the upper pole of the vertical midpoint of each eye. Incyclotorsion – An inward rotation of the upper pole of the vertical midpoint of each eye. Myosin heavy chain (MyHC) isoforms – The major contractile protein in muscle is myosin, which in turn is composed of two heavy and four light chains. MyHC isoforms are responsible for the shortening velocity of muscle fibers during muscle contraction. Oculomotor nerve (CNIII) – The cranial motor nerve which controls contraction of the levator palpebrae superioris muscle, the inferior, medial, and superior rectus muscles, as well as the inferior oblique muscle. It also contains parasympathetic preganglionic axons that are destined for the ciliary body and iris sphincter muscles of the eye. Ora serrata – The junction between the neurosensory retina and the ciliary body. Satellite cells – Myogenic precursor cells that reside between the sarcolemma of the muscle fiber and its surrounding basal lamina. They are responsible for myofiber repair and regeneration after injury or in disease. Strabismus – A disorder characterized by altered tonus or restrictive disease of the extraocular muscles resulting in loss of conjugate binocular vision. Trochlear nerve (CNIV) – The cranial motor nerve which controls contraction of the superior oblique muscle.

The extraocular muscles (EOMs) have an extremely complex anatomy, both at the gross anatomical and histological levels. The main function of the EOM is to move the eyes in the orbit such that the eyes can be precisely positioned to allow focusing of the visual world on corresponding regions of each retina. Within each bony orbit the EOM includes four rectus muscles (superior,

lateral, inferior, and medial) and two oblique muscles (superior and inferior; Figure 1). A seventh skeletal muscle within the human orbit is the levator palpebrae superioris (LPS) muscle. It is the superior-most muscle in the orbit directly inferior to the frontal bone forming the orbital roof. The levator lies directly inferior to the periorbita and inserts via a large aponeurosis into the eyelid skin. The descriptions of the EOM will be primarily based on human muscles for ease of presentation. The general characteristics of size, shape, fiber type, and the like are similar for all EOM in principle, although they vary in detail for each specific animal that has been examined.

Gross Anatomy of the EOM within the Orbit The four rectus muscles originate from the apex of the bony orbit by a common tendinous annulus (of Zinn). The tendinous annulus attaches to the greater and lesser wings of the sphenoid bone as well as to the periosteum, the dense connective tissue lining the orbit. The annulus crosses over the inferior portion of the superior orbital fissure and runs superior and medial to the optic foramen (Figure 2). The superior (Figure 3) and medial rectus muscles arise from the superior part of this annulus, while the inferior and lateral rectus muscles arise from its inferior part. These muscles are all surrounded by a connective tissue capsule called Tenon’s capsule and are described as forming the muscle cone. The superior oblique (SO) muscle originates from the periosteum slightly superior and medial to the tendinous annulus. The inferior oblique (IO) muscle is the only EOM that does not arise from the orbital apex, but rather originates from the lateral border of the lacrimal fossa, which is anterior and nasal within the orbit. The rectus muscles run anteriorly to insert on the sclera on the anterior pole of the globe, at a location superficial to the ora serrata. The lateral and medial rectus muscles in human adults are approximately 41 mm in length. The superior rectus (SR) is the longest, averaging 42 mm, while the inferior rectus is the shortest averaging 40 mm. The insertions of the muscles onto the globe vary in their distance from the corneal limbus, with the SR furthest and the medial rectus closest. According to a study by Fuchs in 1884 on cadaver eyes, the distance from the limbus of rectus muscle insertions onto the globe

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Superior

LPS/SR SO ON LR

MR IR

Figure 1 Magnetic resonance imaging view of the orbit in cross section. LPS/SR: levator palpebrae superioris and superior rectus muscles. SO, superior oblique muscle; MR, medial rectus muscle; IR, inferior rectus muscle; LR, lateral rectus muscle; ON, optic nerve. MRI generously provided by Dr. Michael S. Lee at the University of Minnesota.

Superior

Medial

Figure 2 Bony orbit with the tendinous annulus indicated in the orbital apex by the black oval. It encloses the optic foramen superiorly and crosses the inferior portion of the superior orbital fissure.

decreases as one progresses sequentially in the following order: SR at 7.7 mm, lateral rectus at 6.9 mm, inferior rectus at 6.5 mm, and medial rectus at 5.5 mm. However, when measured in vivo during strabismus surgery the distances were shorter for all muscles: SR at 6.7 mm, lateral rectus at 6.2 mm, inferior rectus at 5.89 mm, and medial rectus at 4.5 mm. The insertions typically are circumferential to the limbus. However, wide variations in obliquity and regularity of the insertions are common, both from patient to patient and from muscle to muscle, even in the same eye. These differences are important in the context of planning surgery on the EOM for treatment of eye motility disorders, such as strabismus. Recent studies convincingly show that the distance from muscle

Figure 3 Dissection of the human orbit from the superior view with the orbital bony roof and the periorbita removed. The most superficial structure is the frontal nerve. Directly inferior to the frontal nerve is the levator palpebrae superioris (LPS, elevated by scissor tips). Inferior to the LPS is the superior rectus muscle.

insertion to the limbus has a high inter-individual variability, and there appears to be no correlation between insertional distance and the amount of deviation in strabismus patients. The lateral rectus is innervated by the abducens nerve (CNVI) on its intraconal or deep surface (Figure 4). The SR muscle is innervated on its intraconal surface by the superior division of the oculomotor nerve (CNIII), while the inferior and medial rectus muscles and the IO muscles

Extraocular Muscles: Extraocular Muscle Anatomy

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Figure 5 Deep orbital dissection from the superior view. The levator palpebrae superioris and superior rectus muscles are reflected medially to allow visualization of the superior and inferior divisions of the oculomotor nerve (CNIII). Figure 4 Dissection of the human orbit from the superior view. The bony roof, periorbita, levator palpebrae superioris, and superior rectus muscles are removed. The abducens nerve can be traced from where it pieces the dura in the posterior cranial fossa, through the superior orbital fissure, which has been opened in this dissection, to where it enters the lateral rectus muscle which it innervates.

are innervated by the inferior division of CNIII (Figure 5). All the cranial motor nerves except for the trochlear nerve (CNIV) enter the muscles intraconally, and all the motor nerves enter at approximately one-third of the muscle’s length from the orbital apex (Figure 6). The six EOMs control the position of the eye in the orbit while orbital fat and fascia constrain the paths of the muscles within the orbit. The two horizontal rectus muscles of each eye are agonist–antagonist pairs with relatively straightforward function; the medial rectus adducts the eye and the lateral rectus abducts it (Figure 7). By contrast, the SO and the IO muscles and the two vertical rectus muscles have far more complex functions. The primary direction of movement caused by the superior and inferior rectus muscles is elevation and depression, respectively. However, due to the shape of the bony orbit and their sites of origin and insertion, the vertical recti have secondary and tertiary actions that are torsional and horizontal, respectively. The SR, for example, is a secondary incyclorotator, moving

the superior pole of the eye toward the nose; its tertiary function is adduction. The inferior rectus muscle, by contrast, is also a secondary excyclorotator, but similar to the SR is a tertiary adductor (Figure 8). Thus, the SR, if acting unopposed, would elevate, adduct and incyclotort the eye such that the eye would be looking up and in. The inferior rectus, if acting unopposed, would depress, adduct, and excyclotort the eye such that the eye would be looking down and in. The superior and IO muscles have a similarly complex cyclovertical functions. The primary action of these two muscles is rotation or torsion, but due to the angle which they take in the orbit toward their insertion on the sclera, they will also elevate (IO) or depress (SO) the eye; both abduct the eye. The remarkable balance and integrity of the ocular motor plant becomes evident when one considers that to look straight superiorly without moving the head, both the IO (a primary excyclorotator, secondary elevator, and tertiary abductor) and the SR (a primary elevator, secondary incyclorotator, and tertiary adductor) coordinately contract. The same is true for the inferior rectus and SO muscles moving the eye straight inferiorly. The SO muscle is the thinnest, roundest, and longest of the EOM. The muscle runs 32 mm along the border of the medial wall and roof of the orbit, and 10–15 mm from the orbital margin it becomes tendinous and passes through

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SR

IO

Figure 8 The primary role of the superior rectus (SR) muscles is to elevate the eye; however, they have a secondary role in adducting the eye. The primary role of the inferior oblique (IO) muscles is to extort the eye, rotating the eye upward (elevation) and outward (abduction).

Figure 6 Deep orbital dissection from the superior view. The lateral, inferior, and medial rectus muscles are clearly visible, as are the nerves entering each muscle. The globe has been retracted anteriorly (by scissors).

Medial rectus adducts

Lateral rectus abducts

Figure 7 The lateral rectus (LR) and medial rectus (MR) muscles move the eyes in the horizontal plane. The MR adducts the eye, moving the eye toward the midline. The LR abducts the eye, moving the eye away from the midline. These muscles are antagonists; they have opposite functions.

a fibro-fascial structure called the trochlea, located anteriomedially under the orbital roof. The trochlea changes the orientation of the muscle, becoming the functional origin of this muscle. The tendon of the SO courses deep to the SR muscle to insert into the sclera laterally on the posterior pole of the globe. The position of the SO tendon insertion, posterior and temporal to the center of rotation of the globe, explains the complex cyclovertical function

Figure 9 The superior oblique muscle can be seen along the medial wall of the orbit. The trochlear nerve (CNIV) is seen coursing along the muscle’s superior surface and enters into the posterior 1/3 of the muscle on its medial side.

of the muscle. The trochlear nerve (CNIV) innervates this muscle, coursing first on the muscle’s superior side, and finally entering the muscle superiomedially at about the proximal one-third of the muscle’s length (Figure 9). The IO muscle is approximately 37 mm in length and travels in a similar orientation to the reflected tendon of the SO distal to the trochlea. The muscle runs inferior to


the inferior rectus muscle and inserts into the sclera of the posterior pole on the lateral side of globe. The insertion is relatively close to the macula of the retina. The IO is innervated by the inferior division of the oculomotor nerve (CNIII) on its superior surface with the nerves entering the muscle at approximately the posterior one-third.

Histological Anatomy of the EOMs Overall Organization The EOMs have a complex anatomical organization at the microscopic level. These muscles differ from those in the limbs and body in that the muscle fibers themselves have extremely small cross-sectional areas, with an average in human EOM of 340 200 mm2 (Figure 10). In addition, there is a great deal of variability in the myofiber crosssectional areas; this heterogeneity is quite striking. In cross-section, two distinct layers can be seen in all six EOMs, the orbital and the global layers. The orbital layer faces the bony orbit and is composed of myofibers with extremely small cross-sectional areas, with a mean of

Orbital

21

260 160 mm2. The global layer faces the globe, and the myofibers are markedly bigger than those in the orbital layer with a mean of 440 200 mm2. However, they are still very small compared to body and limb skeletal muscle myofibers, which typically range from 3500 to 4000 mm2 in the human soleus muscle, as an example. The total number of myofibers found within each of the six EOMs varies significantly. When measured in the mid-belly region of the muscles, the numbers of myofibers in the orbital layer in human EOM range from 7400 to 14 600 and in the global layer the numbers range from 8000 to 16 400 myofibers. This variation in myofiber number is seen in other species where the fiber number has been examined, although the range varies significantly from human. For example, monkey EOMs have approximately half the number of myofibers compared to human EOM. In addition, total myofiber number decreases along the length of each muscle as the insertions are approached. This variation in fiber number is due to the fact that the majority of myofibers within the EOM does not run tendon to tendon, as has been shown by a number of investigators. This can be demonstrated quite easily by serially sectioning muscles and following individual myofibers in consecutive sections (Figure 11). This is also supported by electrophysiological evidence demonstrating that the force produced by stimulating two separate motor unit groups individually is often more than the force produced by stimulating both motor groups simultaneously. This nonlinearity, or loss of force in summated motor units, is postulated to be caused by the lateral dissipation of force due to myofibers that do not reach the tendon ends.

Innervation

Global

Figure 10 A cross section of the medial rectus muscle from a Rhesus monkey immunostained for the presence of the fast myosin heavy chain (MyHC) isoform, which labels all forms of the fast MyHC. The orbital layer is composed of myofibers with extremely small cross-sectional areas, while myofibers in the global layer are somewhat larger. Bar is 50 mm.

Most skeletal muscles receive their motor nerve innervation in approximately the middle, and the neuromuscular junctions form a single endplate zone. Neuromuscular junctions are a pentomer composed of four distinct subunits: a (2), b, g, and d. During maturation, the g subunit is replaced with an e subunit, forming the adult acetylcholine receptor. In contrast to limb skeletal muscle, the EOM maintains a subpopulation of neuromuscular junctions with the immature subunit configuration. While the vast majority of EOM muscle fibers is fasttwitch fibers and receive a typical en plaque type of neuromuscular junction somewhere along their length, the EOM also contains two types of multiply innervated myofibers. These account for approximately 10% of the fibers in the global and orbital layers. In the global layer, these multiply innervated myofibers contain slow-tonic myosin and appear to be innervated by small en grappe endplates along their length. These en grappe neuromuscular junctions retain the g subunit of the acetylcholine receptor, rather

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(a)

(b)

Figure 11 Two serial sections from a normal rabbit superior rectus muscle. Several sets of muscle fibers in cross section are numerically identified. (a) Note two small myofibers present in this section, one to the right of group 5, 6, and 7 (black arrow), and one between fibers 3 and 4 (black arrow). (b) Note that in a section 20 mm from the section in (a) that those two small fiber ends have ended and thus are no longer present in the cross-section. Muscle sections immunostained to visualize dystrophin on the sarcolemma. Bar is 40 mm.

than the e subunit of the mature neuromuscular junction. There are reports in the literature that the en plaque endings in mammalian EOM can also express the immature g subunit of the acetylcholine receptor. This type of multiply innervated myofiber develops a slow-graded or tonic tension when the nerves are stimulated. The orbital layer has a second type of multiply innervated myofiber. These fibers have a neuromuscular junction in the central one-third of their length with typical en plaque structure. In addition, however, there are multiple en grappe endings at the myofiber ends. Thus, the central region displays fast-twitch properties with nerve stimulation while the fiber ends display slow-tonic contractile properties. As a result of fibers that do not run the origin-toinsertion length of the muscles and the presence of neuromuscular junctions on fiber ends, neuromuscular junctions are found throughout the length of the EOM. This has important implications for pharmacologic and surgical manipulations of the muscles in the treatment of eye motility disorders such as strabismus and nystagmus.

Skeletal Muscle Fiber Types From a physiological perspective, the EOMs have extremely fast contractile properties, produce low levels of force, and are relatively fatigue resistant. These properties are conferred on muscle fibers by their expression of specific contractile proteins such as the myosin heavy chain isoforms (MyHC), as well as differences in cellular organelles such as mitochondria and other cellular metabolic pathways. One unusual aspect of EOM histology is the expression, within and between these two layers, of a complex pattern of MyHC expression.

Myosin Isoform Complexity in the EOM Skeletal muscle fibers in body and limb muscles have generally been described as fast, expressing MyHC type IIa, IIb (nonhuman), and/or IIx/d, or slow, expressing MyHC type 1. However, this fiber type organization breaks down in the EOM, as was described by Mayr in 1971. On average, only 16% of the myofibers within the orbital layer are positive for the slow MyHC (MYH7), while 14% of the myofibers in the global layer are positive for slow MyHC. Thus, the vast majority of myofibers expresses the fast MyHC2a isoform (MYH2) which is responsible for the extremely fast contractile properties of the EOM. However, the EOMs express, in total, at least eight distinct MyHC isoforms: 2a, 2x/d, 1 (b-cardiac), developmental (embryonic), neonatal (perinatal), acardiac, slow-tonic, and EOM-specific. If serial sections of individual myofibers are examined immunohistochemically, it quickly becomes evident that single myofibers express more than one isoform (Figure 12). In the Kjellgren et al. study of adult human EOMs, single fibers immunostained for the slow or MyHC type 1 isoform can co-express either or both slow-tonic MyHC and acardiac MyHC. If just the slow myofibers are considered, they can express: 1. 2. 3. 4. 5. 6. 7. 8. 9.

only MyHC type 1, only slow-tonic, only a-cardiac, only EOM-specific, MyHC type1 and slow-tonic, MyHC type 1 and a-cardiac, myosin type 1 and EOM-specific, slow-tonic and a-cardiac, slow-tonic and EOM-specific,


Fast myosin

Developmental myosin

Neonatal myosin

10. 11. 12. 13. 14.

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a-cardiac and EOM-specific, slow type 1, slow-tonic, and a-cardiac, slow type 1, slow-tonic, and EOM-specific, slow-tonic, a-cardiac, and EOM-specific, or all four isoforms (Figure 13).

These combinations may result in 14 types of fibers. It is also known that embryonic (developmental) and neonatal-specific MyHC isoforms are also expressed by some of these fibers as well. The same complexity is seen with the myofibers positive for the fast MYHC type 2A. Single fibers can also express one of one or more of these isoforms: developmental, neonatal, EOM-specific, and 2x/d. These types of myofibers are referred to as hybrid fibers, and they can be seen, albeit to a lesser extent, in other muscles such as the diaphragm. Even when only MyHC expression characteristics are considered, the high degree of individual myofiber polymorphism seen in the collective data from many laboratories strongly supports the view that there is a continuum of myofiber types within the EOM. In some ways, trying to fit the EOM into the classical fiber typing scheme is misleading, as it does not deal effectively with the hybrid and mismatched fibers. This complexity has significant ramifications for muscle function. MyHC isoforms control muscle-shortening velocity, and it has been proposed that this type of polymorphism allows for fine-tuned control over a wide range of forces, velocities, and fatigue properties. From a teleological perspective, these coexpression patterns would allow the EOM to contract at high velocities but with minimal fatigue, a characteristic that is important for muscles that are continually functioning in order to maintain fixation of gaze on the fovea in an infinite number of eye positions. Additionally, studies have shown that the EOMs show rapid alterations in MyHC isoform expression in response to stretch, alterations in hormones, botulinum toxin treatment, denervation, and the like. Nonuniform Expression of MyHC Isoforms along the Muscle Length

Figure 12 Three serially cut cross sections from a region of a rabbit lateral rectus muscle approaching the anterior 1/3 of the muscle. They have been immunostained for fast, developmental, and neonatal myosin heavy chain (MyHC) isoforms. One group of muscle fibers has been circled in yellow with two fibers identified by a light-green arrow and a fiber numbered 2. Note that the fiber indicated by the green arrow is positive for fast and neonatal MyHC but negative for developmental MyHC. Note that fiber 2 is negative for fast MyHC but positive for developmental and neonatal MyHC. A second group of muscle fibers has been circled in red with two fibers identified as fiber 3 and 4. Fiber 3 is positive for fast MyHC but negative for both developmental and neonatal MyHC, while fiber 4 is positive for all three of these isoforms. The fiber indicated by the large blue arrow is negative for fast MyHC, but positive for both developmental and neonatal MyHC.

Immunohistological examination of cross-sections taken from the tendon ends and the middle region of an EOM shows that the overall percentages of specific MyHC isoforms change dramatically depending on the location along the muscle length (Figure 14). For example, in a study of rat EOM, within the orbital layer the mid-belly region expresses EOM-specific MyHC but this isoform is completely excluded from the tendon ends where the embryonic (developmental) MyHC isoform is present. This is seen even at the level of single isolated myofibers, where the fiber ends express neonatal MyHC and the mid-region of the same myofiber expresses fast MyHC. This type of non-uniform expression of MyHC along the length of single fibers also is seen in the intrafusal muscle

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Type 1

Type 1 α-cardiac

Type 1 slow tonic

Type 1 slow tonic a-cardiac

α-cardiac

Slow tonic

Type 1 eom-specific

Type 1 slow tonic eom-specific

Slow tonic α-cardiac

Type 1 α-cardiac eom-specific

eom-specific

Slow tonic eom-specific

Slow tonic α-cardiac eom-specific

α-cardiac eom-specific

Type 1 slow tonic a-cardiac eom-specific

Some possible hybrid fibers in extraocular muscle Figure 13 Diagrammatic representation of possible co-expression patterns when four myosin heavy chain isoforms are considered. Each red rectangle represents a single myofiber. Based on Kjellgren, D., Thornell, L. E., Andersen, J., and Pedrosa-Domellof, F. (2003). Myosin heavy chain isoforms in human extraocular muscles. Investigative Ophthalmology and Visual Sciences 44: 1419–1425.

(a)

(b)

(c)

(d)

Figure 14 Cross section from a single superior rectus muscle of an adult monkey taken from the orbital (a, b) and global (c, d) regions of the mid-region of the muscle (a, c) and the tendon end (b, d) immunostained for fast myosin heavy chain isoform (MyHC) (a, b) and neonatal MyHC (c, d). The orbital region is at the top of the photomicrographs in (a) and (b). Note that the orbital layer is thicker in (a), but that the individual myofibers have a smaller cross-sectional area in the mid-region compared to the tendon end. Additionally, there are more fast-negative fibers in the tendon region of the orbital layer. In the global layer, the mid-region has 20% of its myofibers positive for the neonatal MyHC, while the tendon end is almost devoid of this isoform. Bars is 50 mm.


fibers found in muscle spindles, another specialized myofiber structure. Using single myofiber reconstructions to localize neonatal MyHC isoform expression in single fibers, myofibers are found that are neonatal MyHCpositive from fiber tip to fiber tip, but it is more common to find single myofibers with variable percentages of the total fiber length expressing this isoform, including fibers where the expression is discontinuous. It is emerging that these types of hybrid fibers are present in other craniofacial muscles, such as jaw and laryngeal muscles. In limb and body skeletal muscles, innervation, neuromuscular activity, exercise (use/disuse), mechanical loading or unloading, hormones, and aging all cause adaptive changes in contractile properties and metabolic profiles. This supports the view that expression of contractile proteins within muscle fibers is extremely dynamic and possesses an incredible adaptability to meet the physiological demands placed upon them. EOMs are constantly active and appear to represent the far end of a continuum of skeletal muscle types relative to their ability to react and adapt to changing functional needs. As the MyHC controls shortening velocity, this MyHC polymorphism would result in a gradation of function within populations of single myofibers. In other words, a continuum of force and movement would be possible, as the contractile properties of each myofiber would reflect its particular subset of contractile proteins. An example of this is shown in Figure 15, where force was determined using a single fiber preparation and the MyHC content was analyzed for each single fiber using polyacrylamide gel separation. As can be seen, fibers that produce the same amount of force can have significantly different MyHC isoform expression patterns. While it has not been demonstrated specifically within EOM myofibers, it is a well-known characteristic of multinucleated myofibers that each myonucleus controls

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the expression of proteins in what is called its myonuclear domain. An elegant study by the Hardeman laboratory showed that transcription occurs in pulses within individual myonuclei, and the activities of single nuclei are not in sync with each other. Each nucleus, and thus each myonuclear domain, is individually controlled, and protein synthesis is a dynamic process that is altered at the local level to respond to the particular stress or strain. The advantage conferred by this complexity within populations of individual EOM myofibers can be hypothesized as an adaptation to the functional needs of eye movements in maintaining binocular vision and highly coordinated vergence movements, where a continuum of contraction forces and speeds would be required. Functionally, the complex MyHC co-expression patterns, and their presumed continuous modulation, would allow finely tuned control over these movements, as the kinetics of the EOM would cover a wide range of eye positions and velocities. Other Molecules Heterogeneously Expressed The heterogeneity of individual myofibers is made even more complex not only by differences in other contractile proteins, such as myosin light chains and troponin, but also by other metabolic differences. Of the molecules that have been specifically examined, the EOM often has patterns of expression not seen in limb muscle. For example, myosinbinding protein C has three isoforms. Despite the fact that the vast majority of myofibers within human EOM is positive for fast MyHC, the EOM does not express the fast form of myosin-binding protein C. Recent work showed that, in contrast to limb skeletal muscles, high levels of glycolytic and oxidative pathways coexist within single myofibers in the EOM. This molecular mismatch would provide these very active muscles with both fatigue resistance and fast contractile properties.

Continuous Remodeling in Normal Adult EOM

Fiber 2

Fiber 1

0

10

20 IIA

30 40 50 60 70 MyHC isoform (%) IIX

Neonatal

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90 IIB

Figure 15 Relative percentages of four myosin heavy chain isoforms from two single-skinned myofibers with the same shortening velocity of 9.4 fiber lengths per second as determined using single-skinned fiber physiology in the EOM of rabbits. Note that fiber 1 has three isoforms expressed, and it contains type IIX as its main isoform, while fiber 2 has only two isoforms expressed, and IIB is the isoform with the greatest amount of expression.

Early studies by Moss and LeBlond demonstrated that the myonuclei within mature, multinucleated myofibers are postmitotic. However, muscle has regenerative capacity that resides in myogenic precursor cells called satellite cells, and these cells become activated, divide, and are responsible for muscle repair and/or regeneration of new fibers in disease and after injury. The EOM in normal adult mammals maintain an elevated number of satellite cells throughout life (Figure 16), which divide and integrate continuously into apparently normal muscle fibers. Concomitantly with nuclear addition, apoptosis of individual myonuclei is seen with apparent segmental cytoplasmic remodeling. The factors that control this process are unknown, but this process represents another dynamic

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Figure 16 Several longitudinally sectioned myofibers from the inferior oblique muscle of a human cadaver donor. Note three satellite cells positive for Pax-7, a marker of all satellite cells. Two satellite cells in close proximity to each other is a common observation in the extraocular muscles.

physiological property of the EOM that would allow continuous adaptation of single fiber functional properties in response to physiological needs. The existence of ongoing remodeling in normal adult EOM suggests that there may be ways to modulate this process in vivo to alter muscle size, force, or response to injury or disease. In particular, it suggests new hypotheses to explain the preferential sparing or involvement of the EOM in skeletal muscle disease.

Acknowledgments This work was supported by EY15313 and EY11375 from the National Eye Institute, the Minnesota Medical Foundation, the Minnesota Lions and Lionesses, Research to Prevent Blindness (RPB) Lew Wasserman Mid-Career Development Award (LKM), and an unrestricted grant to the Department of Ophthalmology from RPB. See also: Extraocular Muscles: Extraocular Muscle Metabolism; Extraocular Muscles: Functional Assessment in the Clinic; Eyelid Anatomy and the Pathophysiology of Blinking.

Further Reading Asmussen, G., Punkt, K., Bartsch, B., and Soukup, T. (2008). Specific metabolic properties of rat oculorotatory extraocular muscles can be linked to their low force requirements. Investigative Ophthalmology and Visual Sciences 49: 4865–4871.

Caiozzo, V. J., Baker, M. J., Huang, K., et al. (2003). Single-fiber myosin heavy chain polymorphism: How many patterns and what proportions? American Journal of Physiology – Regulatory, Integrative and Comparative Physiology 285: R570–R580. Harrison, A. R., Anderson, B. C., Thompson, L. V., and McLoon, L. K. (2007). Myofiber length and three-dimensional localization of NMJs in normal and botulinum toxin-treated adult extraocular muscles. Investigative Ophthalmology and Visual Sciences 48: 3594–3601. Jacoby, J., Chiarandini, D. J., and Stefani, E. (1989). Electrical properties of multiply innervated fibers in the orbital layer of rat extraocular muscles. Journal of Neurophysiology 61: 116–125. Kallestad, K. M. and McLoon, L. K. (2008). Myogenic precursor cells in the extraocular muscles. In Low, W. C. and Verfaillie, C. M. (eds.) Stem Cells and Regenerative Medicine. Hackensack, NJ: World Scientific. Kaminski, H. J., Kusner, L. L., and Block, C. H. (1996). Expression of acetylcholine receptor isoforms at extraocular muscle endplates. Investigative Ophthalmology and Visual Sciences 37: 345–351. Kjellgren, D., Thornell, L. E., Andersen, J., and Pedrosa-Domellof, F. (2003). Myosin heavy chain isoforms in human extraocular muscles. Investigative Ophthalmology and Visual Sciences 44: 1419–1425. Li, Z. B., Rossmanith, G. H., and Hoh, J. F. Y. (2000). Cross-bridge kinetics of rabbit single extraocular and limb muscle fibers. Investigative Ophthalmology and Visual Sciences 41: 3770–3774. Mayr, R. (1971). Structure and distribution of fiber types in the external eye muscles of the rat. Tissue and Cell 3: 433–462. McLoon, L. K., Rowe, J., Wirtschafter, J. D., and McCormick, K. M. (2004). Continuous myofiber remodeling in uninjured extraocular myofibers: Myonuclear turnover and evidence for apoptosis. Muscle and Nerve 29: 707–715. Shall, M. S., Dimitrova, D. M., and Goldberg, S. J. (2003). Extraocular motor unit and whole-muscle contractile properties in the squirrel monkey. Summation of forces and fiber morphology. Experimental Brain Research 151: 338–345. Stephenson, G. M. M. (2001). Hybrid skeletal muscle fibers: A rare or common phenomenon? Clinical and Experimental Pharmacology and Physiology 28: 692–702.

Extraocular Muscles: Extraocular Muscle Metabolism F H Andrade, University of Kentucky Medical Center, Lexington, KY, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Adenine nucleotide translocator – An inner mitochondrial protein that exchanges adenosine diphosphate (ADP) and adenosine triphosphate (ATP) between the mitochondrial matrix and the cytoplasm. It is also known as the ADP/ATP translocator. Chronic progressive external ophthalmoplegia – A syndrome characterized by progressive inability or difficulty to move the eyes and elevate the eyelids. It is a common manifestation of some mitochondrial diseases. Electron transport chain – The set of mitochondrial protein complexes that couples the oxidation of electron donors (such as NADH) to the reduction of electron acceptors (such as oxygen) in order to produce ATP. Gene expression profiling – The measurement of the activity (or expression) of large numbers of genes simultaneously. Glycolysis – The metabolic pathway that converts glucose to pyruvate, with a net result of 2 ATP and 2 NADH. M line – A dark band or line seen in the center of the sarcomeres, using electron microscopy. NADH – Nicotinamide adenine dinucleotide (NAD+) and its reduced form NADH are coenzymes involved in oxidation/reduction reactions as electron acceptors and donors. Oxidative phosphorylation – The metabolic pathway that uses the energy released during the oxidation of nutrients to produce ATP.

The extraocular muscles exhibit the greatest diversity among mammalian skeletal muscles, a likely consequence of the varied functional requirements imposed by the ocular motor system. Extraocular muscle fibers differ from typical limb and respiratory skeletal muscles in mitochondrial content, innervation/contractile patterns, contractile protein isoforms, hormone receptors, cell surface markers, and a variety of other cell and molecular properties that may relate to their unique functions. The divergence from the skeletal muscle stereotype is further exemplified by the fact that extraocular muscles do not conform to traditional fiber type classifications, which are based primarily on myosin isoform expression. The most

accepted fiber-type classification scheme for extraocular muscles includes six fiber types based upon: (1) distribution into orbital and global layers, (2) innervation status, single versus multiple nerve contacts per fiber, and (3) mitochondrial/oxidative enzyme content. Figure 1 shows representative micrographs of extensor digitorum longus (EDL, which is a predominantly type IIB fiber – fast, fatigable – limb skeletal muscle), diaphragm (mixed fiber type, fatigue-resistant respiratory muscle), and the extraocular muscle from rats. EDL is recruited sporadically and diaphragm is constantly active as is the extraocular muscle. Despite the wide difference in activity, EDL and diaphragm sections are mostly indistinguishable. For sure, there are important biochemical differences between the two muscles that reflect their specific adaptations to their respective activation patterns. However, the divergent requirements of EDL and diaphragm motor systems are met with fairly stereotypical muscle fibers, as evident from the micrographs. In contrast, the extraocular muscle fibers are very different: small round fibers with prominent mitochondria, suggestive of atypical contractile and metabolic properties. This article outlines newly identified unique aspects of extraocular muscle metabolism and how they may correspond to contractile function.

Insights from Gene Expression Profiling The fast and constant contractions of the extraocular muscles necessitate well-developed energy supply systems. It might be expected a priori that these muscles would upregulate all the main energy-supply metabolic pathways, from glycolysis to mitochondrial metabolism. Surprisingly, this may only apply to mitochondrial content, which is the highest reported in mammalian skeletal muscles. Studies comparing the gene expression profile of extraocular and limb muscles found that genes coding for key enzymes of glycogen synthesis and breakdown were repressed in the extraocular muscles. Glycogen content in the extraocular muscles is correspondingly reduced. These findings indicate that the extraocular muscles are seemingly less dependent on stored glycogen as a metabolic fuel than other skeletal muscles. They also suggest that the extraocular muscles rely, instead, on constant transport of blood-borne glucose and fatty acids through their extensive microvascular network. Interestingly, the expression of the lactate dehydrogenase (LDH) isoform that preferentially oxidizes lactate to pyruvate is increased

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Figure 1 The extraocular muscles are not typical skeletal muscles. The figure shows representative micrographs of limb (left, extensor digitorum longus), respiratory (middle, diaphragm), and extraocular (right) muscle sections stained with Gomori’s trichrome. This technique stains mitochondria (and sarcoplasmic reticulum) a darker reddish-blue. Despite the differences in functional profiles (occasionally active limb muscle vs. constantly active diaphragm), notice the similarity in fiber size and shape: large polygonal muscle fibers with peripheral myonuclei (left and middle panels). Some fibers have darker cytosolic staining indicating higher mitochondrial content. In contrast, the extraocular muscle shows significantly smaller fibers with clumpy cytosolic and subsarcolemmal staining due to abundant mitochondria (right panel). Scale bars ¼ 25 mm.

in the extraocular muscles, compared to typical skeletal muscles, and that may allow them to use lactate as a fuel for aerobic pathways. The importance of these alternative metabolic pathways is now being tested in extraocular muscles.

Lactate: An Oxidizable Substrate for the Extraocular Muscle In limb skeletal muscles, glycogen breakdown drives glycolysis only during brief bursts of intense activity (Figure 2). In most muscles, metabolic demand during moderate activity is met by aerobic (mitochondrial) pathways. During periods of sustained peak activity, when mitochondrial capacity is exceeded in skeletal muscle, lactate is the end product of glycolysis in a reaction that reduces pyruvate at the expense of NADH and is catalyzed by LDH. For this and other reasons, increased production and accumulation of lactic acid during exercise has been associated with muscle fatigue. However, cells can also use the LDH reaction in the reverse direction from lactate oxidation to pyruvate, and lactate then goes on to become a substrate for aerobic metabolism. As mentioned above, the expression of the LDH isoform that preferentially oxidizes lactate to pyruvate is higher in the extraocular muscles. Combined with their high aerobic capacity, this reaction would allow the extraocular muscles to use lactate as a metabolic substrate. Cinnamate, a blocker of lactate transport, alone or in combination with exogenous lactate can be used to evaluate the role of lactate on fatigue resistance. Cinnamate accelerates fatigue in the extraocular muscles significantly: treated muscles lose their ability to generate force at a faster rate than untreated extraocular muscles. Conversely, cinnamate treatment does not affect the endurance or residual force of limb muscles. Replacing glucose with exogenous

lactate increases limb-muscle fatigability but has no effect on the extraocular muscles. However, the extraocular muscles fatigue faster when exposed to exogenous lactate combined with cinnamate treatment. These results indicate that LDH oxidation of lactate to pyruvate seems to be an important source of metabolic substrate for aerobic metabolism in the extraocular muscles. This conclusion is a significant deviation from the traditional view of lactate as a final waste product of glycolysis; increased lactate production and accumulation during vigorous contractile activity is typically associated with fatigue. Muscle fatigue is a complex phenomenon: substrate depletion, metabolite accumulation, and ionic imbalances are some of the factors that combine to reversibly impair contractile function. In the particular case of the extraocular muscles, lactate can be used via the LDH reaction as an additional substrate source for aerobic metabolism, a concept developed recently for other aerobic muscles and one that also applies to the nervous system.

Creatine Kinase, the Missing ATP Buffer in the Extraocular Muscle Skeletal muscles and other tissues with fluctuating metabolic needs rely on the creatine–phosphocreatine system to buffer intracellular ATP concentration: creatine kinase (CK) catalyzes the reversible transfer of the phosphoryl group from phosphocreatine to ADP in order to maintain constant ATP levels. Cellular CK activity is due to a family of oligomeric enzymes: two cytosolic, ubiquitous brain-type CK-B and muscle-type CK-M, and two mitochondrial isoforms, ubiquitous mitochondrial CK (uCK) and sarcomeric mitochondrial CK (sCK). In differentiated skeletal muscle, CK-MM and sCK are the predominant isoforms. In fast-twitch muscles, most CK activity is due to the CK-MM isoform, some of which is found

Extraocular Muscles: Extraocular Muscle Metabolism

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Glycolysis Glucose ATP ADP Glucose 6-phosphate

Fructose 6-phosphate ATP ADP Fructose 1,6-diphosphate

Glyceraldehyde 3-phosphate

Glyceraldehyde 3-phosphate

NAD

2 ADP

2 ADP

NAD

NADH

2 ATP

2 ATP

NADH

NADH

Pyruvate

Pyruvate

NAD

NAD Lactate

NADH

(Krebs cycle)

Lactate

Figure 2 Glycolysis, the anaerobic breakdown of glucose. The figure presents a diagram showing the sequence of steps in glycolysis, from glucose to pyruvate. The end product, pyruvate, may move on to the Krebs cycle (inside the mitochondria) to continue substrate oxidation, or it may be reduced to lactate in the reaction catalyzed by LDH that restores NAD. Dashed arrows represent omitted steps.

associated with the sarcomeric M line, the sarcoplasmic reticulum, and T-tubules. This arrangement couples the CK-dependent ATP-buffering system to the cellular sites with the highest ATPase activity, and is needed for normal contractile function. Given the predicted need for ATP buffering in the extraocular muscles, we proposed that (1) CK isoform expression and activity in rat extraocular muscles would be higher and (2) the resistance of these muscles to fatigue would depend on CK activity. Instead, we found that messenger ribonucleic acid (mRNA) and protein levels for all (cytosolic and mitochondrial) CK isoforms are lower in the extraocular muscles than in limb muscles. The muscle-enriched isoforms, CK-M and sCK, are less abundant in extraocular muscle, despite the fact that the extraocular muscles have a higher mitochondrial content than limb muscles. Total CK activity is also correspondingly decreased in the extraocular muscles. Moreover, cytoskeletal components of the sarcomeric M line, where a significant fraction of cytosolic CK activity is found, are downregulated in the extraocular muscles as was initially suggested by gene expression profiling. To explore the role of CK activity on muscle function, the CK inhibitor 2,4-dinitro-1-fluorobenzene (DNFB) was used during an in vitro fatigue protocol. Treatment with DFNB accelerates the development of fatigue in limb muscle, but has no detectable effect on the

extraocular muscles. These data support the conclusion that CK activity is not an important ATP buffer in the extraocular muscles. The myokinase reaction (2 ADP ! ATP þ AMP), catalyzed by adenylate kinase (AK), serves as an additional ATP-buffering system in skeletal muscle. While total AK activity is similar in extraocular and limb muscles, the mRNA content for two putative mitochondrial AK isoforms (AK3 and AK4) is over 13-fold more abundant in the extraocular muscles. This suggests that the relative lack of CK in the extraocular muscles may be compensated by upregulation of selected AK isoforms.

Mitochondrial Content in the Extraocular Muscles Aerobic capacity is typically measured by mitochondrial volume density (percentage of muscle fiber volume occupied by mitochondria). In general, mitochondrial volume density is well matched to the metabolic needs of skeletal muscle and it scales almost linearly with maximal oxygen uptake among muscles and across mammalian species. In other words, the consensus is that changes in the oxidative (aerobic) capacity of mammalian skeletal muscles are met by corresponding increases or decreases in mitochondrial volume density. Since the mitochondrial content and the

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activity of respiratory complexes and enzymes of mitochondrial metabolic pathways change in parallel, enzymatic activities are used as indices of mitochondrial content and aerobic capacity. Highly aerobic muscle groups in mammals have abundant capillaries and elevated mitochondrial volume density. The extraocular muscles have, arguably, the highest mitochondrial content of all mammalian skeletal muscles. However, the mechanism responsible for maintaining mitochondrial abundance in the extraocular muscles remains unclear. We have identified a number of transcription factors that influence mitochondrial biogenesis and that are upregulated in the extraocular muscles. Surprisingly, these factors are different from the mitochondrial biogenesis program initiated in response to endurance training.

Mitochondria as Calcium Sinks in the Extraocular Muscle The fast-contracting extraocular muscles rely on tight regulation of free cytosolic calcium concentration ([Ca2+]i). In principle, the extraocular muscles have the profile of very efficient calcium handling capacity: extensive and welldeveloped sarcoplasmic reticulum and the expression of fast calcium ATPase isoforms. Moreover, the extraocular muscles contain parvalbumin, a low-weight calcium-binding protein that serves as a temporary buffer to accelerate the removal of calcium off its binding sites on the myofilaments and facilitates muscle relaxation. Other investigators have already shown that the kinetics of calcium flux into mitochondria are fast enough to influence very rapid events such as neurotransmitter release from motor nerve terminals. The specific inhibition of mitochondrial calcium transport slows the relaxation of mitochondria-rich skeletal muscles. We recently reported that the magnitude and speed of calcium uptake by mitochondria are sufficient to influence contractile function. This property of the extraocular muscles appears to serve at least two complementary functions. First, it couples metabolic supply to demand because higher mitochondrial calcium stimulates enzymes that control substrate oxidation: pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, isocitrate dehydrogenase, and glycerol 3-phosphate dehydrogenase. The combined activity of these enzymes sustains a high NADH/NAD+ level and maximizes oxidative phosphorylation and ATP production. Second, by limiting the [Ca2+]i increase during contractions in response to submaximal stimulation frequencies, mitochondria widen the dynamic range of the extraocular muscles. In other words, the capacity of the extraocular muscles to produce force is spread over a wider stimulation frequency range, increasing the fine control of the effector arm of the ocular motor system.

Are Extraocular Muscle Mitochondria Different? The adenine nucleotide transporter 1 (Ant1) gene encodes an inner mitochondrial membrane protein that transports ADP into mitochondria and ATP from mitochondria to the cytosol. Mutations within Ant1 have been shown to produce a syndrome of chronic progressive external ophthalmoplegia (CPEO) in humans. Ant1 knockout (Ant1–/–) mice develop cardiomyopathy and severe exercise intolerance. Despite this dramatic phenotype, the extraocular muscles are mostly unaffected. Histologically, the extraocular muscles from Ant1–/– mice present a relatively mild mitochondrial myopathy. There are no measurable ocular motor abnormalities in Ant1–/– mice, and their peak eye velocities overlap with those measured in control mice. Moreover, their extraocular muscles do not show evidence of increased fatigability. In addition, the extraocular muscles have higher levels of Ant2 mRNA compared to the limb muscles. Ant2 is a nonskeletal muscle isoform previously described in the heart. Its presence in the extraocular muscles may explain the lack of effects of Ant1 loss, and it was the first documented difference between extraocular muscle and limb muscle mitochondria. The ability of muscles to perform aerobic work depends on their mitochondrial volume density, with the assumption that the composition of these organelles is fairly constant across muscle types and mammalian species. One of these components is the electron transport chain, a series of multimeric complexes (complexes I–IV, plus the ATP synthase which is sometimes called complex V) in the inner mitochondrial membrane responsible for most of the aerobic ATP generation (Figure 3). Recently, we found that the extraocular muscle mitochondria have lower content or lower activity of some enzyme complexes of the electron transport system, causing them to respire at slower rates. This is puzzling given that the extraocular muscles are constantly active and aerobic capacity was predicted to be elevated, given their high mitochondrial content. These findings are not explained by differences in the ultrastructure of extraocular muscle mitochondria: the surface area of their inner membrane is comparable to values reported for other skeletal muscle. Furthermore, the differences are not generalized or systematic: complex II content and activity, and complex III content are similar in mitochondria from triceps surae (a limb skeletal muscle) and extraocular muscle. Complexes I and IV give a more puzzling result: their activities are lower, but their content is higher in the extraocular muscle mitochondria. These are multimeric protein complexes, and differential expression of isoforms of some subunits has been described in skeletal muscle and other tissues.

Extraocular Muscles: Extraocular Muscle Metabolism

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Outer membrane +

H+

H

H+ Intermembrane space

lll

l

lV

ll ATP synthase

H ADP + Pi

+

ATP

H2O

Krebs cycle

Matrix Fumarate

H2O

O2

NADH

NAD+ + H+

Succinate

O2 lV

lll

H+

ll

Inner membrane l

H+

Figure 3 Mitochondrial electron transport chain and ATP synthase. The figure presents a drawing showing the oxidative phosphorylation steps that couple the final substrate oxidation to the reduction of oxygen to water, pumping hydrogen ions (protons, Hþ) from the mitochondrial matrix to the intermembrane space. The substrates for this chain are NADH or succinate, shown here as originating from the Krebs cycle. Complex I (NADH dehydrogenase) oxidizes NADH and transfers the electrons to complex III, which in turn transfers the electrons to complex IV (cytochrome c oxidase). The latter is the complex that reduces oxygen to water. Complex II (succinate dehydrogenase) is not an Hþ pump; it funnels electrons from succinate to complex III, and then complex IV. The ATP synthase (complex V) is driven by the trans-inner membrane electrochemical potential generated by the movement of Hþ to the intermembrane space.

Therefore, the content of some electron transport chain complexes (I, IV, and V) and the subunit composition of some others (I and IV) may not be the same in the extraocular muscles compared to limb muscles. This demonstrates that the metabolic divergence between extraocular and limb muscles includes major differences in the composition and basic function of their respective mitochondrial populations. Intrinsic differences in mitochondrial structure and function may explain the susceptibility of the extraocular muscles to some hereditary and acquired mitochondrial myopathies such as CPEO and related syndromes. For example, the extraocular muscles present the most severe age-dependent loss of mitochondrial respiratory complex activity among muscles. There is a significant increase in the number of fibers with cytochrome c oxidase defects in the extraocular muscles of humans and other primates, even when compared to other highly aerobic muscles such as the diaphragm and heart. This can be at least partially explained by mitochondrial DNA mutations, presumably due to reactive oxygen species generated during mitochondrial respiration or present as part of a more generalized cellular oxidative stress.

Matching Mitochondrial Capacity to Contractile Function The primary role of mitochondria is to generate ATP. Recent studies lead to an obvious question: How do extraocular muscles sustain their contractile function with mitochondria that respire half as fast as mitochondria from other muscles? The content of respiratory complexes is one parameter behind tissue variations in mitochondrial respiration, although some argue that it is not particularly relevant for metabolic control. Under experimental conditions, mitochondrial respiration in the skeletal muscle and heart is regulated at the level of the respiratory chain, while in the liver, kidney, and brain it is controlled mainly at the phosphorylation level by ATP synthase (complex V) and phosphate carrier. That may not be the case in vivo, where different parameters such as cellular steady state, the energy demand, and the energy supply of the tissue may also regulate mitochondrial respiration. In the case of the extraocular muscles, allosteric regulation of respiratory complexes may combine with changing metabolite concentrations to maintain mitochondrial respiration closer to its theoretical maximum.

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For example, a mechanism to enhance energy production in the extraocular muscle is mitochondrial calcium influx during contractile activity in order to activate enzyme systems that exert strong control on substrate oxidation, as mentioned above.

Acknowledgements The author’s work in this field is supported by the National Eye Institute (grant R01 EY012998). See also: Extraocular Muscles: Extraocular Muscle Anatomy.

Matching Energy Supply to Demand Initially inspired by morphological characteristics and gene-expression-profiling results, a more global perspective of extraocular muscle metabolism is beginning to emerge. First, glycogen content is low and the glycogenolysis pathway seems to be downregulated in the extraocular muscles. Second, CK activity and content, including the mitochondrial isoform, are lower in the extraocular muscles, indicating that phosphocreatine may be a less important temporal and spatial ATP buffer in these muscles. In other words, mitochondrial ATP production may be sufficiently high and close to cellular sinks as to obviate the need for an energy buffer. Third, the extraocular muscles can use lactate as an oxidizable substrate due to the presence of a LDH isoform that catalyzes the conversion of lactate to pyruvate that then goes to the Krebs cycle. Finally, the mitochondrial population in extraocular muscles appears to respond to a different biogenesis program, and exhibits atypical functional characteristics that may influence the contractile activity of these muscles significantly.

Further Reading Andrade, F. H. and McMullen, C. A. (2006). Lactate is a metabolic substrate that sustains extraocular muscle function. Pflu¨gers Archiv-European Journal of Physiology 452: 102–108. Andrade, F. H., McMullen, C. A., and Rumbaut, R. E. (2005). Mitochondria are fast Ca2+ sinks in rat extraocular muscle: A novel regulatory influence on contractile function and metabolism. Investigative Ophthalmology and Visual Science 46: 4541–4547. McMullen, C. A., Hayeß, K., and Andrade, F. H. (2005). Fatigue resistance of rat extraocular muscles does not depend on creatine kinase activity. BMC Physiology 5: 12. Porter, J. D., Khanna, S., Kaminski, H. J., et al. (2001). Extraocular muscle is defined by a fundamentally distinct gene expression profile. Proceedings of the National Academy of Sciences of the Unites States of America 98: 12062–12067. Spencer, R. F. and Porter, J. D. (2006). Biological organization of the extraocular muscles. Progress in Brain Research 151: 43–80. Yin, H., Stahl, J. S., Andrade, F. H., et al. (2005). Eliminating the Ant1 isoform produces a mouse with CPEO pathology but normal ocular motility. Investigative Ophthalmology and Visual Science 46: 4555–4562.

Extraocular Muscles: Proprioception and Proprioceptors R Blumer, Medical University of Vienna, Vienna, Austria ã 2010 Elsevier Ltd. All rights reserved.

Glossary Choline acetyltransferase – The enzyme responsible for the synthesis of the neurotransmitter acetylcholine, causing the transfer of acetate to choline. Choline transporter – These recapture choline from the synaptic cleft after acetylcholine release and degradation. This process is critical for new acetylcholine synthesis at the synapse. Golgi tendon organ – A proprioceptive organ that provides information to the brain about changes in muscle tension. In contrast to muscle spindles, these are in series with muscle fibers, interwoven with the collagen in the muscle tendon. Tension on the tendon caused by muscle contraction activates these proprioceptors. Muscle spindles – Proprioceptive organs that provide the brain with information about changes in muscle length and are organized in parallel with the skeletal muscle fibers. They contain modified muscle fibers called intrafusal fibers, in contrast with the muscle fibers themselves, which are called extrafusal fibers. They have a complex structure; they are surrounded by a connective tissue capsule, and contain several types of modified myofibers within them. They are innervated by sensory afferents. Myotendinous cylinders or palisade endings – Proprioceptive organs found at the myotendinous junction consisting of dense axonal branching which invests the tips of single muscle fibers. These are unique to the extraocular muscles. The nerves establish synaptic contacts with both collagen fibrils and muscles fibers at the myotendinous junction. The function of these structures is unknown. Proprioception – The sense that provides information about the location of various parts of the body in relation to each other and in relation to the space. Proprioceptors – Sensory receptors which are found in muscles and tendons that bring sense of body position to the brain. Vesicular acetylcholine transporter – A membrane protein which is necessary for the uptake of acetylcholine into synaptic vesicles.

Proprioception Proprioception refers to a sense that provides information about the location of various parts of the body in relation to each other and in relation to space. It is of practical importance for activities in everyday life and allows a person to use the foot pedal of a car properly while driving or to learn to walk in darkness. Moreover, sportsmen use specific training devices to sharpen their proprioceptive sense. Proprioceptive signals come from specialized sensory nerve endings called proprioceptors that occur throughout skeletal muscle. Typical proprioceptors in skeletal muscle are muscle spindles and Golgi tendon organs which constantly transmit information to the brain. In this way the brain knows, at any given time, the spatial position of our body parts. The eyes are the most mobile organs of the body, and vision is useful only if the brain knows the position of the eyes in the orbit. By knowing where the eyes are pointing, the brain is aware of the position of objects in the surrounding space: if objects are leftwards, straight ahead, or rightwards. Several studies indicate that the brain has access to proprioceptive information from the extraocular muscles (EOMs). Specifically, neuronal tracing experiments have demonstrated projections from the EOM to various peripheral and central nervous system structures, including the trigeminal ganglion, the mesencephalic trigeminal nucleus, the superior colliculus, the vestibular nuclei, and the cerebellum. A recent physiology experiment showed that the primary somatosensory cortex, which receives proprioceptive input from all other skeletal muscles, also receives signals from EOM. This new finding completes the somatotopic representation of the body in the primary somatosensory cortex which, thus far, had lacked a map of the eye muscles. Indication that there is proprioceptive input from the EOM has also come from psychophysical investigations. Patients suffering from strabismus were tested after surgery, and it was detected that they had deficits in spatial perception. These results were interpreted to mean that the surgical intervention has damaged the proprioceptors at the myotendinous junction resulting in a loss of eye position signals. Despite this evidence for EOM proprioception, there are also counterarguments. Specifically, no stretch reflex has been observed in the EOM of monkey. By cutting the ophthalmic nerve, which is supposed to carry the afferent fibers from EOM, deficits in eye movements would be

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expected. However, findings indicate that deafferentation does not affect ocular alignment or eye movements, including saccades and smooth pursuit. Such observations led scientists to doubt whether EOM proprioception really exists. Instead, it has been hypothesized that the motor command that is sent to the EOMs is copied, called efference copy, and this copy provides the necessary information for the brain to be aware of the eye’s position. If there is sensory feedback from EOM, the eye muscles should have proprioceptors. In the last century, the EOMs of several mammalian species and man have been screened for muscle spindles and Golgi tendon organs. Interestingly, the endowment with classical proprioceptors varies widely among the species, and there are even some species that do not have proprioceptors at all. In view of these interspecies variations, it is not clear where the source of EOM proprioception lies. By searching for alternative sensory organs in the EOMs, palisade endings (also called myotendinous cylinders) have been detected, and so far, palisade endings have been observed in each species investigated. In the following section, we discuss EOM proprioceptors, including muscle spindles, Golgi tendon organs, and palisade endings. We give an overview about the occurrence, distribution, number, and structure of these organs and speculate about their putative function. Recent studies have focused on the molecular characteristics of palisade endings, and we also refer to these findings.

Muscle Spindles Occurrence, Distribution, and Number of Muscle Spindles Muscle spindles are regularly observed in the EOMs of even-toed ungulates (sheep, cow, camel, goat, and pig) and in the EOMs of primates (monkey and man). In other animal species, including fellidae (cat), rodents (rat and guinea pig), odd-toed ungulates (horse), and lagomorphs (rabbit), muscle spindles have not been found (Table 1). In the EOMs of even-toed ungulates, muscle spindles are uniformly distributed throughout the entire muscle length. The number of muscle spindles is remarkably high, and counts per muscle yield between 146 and 333 muscle spindles in pig; between 100 and 181 muscle spindles in camel; and more than 200 muscle spindles in cow. In man the distribution of muscle spindles exhibits differences when compared with that in even-toed ungulates. Specifically, muscle spindles are located predominantly in the proximal and distal parts of the EOM, and each muscle has a spindle-free zone approximately in the middle. The number of human EOM spindles varies between 13 and 42. Only in the inferior oblique muscle has a lower number of muscle spindles been counted (3–7). The density of human EOM spindles is comparable

Table 1 Occurrence of muscle spindles, Golgi tendon organs, and palisade endings in the extraocular muscles of man and mammals

Species

Muscle spindles

Golgi tendon organs

Man Monkey Fellidae Even-toed ungulates Odd-toed ungulates Lagomorphs Rodents

þ þ þ

þ

a

Palisade endings þ þ þa

b

þ þ

So far, palisade endings have only been demonstrated in sheep. So far not analyzed for palisade endings.

b

to that of muscle spindles in finely controlled skeletal muscle such as the hand lumbrical and deep dorsal neck muscles. In monkey (rhesus monkey and cynomolgus monkey), very few muscle spindles (2–6) have been observed in some EOMs, and none in the others. Structure of Muscle Spindles Muscle spindles in the EOMs of even-toed ungulates conform in their structure with those in other skeletal muscles. The muscle spindles have a fusiform shape with a wide central region (equatorial region) and two narrow polar regions. Muscle spindles are ensheathed by a capsule consisting of several layers of perineural cells. The capsule space is filled with a viscous fluid containing acidic mucopolysaccharides. Inside the capsule two types of intrafusal muscle fibers (nuclear chain fibers and nuclear bag fibers) can be distinguished, which both exhibit modifications concerning their myonuclei in the spindle’s equatorial region. Nuclear chain fibers have a single row of centrally arranged nuclei, whereas nuclear bag fibers show an accumulation of nuclei. In the muscle spindle’s equatorial region, a large tissue-free space (periaxial space) separates the intrafusal muscle fibers from the capsule. Muscle spindles in the EOMs of even-toed ungulates receive a double innervation from sensory and motor nerve fibers. In the equatorial region, both types of intrafusal muscle fibers are endowed with sensory nerve endings (annulospiral sensory endings) which are wrapped spirally around the muscle fibers. Whether a second type of sensory nerve ending (flower-spray ending) that is common in other mammalian skeletal muscle spindles is also present in ungulate EOM spindles is unclear. Fine structural analyses have shown that sensory nerve terminals contain mitochondria and a few clear vesicles. The synaptic cleft separating the nerve terminal from the muscle fiber surface is free from a basal lamina. At the muscle spindle’s pole, intrafusal muscle fibers receive motor terminals. Motor terminals contain mitochondria

Extraocular Muscles: Proprioception and Proprioceptors

and dense aggregations of clear vesicles, and the synaptic cleft is filled with a basal lamina. Muscle spindles in EOM of primates exhibit structural differences when compared with those in even-toed ungulates. Specifically, in most muscle spindles of monkey and man the periaxial space exhibits little or no expansion. Only in human infants have some muscle spindles with a wide periaxial space been observed. Thorough analyses of the intrafusal fiber composition have been done in human EOM spindles. The findings indicate that human EOM spindles contain nuclear chain fibers but most of them lack nuclear bag fibers. Only 2% of the spindles contain nuclear bag fibers and, when present, the bag region is poorly developed with only two nuclei lying side by side. In addition to nuclear chain fibers, anomalous muscle fibers are also regularly observed in human EOM spindles. Anomalous muscle fibers exhibit no nuclear modification in the spindle’s equatorial region and are indistinguishable from muscle fibers outside the spindle. The unique morphology of human EOM spindles was initially described in aged persons (67–83 years old) and later was confirmed in infants (Figure 1(a)). The innervation pattern of primate EOM spindles has only been analyzed in humans. In human EOM spindles, sensory nerve endings have been observed on nuclear chain and, when present, on nuclear bag fibers, but only 7% of the anomalous fibers are endowed with sensory nerve terminals. In their fine structure, sensory nerve terminals in human EOM spindles do not differ from sensory nerve terminals in EOM spindles of even-toed ungulates. At the muscle spindle’s pole, intrafusal muscle fibers are equipped with motor terminals. Motor terminals in human EOM spindles are identical in their structure with those in EOM spindles of even-toed ungulates (Figure 1(b)).

35

Function of Muscle Spindles Muscle spindles in mammalian skeletal muscle are stretch receptors which register changes in muscle length. Indications that muscle spindles in the EOMs of even-toed ungulates are capable of monitoring muscle length have come from electrophysiological investigations. Specifically, in goat and sheep the EOMs were stretched and afferent signals were recorded in the sensory trigeminal ganglion. Recorded signals exhibited characteristics that are the same as muscle spindles in other skeletal muscles. There is controversy whether muscle spindles in human EOMs are functional. Due to their unusual morphology, some authors suppose that human EOM muscle spindles are not functional. On the other hand, muscle spindles in human EOMs are numerous, and their nerve terminals exhibit a normal morphology. This is why other authors suggest that human EOM spindles are functional, and their unusual morphology might indicate special functional properties. In particular, as most human EOM muscle spindles lack nuclear bag fibers, muscle spindles might have a predominantly static function and monitor the degree of muscle stretch rather than the contraction velocity of muscle fibers.

Golgi Tendon Organs Occurrence, Distribution, and Number of Golgi Tendon Organs Golgi tendon organs are exclusively found in the EOMs of even-toed ungulates (pig, sheep, camel, and cow). They have not been found in other mammals and man. In even-toed ungulates, Golgi tendon organs are distributed throughout the proximal and distal EOM tendons, their number always being higher in the distal tendons (Table 1). The number of Golgi tendon organs per muscle has been counted to be

C

BL N

AF ST

NC

(a)

(b)

Figure 1 (a) Semi-thin cross section through an extraocular muscle spindle of a 2-year-old human infant and (b) ultra-thin cross section through a nuclear chain fiber. (a) The muscle spindle contains six nuclear chain fibers (NF) and one anomalous fiber (AF). The anomalous fiber is indistinguishable from muscle fibers outside the spindle. N, nerve and C, capsule. Scale bar ¼ 100 mm. (b) Nuclear chain fiber (NF) with a sensory nerve terminal (ST). BL, basal lamina. Scale bar ¼ 1 mm.

36


46–128 and 30–90 in pig and camel, respectively. In both species, Golgi tendon organs are more frequent in the rectus EOMs than in the oblique EOMs. Structure of Golgi Tendon Organs Golgi tendon organs in EOMs of even-toed ungulates exhibit a fusiform shape and are enclosed by a capsule of perineural cells. The capsule space is filled with a viscous fluid containing acidic mucopolysaccharides. The main component of the Golgi tendon organs are collagen bundles that pass through the organ. At one end of the organ the collagen fascicles are attached to muscle fibers, and at the other end the fascicles merge with the tendon of the muscle. Many Golgi tendon organs contain only collagen bundles, but there are others which contain both collagen bundles and muscle fibers. Such intracapsular muscle fibers penetrate the Golgi tendon organ at one end and either terminate in collagen bundles or, more rarely, pass through the tendon organ. All Golgi tendon organs in the EOMs of even-toed ungulates exhibit a wide space in the central region that separates the collagen bundles and muscle fibers, if present, from the capsule (Figure 2(a)). Each Golgi tendon organ is innervated by a single sensory nerve fiber. The nerve fiber penetrates the capsule at various points. Inside the organ, the axon divides into several preterminal branches which finally establish nerve terminals that contact the surrounding collagen fibrils. Nerve terminals are only partly covered with Schwann cells, and at the area of contact only a basal lamina lies between the nerve terminal and the neighboring collagen. Nerve terminals contain mitochondria and a few clear vesicles (Figure 2(b)).

With the exception of intracapsular muscle fibers and a more pronounced capsule space in the central region, Golgi tendon organs in even-toed ungulates share the structural features of Golgi tendon organs found in other mammalian skeletal muscles. Function of Golgi Tendon Organs Golgi tendon organs in mammalian skeletal muscle are sensitive to muscle contraction. During muscle fiber contraction collagen bundles are stretched, and the nerve terminals within the collagen are deformed, thereby generating a receptor potential. Golgi tendon organs in EOMs of ungulates are supposed to function analogously and register muscle fiber contraction. Muscle fibers passing through Golgi tendon organs are supposed to regulate the sensitivity of the organ.

Palisade Endings Occurrence, Distribution, and Number of Palisade Endings Palisade endings (myotendinous cylinders) are sensory end organs that are unique to EOMs. So far, palisade endings have been found in the EOMs of almost all species investigated, including fellidae (cat), lagomorphs (rabbit), even-toed ungulates (sheep), rodentia (rat), and primates (monkey and man). These organs are located at the distal and proximal myotendinous junctions. Palisade endings are plentiful in the EOMs of monkey and cat (Table 1). In the distal myotendon of a monkey medial rectus 350 palisade endings have been counted, and in a

S

N

ST

MF COL C

COL (a)

(b)

Figure 2 (a) Semi-thin cross section through a Golgi tendon organ of a calf extraocular muscle and (b) ultrathin section through a sensory nerve terminal. (a) The Golgi tendon organ is ensheathed by a capsule (C) and contains collagen bundles (COL) and one muscle fiber (MF). Nerve fiber (N). Scale bar ¼ 100 mm. (b) A sensory nerve terminal (ST) which is partly ensheathed by Schwann cells (S) contacts the surrounding collagen bundles (COL). Scale bar ¼ 1 mm.

Extraocular Muscles: Proprioception and Proprioceptors

cat medial rectus 94. A smaller number of this EOMspecific organ have been found in the distal EOM myotendons of rat (27) and human (20–30). Structure of Palisade Endings Innervation for the palisade endings arises from nerve fibers that come from the muscle and extend into the tendon. Within the tendon, the nerve fibers make a 180 loop and return to the muscle. At the muscle–tendon junction, the returning axons divide into preterminal branches. Preterminal axons establish nerve terminals around the muscle fiber tips which have the appearance of a palisade fence (Figure 3), which is also the reason why this formation is called a palisade ending. The whole palisade complex is ensheathed by a capsule of fibroblasts. Palisade ending is exclusively associated with the multiply innervated muscle fibers of the global (inner) layer of the EOMs. Such muscle fibers have several motor contacts along their length, and with respect to contraction they exhibit nontwitch characteristics. The multiply innervated muscle fibers have a unique innervation from small motoneurons located outside the borders of the main EOM nuclei. The fine structure of palisade endings was initially analyzed in cat and monkey and later in sheep, rabbit, and man. It was observed that the majority of palisade nerve terminals contact the collagen fibrils of the tendon, and only a few of them contact the muscle fiber tip. Nerve terminals contacting the collagen fibrils are only partly enwrapped with Schwann cells, and at the area of contact with the collagen only a basal lamina covers the nerve terminals. Such neurotendinous contacts contain dense aggregations of clear vesicles and mitochondria. Palisade nerve terminals contacting the muscle fiber are free from a basal lamina in the synaptic cleft, thereby resembling sensory nerve terminals on intrafusal fibers of muscle spindles. Identical to neurotendinous contacts, neuromuscular contacts contain mitochondria and a large number of clear vesicles. Interestingly, in palisade endings of man and monkey, neuromuscular contacts have a basal lamina in the synaptic cleft which is a feature of motor terminals. Palisade

37

endings in rabbits and rats are an exception. In both species, the palisade endings lack neurotendinous contacts and neuromuscular contacts are present exclusively.

Molecular Characteristics of Palisade Endings In cat and monkey, it has been recently demonstrated that palisade endings have a cholinergic phenotype. Utilizing immunohistochemistry, palisade endings have been labeled with all commercially available cholinergic markers, including antibodies against choline transporter (ChT), choline acetyltransferase (ChAT), and vesicular acetyl choline transporter (VAChT), as well as a-bungarotoxin. In the nervous system, ChT is used for the uptake of choline, ChAT is the synthesizing enzyme of acetylcholine, and VAChT is used to transport acetylcholine into the synaptic vesicles. a-Bungarotoxin is a snake venom that binds to nicotinic acetylcholine receptors, and this neurotoxin is widely used to detect motor terminals in skeletal muscle. In cat and monkey, it has been shown that the nerve fibers supplying palisade endings are ChAT immunoreactive. The palisade complexes, including palisade nerve terminals, are ChAT positive as well. In monkey, it also has been demonstrated that palisade nerve terminals exhibit ChT/VAChT immunoreactivity, and neuromuscular contacts, when present, exhibit a-bungarotoxin binding. Finally, in some cases it has been detected that nerve fibers supplying palisade endings establish a-bungarotoxin-positive neuromuscular contacts outside the palisade complex (Figure 3(b)). Function of Palisade Endings So far, physiological studies on palisade ending are missing, and their function remains speculative. Indication that palisade endings are sensory organs comes from morphological studies and a single neuronal tracing experiment. Specifically, morphological studies show that palisade endings have nerve terminals contacting the tendon, and nerve terminals in apposition to collagen are arguably sensory. Palisade nerve terminals contacting

Figure 3 Palisade endings: (a) three-dimensional reconstruction of a palisade ending and (b) palisade ending labeled with anti-neurofilament (general marker for nerve fibers) and anti-ChAT (marker for cholinergic nerve fibers). Muscle fibers are counterstained with phalloidin. The tendon is not labeled and is continuous with the muscle fiber tip to the right. (a) A nerve fiber (green) coming from the muscle extends into the tendon and turns back to establish nerve terminals (red) around a muscle fiber tip. The muscle fiber is white. (b) This shows a nerve fiber forming a palisade ending. The nerve fiber and the palisade ending are positive for neurofilament (red) and ChAT (green). Muscle fiber (white). Scale bar ¼ 100 mm.

38


the muscle fibers lack a basal lamina in the synaptic cleft which is common with sensory nerve terminals in muscle spindles. Moreover, by injecting neuronal tracer into the sensory trigeminal ganglion, structures resembling palisade endings have been labeled. Palisade endings lie in series to the multiply innervated muscle fibers of the global (inner) EOM layer, and it is supposed that palisade endings register contraction of such muscle fibers. Although a consensus exists that palisade endings are sensory, there are other findings which favor a motor role for palisade endings. In particular, immunohistochemical studies have demonstrated that palisade endings are cholinergic, a feature common with motor nerve terminals. Nerve fibers forming palisade endings also establish motor neuromuscular contacts outside the palisade complex. In a nerve degeneration experiment a lesion of the oculomotor nucleus was performed and, in addition to the expected loss of motor terminals, the palisade endings degenerate as well. The functional significance of palisade endings with a motor nature is difficult to predict. In particular, it is unclear what effect cholinergic neurotendinous contacts would have on the surrounding collagen. At the moment the function of palisade endings is still a matter of discussion, and for clarification physiological studies are highly warranted.

Further Reading Billig, I., Buisseret, D. C., and Buisseret, P. (1997). Identification of nerve endings in cat extraocular muscles. The Anatomical Record 248: 566–575. Blumer, R., Konakci, K. Z., Brugger, P. C., et al. (2003). Muscle spindles and Golgi tendon organs in bovine calf extraocular muscle studied by

means of double-fluorescent labeling, electron microscopy, and three-dimensional reconstruction. Experimental Eye Research 77: 447–462. Blumer, R., Lukas, J. R., Aigner, M., et al. (1999). Fine structural analysis of extraocular muscle spindles of a two-year-old human infant. Investigative Ophthalmology and Visual Science 40: 55–64. Blumer, R., Wasicky, R., Brugger, P. C., et al. (2001). Number, distribution and morphological particularities of encapsulated proprioceptors in pig extraocular muscle. Investigative Ophthalmology and Visual Science 42: 3085–3094. Blumer, R., Wasicky, R., and Lukas, J. R. (2001). Presence and structure of innervated myotendinous cylinders in rabbit extraocular muscle. Experimental Eye Research 73: 787–796. Buisseret, P. (1995). Influence of extraocular muscle proprioception on vision. Physiological Reviews 75: 323–338. Buttner, E. J. A., Konakci, K. Z., and Blumer, R. (2005). Sensory control of extraocular muscles. Progress in Brain Research 15(1): 81–93. Donaldson, I. M. L. (2000). The functions of proprioceptors of the eye muscles. Philosophical Transactions of the Royal Society of London 355: 1685–1754. Konakci, K. Z., Streicher, J., Hoetzenecker, W., et al. (2005). Molecular characteristics suggest an effector function of palisade endings. Investigative Ophthalmology and Visual Science 46: 155–165. Konakci, K. Z., Streicher, J., Hoetzenecker, W., et al. (2005). Palisade endings in extraocular muscles of the monkey are immunoreactive for choline acetyltransferase and vesicular acetylcholine transporter. Investigative Ophthalmology and Visual Science 46: 4548–4554. Lukas, J. R., Aigner, M., Blumer, R., Heinzl, H., and Mayr, R. (1994). Number and distribution of neuromuscular spindles in human extraocular muscles. Investigative Ophthalmology and Visual Science 35: 4317–4327. Lukas, J. R., Blumer, R., Denk, M., et al. (2000). Innervated myotendinous cylinders in human extraocular muscle. Investigative Ophthalmology and Visual Science 41: 2422–2431. Ruskell, G. L. (1989). The fine structure of human extraocular muscle spindles and their potential proprioceptive capacity. Journal of Anatomy 167: 199–214. Ruskell, G. L. (1990). Golgi tendon organs in the proximal tendon of sheep extraocular muscle. The Anatomical Record 227: 25–31. Ruskell, G. L. (1999). Extraocular muscle proprioceptors and proprioception. Progress in Retinal and Eye Research 18: 269–291.

Abnormal Eye Movements due to Disease of the Extraocular Muscles and Their Innervation A Serra, University of Sassari, Sassari, Italy R J Leigh, Case Western University, Cleveland, OH, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Amblyopia – Lazy eye with poor vision because of misalignment of the eyes during development. Chronic progressive ophthalmoplegia (CPEO) – This term describes a number of disorders affecting the extraocular muscles that lead to progressive limitation of eye motion. Diplopia – Double vision. Esotropia – Misalignment of the eyes, one of which turns in (toward the nose)–cross-eyed. Exotropia – Misalignment of the eyes, one of which turns out (away from the nose)–wall-eyed. Kearns–Sayre syndrome – One cause of CPEO (see above) that is inherited from the mother and affects other tissues, such as the heart. Myasthenia gravis – A disorder causing muscle fatigue that is due to failure of the nerves to stimulate the muscles to contract. Nystagmus – An oscillation of the eyes (shimmering or jumping eyes). Optokinetic reflexes – Eye movements induced by moving a visual pattern in front of the eyes. Ptosis – Droopy lids. Saccades – Rapid eye movements that are used to move the point of visual fixation from one feature of interest to the next. Smooth pursuit – Eye movements that smoothly follow a moving object, such as a bird in the sky. Strabismus – Misalignment of the eyes; the eyes point in different directions. Vestibulo-ocular reflexes – Eye movements induced by head movements, which stimulate the balance mechanism in the inner ear.

Introduction In this article we apply current knowledge of the extraocular muscles (EOMs) and their brainstem innervation to develop working hypotheses to account for a range of abnormal eye movements. To be concise, we have mainly selected diseases with well-defined processes that affect

specific sites, from muscle to premotor neurons in the brainstem (Figures 1 and 2). This bottom-up approach is somewhat reductionist and simplified, but we hope that it will provide insights for readers with a broad range of interests. For more comprehensive reviews, readers can turn to sources listed at the end of this article. A prerequisite for understanding disordered ocular motility is that eye movements can be systemically examined (Table 1). Thus, restricting attention to the most evident disturbance (e.g., strabismus) will impoverish interpretation of the underlying disorder. Conversely, considering the properties of saccades, pursuit, vestibular, and vergence eye movements, as well as the presence of any visual deficits, will enrich the understanding of the pathogenesis of the disorder.

Effects of Disorders of the EOMs on Eye Movements The EOMs (Figure 1, site 1) possess unique properties that make them resistant to some diseases and susceptible to others. Thus, on the one hand, the EOMs are spared in Duchenne muscular dystrophy, even when the disease is well advanced, a finding that has prompted much research. On the other hand, EOMs are rich in mitochondria, which is appropriate for the sustained contraction required for precise gaze control, but which makes them susceptible to mitochondrial disorders. Although such disorders may arise in childhood along with involvement of other tissues, such as heart muscle (Kearns–Sayre syndrome), these may present throughout adulthood with the syndrome of chronic progressive ophthalmoplegia (CPEO). Such individuals have ptosis and a limited range of eye movements. The complaint of diplopia is rare in CPEO, and although this had been ascribed to equal involvement of each of the eye muscles, another explanation seems more likely. Thus, slow progression of CPEO allows time for the visual system to adapt, and suppress images from one eye. Another interesting finding is that such patients may also make relatively quick eye movements (saccades or vestibular eye movements), despite a limited range of movement. This anomaly may be due to sparing of fast type myosin heavy chain (MyHC) EOM-specific global EOM fibers, which have fewer mitochondria.

39

40


1 MR

LR

2

Ach CN III

Oculomotor nerve

Nodulus

Vergence

Dentate nucl.

Abducens nerve ATD

3

5 6

ICP

MLF

PPRF EBN

glu/asp

glu/asp

glu SVN

CN VI Abd. nucl.

4

glu

/as

IBN

p

CN VII

gly gly

gly

CTT MLF CN VI

PPRF MVN NPH MLF IVN

ICP

Ach

8

7

NPH

MVN

MRF Inf. olivary nucl.

Figure 1 Anatomic scheme for the synthesis of signals for horizontal eye movements. The abducens nucleus (CN VI) contains abducens motoneurons that innervate the ipsilateral lateral rectus muscle (LR), and abducens internuclear neurons that send an ascending projection in the contralateral MLF to contact medial rectus (MR) motoneurons in the contralateral third nerve nucleus (CN III). From the horizontal semicircular canal, primary afferents on the vestibular nerve project mainly to the MVN, where they synapse and then send an excitatory connection to the contralateral abducens nucleus and an inhibitory projection to the ipsilateral abducens nucleus. Saccadic inputs reach the abducens nucleus from ipsilateral excitatory burst neurons (EBNs) and contralateral inhibitory burst neurons (IBNs). Eye position information (the output of the neural integrator) reaches the abducens nucleus from neurons within the nucleus prepositus hypoglossi (NPH) and adjacent MVN. The medial rectus motoneurons in CN III also receive a command for vergence eye movements. Putative neurotransmitters for each pathway are shown: Ach, acetylcholine; asp, aspartate; glu, glutamate; gly, glycine. The anatomic sections on the right correspond to the level of the arrowheads on the schematic on the left. Abd. nucl., abducens nucleus; CN III, oculomotor nerve; CN IV, trochlear nerve; CN VI, abducens nerve; CN VII, facial nerve; CTT, central tegmental tract; ICP, inferior cerebellar peduncle; IVN, inferior vestibular nucleus; Inf. olivary nucl., inferior olivary nucleus; MVN, medial vestibular nucleus; MRF, medullary reticular formation; SVN, superior vestibular nucleus. Numbers indicate lesion sites that are discussed in the text. Adapted from Leigh, R. J. and Zee, D. S. (2006). The Neurology of Eye Movements, 4th edn. New York: Oxford University Press.

Besides mitochondrial disorders, the EOM may be affected by other genetic diseases such as hereditary disorders of myosin, or acquired disorders that present as a restrictive ophthalmopathy, such as thyroid disease. Thyroid ophthalmopathy, which has been attributed to accumulation of glycosaminoglycans in the orbit, often presents with vertical diplopia that is worse on wakening. Associated lid retraction and exophthalmia are common manifestations.

Effects of Disorders of the Neuromuscular Junction on Eye Movements Eye movements are especially susceptible to a disease affecting the neuromuscular junction (Figure 1, site 2), classical myasthenia gravis, which is due to an abnormal immune attack on the postsynaptic acetylcholine receptor. In half of all patients with myasthenia gravis, diplopia or ptosis is the presenting complaint and, in about 80%

Abnormal Eye Movements due to Disease of the Extraocular Muscles and Their Innervation

41

Superior colliculus Thalamus

PC

CG Cerebellum

TR ND

riMLF EBN

MRF IV

3

MLF

1 INC lBN

MB

CN IV 2

CN VII

III PPRF EBN

CN III

NPH

VI

NRTP CN VI Med RF IBN

Rostral

3 mm

Caudal

Figure 2 A sagittal section of the monkey brain stem showing the locations of premotor burst neurons: excitatory burst neurons for horizontal saccades lie in the paramedian pontine reticular formation (PPRF) and, for vertical and torsional saccades lie in the rostral interstitial nucleus of the medial longitudinal fasciculus (rostral iMLF). Burst neurons project to ocular motoneurons lying in the abducens nucleus (VI), the trochlear nucleus (IV) and the oculomotor nucleus (III). Omnipause neurons (indicated by an asterisk) lie in the midline raphe of the pons between the rootlets of the abducens nerve (CN VI) and gate the activity of burst neurons. CG, central gray; MB, mammillary body; MT, mammillothalamic tract; CN III, rootlets of the oculomotor nerve; CN IV, trochlear nerve; ND, nucleus of Darkschewitsch; NRTP, nucleus reticularis tegmenti pontis; PC, posterior commissure; NPH, nucleus prepositus hypoglossi; TR, tractus retroflexus. The arrow refers to the Horsley-Clarke plane of section. Numbers indicate lesion sites that are discussed in the text. Courtesy of Dr. Jean Bu¨ttner-Ennever).

of patients, movements of the eyes and lids are ultimately abnormal, with fluctuating weakness. Why are the EOMs so susceptible to diseases affecting the neuromuscular junction? One physiological reason arises from the demands made of eye movements to sustain the precise alignment of the eyes required for single binocular vision. It follows that fluctuating weakness due to myasthenia often causes ocular misalignment and diplopia. One morphological reason is that the postsynaptic junction of the EOM is poorly folded, thereby reducing the potential area for acetylcholine receptors. It follows that the EOM will be especially susceptible to loss of acetylcholine receptors. However, one subtype of EOM fibers, the MyHC EOMpositive myofibers (fast twitch/fatigable), which seems important for fast eye movements, does have substantial folding of its postjunctional membranes and, therefore, seems less susceptible to fatigue. Thus, it is interesting to note that patients with severe ocular myasthenia and little residual movement often retain the ability to make fast movements (quiver movements)-presumably due to preserved activity of their MyHC EOM-positive fast twitch myofibers.

Why is diplopia a common complaint in ocular myasthenia but rare in CPEO? The view that the eyes move conjugately in CPEO but not in myasthenia is not supported by measurements of eye movements. A more cogent reason is that the weakness in ocular myasthenia is highly variable (hence the characteristic symptom of fatigue), whereas in CPEO it evolves slowly and steadily. Thus, in CPEO the visual system has time to adapt to the loss of binocular correspondence, whereas in ocular myasthenia visual inputs are continually varying. This is not to state that adaptation of eye movements does not occur in myasthenia: the converse is the case, and is often evident by the occurrence of abnormally large eye movements immediately following pharmacological reversal of the neuromuscular failure by intravenous injection of the acetylcholine esterase inhibitor, edrophonium. Although ocular myasthenia is the most common disease to affect the EOM neuromuscular junction, other disorders that can impair eye movements include systemic botulism, neuromuscular blocking agents, and the Lambert-Eaton myasthenic syndrome (LEMS), which is due to the impaired release of acetylcholine secondary to autoimmune

42


Table 1

Functional classes of human eye movements

Class of eye movement Vestibular

Visual fixation Optokinetic Smooth pursuit

Nystagmus quick phases Saccades Vergence

Main function Holds images of the seen world steady on the retina during brief head rotations or translations Holds the image of a stationary object on the fovea by minimizing ocular drifts Holds images of the seen world steady on the retina during sustained head rotation Holds the image of a small moving target on the fovea; or holds the image of a small near target on the retina during linear self-motion; with optokinetic responses, aids gaze stabilization during sustained head rotation Reset the eyes during prolonged rotation and direct gaze toward the oncoming visual scene Bring images of objects of interest onto the fovea Moves the eyes in opposite directions so that images of a single object are placed or held simultaneously on the fovea of each eye

Adapted from Leigh, R. J. and Zee, D. S. (2006). The Neurology of Eye Movements, 4th edn. New York: Oxford University Press.

both receive increased innervation. One consequence of this adaptation is that movements of the weak eye may improve (unless paralysis is complete). A second consequence is that the strong eye, which also receives increased innervation, would, for example, make leftward saccades that overshoot the visual target. Such behavior is sustained for some time after adaptation even if the weak eye is covered and the strong eye views. This is a special example of plastic adaptation or motor learning, a property that depends heavily on the cerebellum, and which has been a subject of research interest for the past quartercentury. Because of the yoking mechanism by which eye movements are made conjugate in the brainstem circuitry (see the next section), there is a limitation to how much adaptive mechanisms can contribute to the recovery of the weakness due to nerve palsy. Nonetheless, such adaptive mechanisms undoubtedly contribute to the recovery from ocular motor nerve palsies. Paradoxically, in the case of trochlear nerve palsy, there is some recent evidence that such mechanisms may be maladaptive.

Effects of Disorders of the Brainstem Circuitry on Eye Movements Horizontal Movements

attack on presynaptic P/Q voltage-gated calcium channels. As opposed to myasthenia, in LEMS, repetitive saccades may change from hypometric (under-shooting) to hypermetric (over-shooting) as a consequence of the characteristic facilitation of muscle strength.

Effects of Disorders of the Oculomotor, Trochlear, and Abducens Nerves on Eye Movements Palsies of the nerves innervating EOMs are common in clinical practice and cause diplopia and selective patterns of weakness of the muscles they supply. Such paralytic strabismus (misalignment of the visual axes) is greatest when the affected patient attempts to look in the direction of the weak muscle. Thus, in the case of left abducens palsy (Figure 1, site 3), the patient cannot abduct (turn out) the affected eye to the left. However, such weakness and the attendant diplopia is a stimulus to adapt the neural signals that move the eyes. Such adaptive changes are evident if the strong eye is covered and the weak eye forced to view the world. A similar situation occurs naturally if the weak eye is also the visually dominant eye. In either case, the level of innervation is increased in motoneurons supplying muscles that induce corresponding movements in each eye. Thus, in our example, the left lateral rectus, which turns the left eye out, and the right medial rectus, which turns the right eye in, would

The brainstem machinery whereby the eyes are coordinated to move together (conjugately) in the horizontal plane is summarized in Figure 1. The abducens nucleus, which lies in the pons, may be regarded as the horizontal gaze center (Figure 1, site 4). Thus, the abducens nucleus receives inputs for each functional class of eye movements, including saccades, smooth pursuit, vestibular and optokinetic reflexes. It follows that each of these classes of conjugate eye movement (Table 1) may be independently affected by disease. The abducens nucleus contains two main groups of neurons: abducens motoneurons and abducens internuclear neurons. Axons of abducens motoneurons project in the sixth (abducens) cranial nerve to innervate the lateral rectus muscle. Axons of abducens internuclear neurons cross the midline and ascend in the contralateral medial longitudinal fasciculus (MLF, Figure 1, site 5) to contact medial rectus motoneurons in the oculomotor nucleus, which lies in the midbrain. Axons of medial rectus motoneurons project in the third (oculomotor) cranial nerve to innervate the medial rectus muscle. It follows that lesions affecting the abducens nucleus (Figure 1, site 4) will impair movements of both eyes to the side of the lesion (horizontal gaze palsy). It also follows that lesions of the MLF (Figure 1, site 5) will impair the ability of the ipsilateral eye to adduct; this is called internuclear ophthalmoplegia (INO), because the coordination of the abducens motoneurons and oculomotor medial rectus motoneurons is disrupted. Multiple sclerosis (MS) is the

Abnormal Eye Movements due to Disease of the Extraocular Muscles and Their Innervation

most common etiology for an INO in young persons, especially when it is present bilaterally. Patients with INO due to demyelination of the MLF in MS show slowing or absent movements of the adducting eye because the MLF can no longer conduct high-frequency signals between the abducens and the oculomotor nuclei. However, vergence movements may be preserved either in abducens nucleus lesions or in INO, since vergence commands project directly to the oculomotor nucleus (Figure 1). Horizontal saccades depend on premotor burst neurons, which lie in the paramedian pontine reticular formation (PPRF, Figure 1, site 6), and generate a high-frequency pulse of action potentials. Disorders affecting burst neurons of the PPRF selectively slow, or abolish, horizontal saccades. In contrast with abducens nucleus lesions, which cause complete horizontal gaze palsy, lesions of the PPRF usually spare ipsilateral smooth pursuit and vestibular eye movements. The vestibulo-ocular reflex (VOR) for horizontal head rotations depends on vestibular afferents from the lateral semicircular canals, which relay their signal to the contralateral abducens nucleus via the medial vestibular nucleus (MVN, Figure 1, site 7). Wernicke’s encephalopathy, a disorder due to thiamine deficiency that occurs in alcoholics, involves the vestibular nuclei and may impair the horizontal VOR. The nucleus prepositus hypoglossi (NPH , Figure 1, site 8), the adjacent MVN, and the cerebellum play an important role in holding the eyes in an eccentric position (e.g., far right gaze) against the elastic pull of the orbital tissues. This function depends on mathematical integration of premotor (visual, vestibular, saccadic) signals by the NPH/MVN–cerebellar network. Impaired function of this network (leaky integration) due, for example, to intoxication with alcohol, causes the eyes to drift back to the center, leading to gaze-evoked nystagmus. It has also been postulated that the ocular motor neural integrator network may also become unstable causing either increasing velocity drifts away from center position or quasisinusoidal eye oscillations (acquired pendular nystagmus). Vertical Movements The coordination of eye movements in the vertical plane depends heavily upon neural circuits in the midbrain. However, there is no single vertical gaze center similar to the abducens nucleus for horizontal gaze. The oculomotor and trochlear nuclei (Figure 2) house motoneurons that innervate EOMs that rotate the eyes mainly vertically (superior and inferior rectus muscles) or mainly torsionally (around the line of sight-the superior and inferior oblique muscles). These motoneurons receive their saccadic input from the rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF), which lies in the prerubral fields of the rostral midbrain. Bilateral lesions involving the riMLF (Figure 2, site 1) cause slow

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or absent vertical saccades, such as in progressive supranuclear palsy (PSP), a Parkinsonian disorder. Unilateral lesions of riMLF, such as in rostral brainstem strokes, cause loss of torsional rapid movements that rotate the top pole of the eye toward the side of the lesion. The signals for vertical vestibular and pursuit eye movements ascend from the medulla and pons to the midbrain in the MLF and other pathways. Thus, bilateral MLF lesions (Figure 2, site 2), which occur in MS, cause impaired vertical pursuit and vestibular responses (as well as bilateral adduction failure during horizontal saccades, as described in the previous section). The interstitial nucleus of Cajal plays an important role in holding vertical eccentric gaze steady (e.g., far upward gaze); lesions here (Figure 2, site 3) cause gaze-evoked nystagmus on upward or downward gaze, postulated to be due to a leaky ocular motor integrator. The superior colliculus is a midbrain tectal structure that receives inputs from the cortical eye fields, and is important for triggering both horizontal and vertical saccades. Functional imaging studies in humans have demonstrated activation of the superior colliculus during generation of short-latency (express) saccades. Neural circuits important for the generation of vergence eye movements are also located in the pretectum and midbrain, but pontine nuclei and their projections to the cerebellum also contribute. Thus, disturbances of vergence eye movements are encountered with lesions, such as strokes, throughout the brainstem. However, abducens nucleus lesions and INO (Figure 1, sites 4 and 5) usually spare vergence movements.

Congenital Misalignment of the Eyes (Infantile Strabismus) and Attendant Nystagmus Ocular misalignment from infancy may be due to disorders of the orbital tissues, the innervation of EOM, or as a consequence of failure to develop binocular vision. The failure to develop binocular vision usually presents as the fusional maldevelopment nystagmus syndrome (FMNS), which includes amblyopia of one eye, strabismus (commonly esotropia and dissociated vertical deviation, with upward deviation of the covered eye) and latent nystagmus. Latent nystagmus is a jerk nystagmus comprising slow drifts of the eyes off target and a rapid resetting component that is absent when both eyes are viewing but appears when one eye is covered. The quick components of latent nystagmus beat away from the covered eye, and the nystagmus reverses direction upon covering of either eye. In most patients, the nystagmus is present (but low amplitude) when both eyes are uncovered, and is termed manifest latent nystagmus. Thus, although binocular viewing is possible, affected individuals almost invariably choose to fix with one eye and suppress the image from the other. Latent nystagmus can be

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induced experimentally in monkeys, either by depriving them of binocular vision early in life, or by surgically creating strabismus. In monkeys with latent nystagmus, the brainstem nucleus of the optic tract (NOT) shows abnormal electrophysiological properties. In normal monkeys, NOT neurons respond to visual stimuli presented to either eye. However, in monkeys with latent nystagmus, NOT neurons are driven mainly by the contralateral eye. Furthermore, inactivation of NOT with muscimol abolishes latent nystagmus in monkeys who have been deprived of binocular vision. Since the NOT projects to vestibular circuits concerned with gaze control during head rotations, one current view of the pathogenesis of latent nystagmus is that it represents the consequences of imbalance of visual inputs to the vestibular system, as if the subject was being rotated toward the side of the viewing eye.

Effect of Visual System Disorders on Eye Movements Patients with a broad range of retinal disorders causing blindness is often a familial disorder. Both show continuous jerk nystagmus, with components in all three planes, which changes in direction over the course of seconds or minutes. The drifting null point, the eye position at which nystagmus changes direction, probably reflects an inability to calibrate the ocular motor system. Animals raised in a strobe illuminated environment, which deprives them of retinal image motion while still providing position cues, also develop spontaneous ocular oscillations. Gene therapy used to restore vision to dogs blind due to an inherited retinal disease resulted in a decrease in their associated nystagmus. Nystagmus is also a feature of albinism, which is associated with abnormal development of visual pathways and optic nerve hypoplasia.

Infantile Forms of Nystagmus in Individual with Normal Visual Systems Infantile nystagmus syndrome (INS), or congenital nystagmus, may be present at birth but usually develops during infancy. The nystagmus is almost always conjugate and horizontal, even on up or down gaze, with a small torsional component. It is usually accentuated by the attempt to fix upon an object and by attention or anxiety. Up to 30% of patients with INS have strabismus but, even in individuals lacking strabismus, stereovision is usually degraded, partly due to retinal image motion. Head turns are common in INS and are used to bring the eye in the orbit close to the null point or zone, at which nystagmus is minimized. Some patients with INS also show head oscillations; such head movements could not act as an adaptive strategy to improve vision unless the VOR was negated.

It seems possible that the head tremor and ocular oscillations in INS represent the output of a common neural mechanism. Measurements of nystagmus in INS demonstrate typical waveforms with increasing slow-phase velocity and the superimposed presence, during each cycle of oscillations (usually after a quick phase), of a brief period when the eye is still and is pointed at the object of regard. Such foveation periods are probably one reason why many individuals with INS have near-normal vision and why most do not complain of oscillopsia (illusory motion of the seen world), in spite of otherwise nearly continuous movement of their eyes. INS, either with or without associated visual system abnormalities, is often a familial disorder. Both autosomal dominant and sex-linked recessive forms of inheritance have been reported. Although several hypotheses for the pathogenesis of INS have been offered, no animal models exist. At present, it seems possible that genetic studies will identify the underlying molecular mechanisms and point researchers to the neural disturbance causing INS.

Conclusions Recent progress in understanding disorders of the EOMs and their innervation from the viewpoint of molecular biology and genetics is approaching the point where it can be combined with behavioral and electrophysiological studies. For example, recent evidence indicates that each functional class of eye movements (Table 1) is served by a separate population of ocular motoneurons that receive specific premotor inputs. It follows that each functional class of eye movements may depend on distinct molecular mechanisms or morphological characteristics, from premotor neurons to EOM. Human diseases provide many opportunities to study behavioral effects of a disease when the disease process affects a specific site-such as the acetylcholine receptor in myasthenia gravis. In this way, insights from basic science have a growing impact on clinical ophthalmology and neurology, and vice versa. See also: Extraocular Muscles: Extraocular Muscle Anatomy; Extraocular Muscles: Extraocular Muscle Metabolism; Extraocular Muscles: Functional Assessment in the Clinic.

Further Reading Kennard, C. and Leigh, R. J. (2008). Using eye movements as an experimental probe of brain function. A symposium in honor of Jean Bu¨ttner-Ennever. Progress in Brain Research 171: 1–603. Leigh, R. J. and Zee, D. S. (2006). The Neurology of Eye Movements, 4th edn. New York: Oxford University Press. Leigh, R. J. and Devereaux, M. W. (2008). Advances in Understanding Mechanisms and Treatment of Infantile Forms of Nystagmus. New York: Oxford University Press.

Extraocular Muscles: Functional Assessment in the Clinic S P Christiansen, Boston University School of Medicine, Boston, MA, USA L K McLoon, University of Minnesota, Minneapolis, MN, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Binocular vision – The simultaneous perception by both eyes of two slightly disparate images of the same target on corresponding retinal elements resulting in a single three-dimensional image. Cover test – The use of an ocular occluder over one eye or alternately occluding the eyes, either alone or in conjunction with prisms, to detect the presence of an ocular deviation and to measure its magnitude. Diplopia – Double vision caused by a misalignment of the eyes resulting from the same image stimulating noncorresponding retinal elements in the two eyes. Esotropia – A form of strabismus where there is a nasal-ward deviation of the nonfixing eye. Exotropia – A form of strabismus where there is a temporal deviation of the nonfixing eye. Phoria – This is a latent misalignment of the eyes kept under control by fusional mechanisms and is contrasted with a tropia which is a manifest constant or intermittent deviation of the eyes. A phoria can be seen only when fixation is interrupted, as during a cover test. Recession surgery – In recession surgery, the overacting extraocular muscle is surgically removed from the sclera and resutured in a more posterior location on the globe. The goal is to decrease the rotational effect of muscle contraction. Resection surgery – In resection surgery, the underacting extraocular muscle is surgically removed from the sclera, a portion of the insertional end is removed, and the remaining, now shorter, muscle is resutured to its original insertional site on the globe. The goal is to increase the rotational effect of muscle contraction. Strabismus – A latent or manifest misalignment of the eyes. Tropia – A manifest misalignment of the visual axes of both eyes.

Normal Eye Movements There are six extraocular muscles responsible for eye movement within each orbit. These muscles are innervated by

cranial nerves III (superior rectus, medial rectus, inferior rectus, and inferior oblique), IV (superior oblique), and VI (lateral rectus). The medial rectus muscles are primarily responsible for adduction, pulling the eyes toward the nose. The lateral rectus muscles are responsible for abduction, pulling the eyes temporally. These horizontal movements are the most straightforward of the six extraocular muscles. The remaining four are cyclovertical muscles and have more complex function related to the fact that forwardoriented eyes are housed in laterally directed orbits. This means that the midline of these muscles does not consistently lie over the center of rotation of the globe in any position of gaze. If examined from the superior view, the bony medial orbital walls are parallel to each other and are in the sagittal plane. The lateral walls, however, are at a 45 angle from the plane of the medial walls. Since all but the inferior oblique muscles take their origin from the orbital apex, contraction of the superior and inferior rectus and superior oblique muscles will have a rotational or torsional component. The same is true for the inferior oblique, which originates from the anterior and inferior nasal orbital wall and courses posteriorly and laterally to insert onto the globe inferior to the belly of the inferior rectus muscle. The direction of contraction of the inferior oblique muscle thus also results in both a torsional and vertical movement of the eye. The function of the individual extraocular muscles has been more extensively covered elsewhere in this encyclopedia. To summarize, however, the superior rectus muscle and the inferior oblique muscles are the principal elevators of the eye while the inferior rectus muscle and superior oblique muscle are the principal depressors of the eye. The incyclotorters of the eye are the superior oblique and the superior rectus while the excyclotorters are the inferior oblique and the inferior rectus muscles. Each of the cyclovertical muscles also has minor horizontal function. It is important to recognize that the vertical or torsional component of each of the cyclovertical muscles changes depending on whether the eye is held in adduction or abduction. There are two basic kinds of eye movements: saccade and pursuit. Saccades are rapid and subserve fast changes in fixation. They are generated by a pulse-step pattern of innervation from the brainstem. An estimate of saccadic velocity can be gained by clinical observation alone, often with the use of an optokinetic nystagmus (OKN) drum or flag that the examiner uses to drive repeated changes in fixation, first in one direction and then in another.

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RSR

LIO

RLR

LMR

RIR

LSO

RSR

RIR

LSR

LIR

RIO

LSR

RMR

LLR

RSO

LIR

(b) Figure 1 (a) Composite photographs showing a subject displaying the nine cardinal positions of gaze and (b) Chart showing the principal yoked muscles from the right (pink) and left (green) eyes responsible for movement of the eyes into the nine cardinal positions of gaze.

A decrease in saccadic velocity can be seen in patients with extraocular muscle palsy. Pursuit movements are slower, and are driven by smooth following movements in response to a target moved slowly before the patient. There should be no lag or saccadic interlude during pursuit movements. Gaze refers to movement of both eyes together. Gaze is conjugate if both eyes move the same amount, at the same speed, and in the same direction. Thus, in right gaze, both eyes look to the right and reach the intended target at the same time. Gaze is disconjugate if both eyes move in opposite directions or there is substantial failure of one eye to reach the target. Therefore, convergence and divergence movements are disconjugate. Ductions are movements of one eye examined under monocular viewing conditions. For example, we refer to adduction as a nasalward movement of an eye when the other eye is covered, eliminating any binocular adjustment in eye position. In contrast, versions are movements of both eyes examined under binocular viewing conditions. Dextroversion is movement of both eyes to the right; levoversion is movement of the eyes to the left. Version testing is helpful for assessing over- or under-action of a muscle compared to its yoked muscle in the other eye. As conjugate gaze position shifts, yoked muscles contract in response to gaze-evoked increases in innervational frequency (Figures 1(a) and 1(b)). However, innervation to the antagonist muscles is inhibited. Therefore, for gaze right, the right lateral and the left medial muscles

contract, but the right medial and left lateral rectus muscles are innervationally inactive. Because the eyes must move in an accurate, balanced, and coordinated fashion when gaze position is changed, complex central nervous system control mechanisms are required. Input from the frontal and parietal cortex and from the cerebellum is routed through a neural integrator in the brainstem whose function and control is still being investigated. Feedback from the afferent visual system and from proprioceptive input from the extraocular muscles modulates innervational tone to all the extraocular muscles and is important for long-term calibration of eye movements. Ocular Motility Assessment in the Clinic When an individual is looking straight ahead, in what is called primary gaze, a light falling on the eyes from a distance will be perceived by an examiner as a corneal reflex or reflection in approximately the same position in the pupil of each eye (Figure 1(a), central photograph). If the eyes are not aligned, there will be a nasal, temporal, or vertical offset of the corneal reflex in one eye compared to the other eye. The amount of offset of the reflex can be estimated subjectively by the examiner or measured with prisms. Care must be taken in the patient who has a very small degree of misalignment as these tests are not sensitive enough to detect it. To confirm normal alignment of the eyes, a cover test is performed. If there is no refixation movement of either eye

Extraocular Muscles: Functional Assessment in the Clinic

when the other eye is covered, the alignment of the eyes is considered orthotropic. This does not imply, however, that the eyes are normally aligned since latent misalignment of the eyes may be controlled by fusional mechanisms that can be remarkably robust. To determine if an individual’s eyes are normally aligned, an alternate cover test is performed. Here, the eyes are alternately occluded. If no refixation movement of the eye under cover occurs when the cover is moved to the other eye, then the individual is considered to have normal eye alignment and is deemed orthophoric. The alternate cover test can be used in all the cardinal positions of gaze to determine if changes in gaze position result in any misalignment. If refixation movement is detected with alternate cover testing, then prisms may be used with the cover test to measure the magnitude of the misalignment. A convergent misalignment of the eyes is called esotropia; divergent misalignment is called exotropia; and vertical misalignment is called hypertropia. In addition to alignment testing, an examiner performs certain sensory tests to determine the quality of binocular function. These tests detect the presence of normal or abnormal retinal correspondence, the presence of suppression or diplopia, even if not subjectively present, the quality of stereoacuity if present, and the presence of torsion. The nature of this text does not allow an indepth review of such testing, but it is useful to document, as binocular function is an important aspect of normal visual experience, and is critical to the assessment of outcomes after treatment for strabismus. Children with strabismus typically do not experience diplopia unless the onset of misalignment has been rapid as might be seen in an acute CN VI palsy due to increased intracranial pressure or due to a brain tumor. More often, children with strabismus suppress the nonfixing eye, an adaptive mechanism that obviates diplopia, but places the child at risk for the development of amblyopia, a nonorganic loss of vision that may be permanent if not treated during the child’s period of visual plasticity. Therefore, during the examination of children being evaluated for strabismus, careful assessment of visual acuity is essential. During a motility examination, all patients should be carefully observed for the presence of nystagmus (rhythmic to and-fro movements of the eyes), muscle weakness (paresis), muscle restriction, or binocular-gaze deficits along with abnormal head posturing, head nodding, or other adaptive mechanisms that may arise as a result of an abnormal ocular motor condition. Clinical Treatment for Primary Eye Motility Disorders Once strabismus has been diagnosed, then a decision must be made regarding how to treat it. In children, glasses are an important consideration. Children with esotropia who

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are hyperopic (far-sighted) will often have an accommodative component to their strabismus. Control of accommodation by correcting the hyperopia may eliminate or reduce the angle of esotropia. Glasses are occasionally used in children with exotropia as well. Certainly, if a child has a significant refractive error, glasses will be required to optimize acuity. A child with strabismus and amblyopia will likely be given treatment for the amblyopia which may include occlusion therapy or optical penalization with atropine drops. Treatment of amblyopia is usually recommended before more definitive treatment of the strabismus is undertaken. Other nonsurgical treatments for strabismus include prisms, if the angle of misalignment is small or, in less common scenarios, exercises such as convergence training. When a decision has been made to proceed with surgery, there are several available options. Typical incisional surgery addresses the misalignment mechanically. If a muscle is overacting, then it is weakened. This is usually done by recession surgery in which the muscle insertion on the sclera is transected and then attached with sutures more posteriorly on the globe. This decreases the mechanical advantage of the muscle by reducing the arc of contact of the muscle on the globe. By contrast, if a muscle is underacting relative to its antagonist, then the muscle insertion is resected. In this surgery, a portion of the insertion is removed and the shortened muscle is reattached to the original muscle insertion on the sclera. This surgery works by shortening the tether length of the muscle and increasing its mechanical advantage relative to its antagonist. The amount of recession or resection is titrated to the angle of the strabismus with larger amounts of surgery for larger angles of misalignment. There are numerous variations on this theme, and the literature is full of unique means of weakening or strengthening muscles. In certain situations, such as when a muscle is significantly paralyzed, healthy muscle insertions may be transposed to approximate the insertion of the paralyzed muscle to assist its function. Sometimes, this is done in conjunction with botulinum toxin injection into the antagonist muscle to weaken it and to improve the rotation of the globe in the direction of the paralyzed muscle. Botulinum toxin A has heralded the beginning of a new era in strabismus surgery. First introduced into clinical use in the early 1980s by Dr. Alan Scott at SmithKettlewell in San Francisco, botulinum toxin weakens muscle by chemical denervation of the muscle. Release of the neurotransmitter, acetylcholine, into the synaptic cleft of neuromuscular junctions (NMJs) of treated skeletal muscle (including extraocular muscle) is blocked. This temporary paralysis of the synapses of the NMJs results in a spread of NMJ sites across the surface of the muscle. Treatment effect is maximal for approximately 6 weeks and then begins to diminish as the terminal nerves regrow and form new NMJs. Ultimately, there is a return of

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function at the sites of the original NMJs and retraction of the sprouted nerves. Treatment effect is essentially void by 3 months postinjection. Botulinum injection has been used by some clinicians for the treatment of infantile and childhood forms of strabismus, especially esotropia, but its use has not been widely adopted because of the frequent need for reinjections, especially for larger angles of misalignment. However, pharmacologic treatment of strabismus is attractive because of decreased operative times required for injection compared with typical incisional surgery, decreased scarring, and preserved biomechanical relationships of the muscle, and orbital soft tissues. In the recent literature, there have been a number of reports of the use of new candidate drugs for both weakening and strengthening extraocular muscle in experimental animals. Although much research is still needed, the era of drug treatment for strabismus is dawning, and heralds the possibility of both reducing the short-term risks of strabismus surgery and improving the long-term outcomes of our interventions.

Acknowledgments This work was supported by EY15313 and EY11375 from the National Eye Institute, the Minnesota Medical

Foundation, the Minnesota Lions and Lionesses, Research to Prevent Blindness (RPB) Lew Wasserman Mid-Career Development Award (LKM), and an unrestricted grant to the Department of Ophthalmology from RPB. See also: Abnormal Eye Movements due to Disease of the Extraocular Muscles and Their Innervation; Extraocular Muscles: Extraocular Muscle Anatomy; Extraocular Muscles: Extraocular Muscle Metabolism.

Further Reading Anderson, B., Christiansen, S. P., and McLoon, L. K. (2008). Myogenic growth factors can decrease extraocular muscle force generation: A potential biological approach to the treatment of strabismus. Investigative Ophthalmology and Visual Science 49: 221–229. Leigh, R. J. and Zee, D. S. (1999). The Neurology of Eye Movements, 3rd edn. New York: Oxford University Press. McLoon, L. K., Anderson, B., and Christiansen, S. P. (2006). Sustained release of insulin growth factor-I results in stronger extraocular muscle. Journal of the American Association of Pediatric Ophthalmology and Strabismus 10: 424–429. McLoon, L. K. and Christiansen, S. P. (2005). Pharmacological approaches for the treatment of strabismus. Drugs of the Future 30: 319–327. Wong, A. (2008). Eye Movement Disorders. New York: Oxford University Press.

II. STRUCTURE AND FUNCTION OF THE TEAR FILM, OCULAR ADNEXA, CORNEA AND CONJUNCTIVA IN HEALTH AND PATHOGENESIS IN DISEASE


Tear Film J P Craig, University of Auckland, Auckland, New Zealand A Tomlinson and L McCann, Glasgow Caledonian University, Glasgow, UK ã 2010 Elsevier Ltd. All rights reserved.

Glossary Fluorescein sodium – A topical agent used extensively as a diagnostic tool in ophthalmology to enhance tear film visibility or to highlight epithelial cell loss. The molecule is highly fluorescent, with excitation and emission occurring at 494 and 521 nm, respectively. Interference bandpass filters are commonly combined with the observation systems used in ophthalmology to optimize visualization of the fluorescence alone. Lacrimal gland – The lacrimal gland is a compound tubuloalveolar gland, similar to the salivary gland, situated superotemporally in the orbit, which secretes aqueous tear fluid. Lacrimal sac – The lacrimal sac forms part of the tear drainage system, collecting tear fluid from the ocular surface via the puncta and canaliculi. Blinking controls the pumping action of the lacrimal sac into the nasolacrimal duct for drainage into the nasal cavity. Meibomian gland – Vertically oriented tubulo-acinar glands, embedded in the upper and lower tarsal plates, which release meibum (lipid). Videokeratoscopy – A computerized, dynamic technique, based on the principle of keratoscopy, used traditionally to assess the shape of the anterior surface of the cornea (corneal topography) from the reflection of a series of projected concentric rings.

The tear film is a thin film of fluid, which covers the exposed ocular surface. Essential for the health and normal function of the eye and visual system, any abnormality in quantity or quality of the tear film can lead to signs and symptoms of dry eye disease and ultimately to a loss of vision.

The Role of the Tear Film The tear film has a number of important functions, the first of which, as the most anterior element of the visual system, is maintenance of high-quality vision. Alterations in the stability of the tear film due to abnormal tear evaporation, production, and/or drainage can cause optical aberrations and adversely affect retinal image quality.

Secondly, the tear film plays an important role in ocular surface defence. Environmental challenges such as extremes of temperature or humidity, and exposure to irritants such as pollutants and allergens, can have a detrimental effect on the tear film. The tear film must be sufficiently robust to be able to withstand these challenges and be capable of responding rapidly with reflex tearing to help flush out irritants when required. External and adnexal infectious agents pose an additional risk to the exposed ocular surface. Antimicrobial components of the tear film, which include lysozyme, lactoferrin, and immunoglobulin A, help to protect the ocular surface from microbial infection. Lubrication is another important tear film function. The non-Newtonian rheological properties of the tear film mucins enable the tear film to lubricate the corneal and mucosal surfaces. The normal blinking mechanism draws the tear film across the ocular surface, enhancing comfort and cushioning the ocular surfaces from the shearing forces present during the blink, while the mucins that trap and coat foreign particles in the tear film for removal at the caruncle, confer further epithelial surface protection. Finally, the tear film plays a vital nutritive role in the transport of substances necessary for corneal metabolism and regeneration. Uniquely avascular for transparency, the cornea requires a nonvascular route for the supply of oxygen, electrolytes, growth factors, and nutrients to, and for the removal of metabolic by-products such as carbon dioxide from the ocular surface. While glucose diffuses primarily from, the aqueous humor, oxygen must be transported to the tissue through the tear film, either from the air in the open eye state or via the palpebral conjunctival vessels in the closed eye state.

Structure and Thickness of the Tear Film Initial reports described the tear film as trilaminar in structure, consisting of a thin superficial lipid layer, an intermediate aqueous layer, and an underlying mucous layer. Each of these layers has the potential to be affected by different conditions resulting in qualitative and quantitative changes. Almost half a century later, it was proposed that interfaces existed between the layers, giving rise to a six-layer model, with an oily layer, a polar lipid monolayer, an absorbed mucoid layer, an aqueous layer, and a mucoid layer on a glycocalyx base. The carbohydrate-rich

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Structure and Function of the Tear Film, Ocular Adnexa, Cornea and Conjunctiva in Health

Lipid component

Aqueous component

Mucous component Corneal epithelium

MUC5AC

SP-A

MUC5B

MUC7

MUC1

SP-D

MUC4

SP-B

MUC16

SP-C

Figure 1 Diagrammatic representation of our current understanding of tear film structure. The tear film comprises a thin superficial lipid layer, and an aqueous-mucin continuum increasing in mucin concentration toward the glycocalyx, adjacent to the ocular surface epithelium. Adapted from Braüer, L. and Paulsen, F. P. (2008). Tear film and ocular surface surfactants. Journal of Epithelial Biology and Pharmacology 1: 62–67, with permission.

glycocalyx, produced by the surface cells of the corneal epithelium and subsurface vesicles of the conjunctival epithelium, is believed to attach the tear film to the surface of the epithelial cells. The most recent studies do not differentiate this number of distinct layers, but instead suggest the existence of an aqueous-mucin continuum that contains a decreasing concentration of dissolved mucus toward the superficial lipid layer, and is anchored to the epithelium by glycocalyx (Figure 1). The thickness of the precorneal tear film has proven to be a subject of great debate. Early estimates placed the thickness of the tear film in the region of between 4 and 8 mm. Later, on the basis of noninvasive techniques such as interferometry, it was proposed that due to a previously underestimated contribution from the mucous layer, the tear film thickness was closer to 40 mm in thickness. However, the most recent findings using techniques such as tomography and reflectance spectra propose values closer to the original measurements, suggesting that the tear film thickness is approximately 3 mm.

The Lipid Layer The superficial lipid layer of the tear film forms the initial barrier between the ocular surface and the environment. This thin, oily layer approximates 100 nm in thickness, although values ranging between 10 and 600 nm have

been reported. It is derived primarily from the meibomian glands, with additional lipid secreted by the eyelid glands of Moll and Zeiss. The lipids are excreted as meibum onto the ocular surface through the gland orifices located at the mucocutaneous junction of the lid margins. Between blinks, the lipid layer forms in two distinct phases. An inner, thin, polar layer spreads as a monolayer across the aqueous in the initial phase after the blink, then a thicker, outer, nonpolar layer follows, creating a final lipid structure with multiple layers. The lipid layer must be spread evenly by the blink to form a continuous layer without excessively thin or thick patches in order to inhibit evaporation and to prevent accelerated tear breakup from mucin contamination, respectively. Table 1 describes the proportions of the major lipid components of meibum. The polar layer consists of phospholipids, free fatty acids, and cerebrosides, while the less surface-active, nonpolar layer comprises mainly wax esters and sterol esters. The lipid layer confers a number of important protective functions including the formation of a hydrophobic barrier to prevent tear overflow onto the lids and to provide a water-tight seal during overnight lid closure, and the prevention of tear film contamination by skin lipids. However, arguably one of the most critical roles of the superficial lipid layer is to retard evaporation from the ocular surface. The polar lipids of the ocular tear film in the normal eye are capable of reducing its rate of evaporation by about 80–90%.

Tear Film

Tear Evaporation Numerous investigators have measured evaporation of fluid from the tear film, since it was established that the lipid layer retarded evaporation in a rabbit model, in 1961. Later work, also in a rabbit model, passed dry air over a cornea enclosed within a chamber. From the weight of water collected, the evaporative rate was measured as 10.1 10–7 g cm–2 s–1, and a fourfold increase in evaporation was found to occur with the removal of the rabbit tear film lipid layer. A similar increase in human tear film Table 1

Major lipid components of meibum

Component Synthesised lipids Wax esters Sterol esters Triglycerides Diglycerides Monoglycerides Fatty alchohol Hydrocarbons Membrane-derived lipids Cerebrosides Ceramides Phospholipids Degeneration products Free fatty acids

Percentage (%) 44 33 5 2 Trace Trace 2 4 Trace 8 2

Adapted from McCulley, J. P. and Shine, W. E. (2003). Meibomian gland function and the tear lipid layer. Ocular Surface 1(3): 97–106, with permission.

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evaporation has since been confirmed in patients with incomplete or absent lipid layers (Figure 2). The use of different techniques for measurement of tear film evaporation makes comparison of evaporation rates in different studies difficult because the absolute values recorded are technique-dependent. However, a pattern to the observations reported in the literature does exist, making evaporation rate a useful measurement in the differential diagnosis of dry eye. In most cases, significant increases from normal tear film evaporation are seen in patients with aqueous deficient dry eye (ADDE), evaporative dry eye (EDE), and meibomian gland dysfunction (MGD). The evaporation in normal eyes averages 13.57 6.52 10–7 g cm–2 s–1, while in ADDE the values average 17.91 10.49 10–7 g cm–2 s–1, and in EDE, 25.34 13.8 10–7 g cm–2 s–1.

The Aqueous Layer The aqueous component of the tear film is a watery phase, bordering the lipid layer and comprising most of the tear film thickness. It is produced principally by the main lacrimal gland and accessory lacrimal glands of Krause and Wolfring although additional water and electrolytes are secreted by the epithelial cells of the ocular surface. The typical or basal level of tear flow present is believed to originate mainly from the accessory glands while the reflex tears, produced in response to mechanical, noxious, or emotional stimuli, arise from the main lacrimal gland.

Relative humidity sensor

Temperature sensor

Water vapour from ocular surface

Figure 2 Tear film evaporation rate measured by a modified ServoMed EP-3 Evaporimeter (Kinna, Sweden). This technique involves the measurement of the vapor pressure gradient from recordings of relative humidity and temperature at two points a known distance above the ocular surface. Reprinted from The Ocular Surface (www.theocularsurface.com), with permission.

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During sleep, tear production is minimal but in the normal eye, in the open eye state, sensory stimulation of the exposed ocular surface induces tear production at a rate that varies according to the demands of the external environment. The secretion of electrolytes, protein, and water onto the ocular surface serves to nourish and protect the epithelia and convey messages between the structures bathed in aqueous. Corneal innervation is denser than that of any other part of the body, resulting in extreme pain if the corneal epithelium is damaged. Sensory nerve supply to the ocular surface arises from the trigeminal nerve. Stimulation of these nerve endings causes the release of neuropeptides such as substance P and calcitonin gene-related peptide (CGRP), which, through initiation of the inflammatory cascade, is believed to be an important step in the pathogenesis of many cases of dry eye. The lacrimal and meibomian glands are innervated by parasympathetic efferent nerve fibers (muscarinic and vaso-intestinal peptide (VIP)-ergic fibers) and to some extent they, and the blood vessels supplying them, are sympathetically innervated through tyrosine hydroxylase (TH) and neuropeptide Y fibers. Parasympathetic efferent nerve terminals surrounding the goblet cells suggest that conjunctival secretions are also under neurogenic control. The aqueous phase has a number of important responsibilities. These include creating a nurturing environment for the epithelial cells of the ocular surface, carrying essential nutrients and oxygen to the cornea, allowing cell movement over the ocular surface, and washing away epithelial debris, toxic elements, and foreign bodies. The major electrolytes present in the tear film are sodium, potassium, bicarbonate, and chloride, with magnesium, calcium, nitrate phosphate, and sulfate present in smaller quantities. The electrolytes dictate the osmolarity of tears, besides acting as a buffer to maintain pH and playing a role in maintaining epithelial integrity. An increase in the electrolyte concentration, described as hyperosmolarity, can cause damage to the ocular surface. The tear film protein concentration is approximately 10% that of plasma. The proportion of lacrimal gland versus serum-derived proteins and enzymes varies with tear flow rate, epithelial surface stimulation, blinking, and ocular surface disease. The tear proteins are involved in defense of the ocular surface and the maintenance of tear film stability. Electrophoresis has confirmed the presence of approximately 80 different components of human tear proteins. Around 30 proteins have been identified, half of which are enzymes. The principal tear proteins are lysozyme, lactoferrin, albumin, tear-specific pre-albumin, and globulins. Table 2 shows typical concentrations of the most significant tear proteins. The tear film also contains antioxidants such as vitamin C and tyrosine, which scavenge free radicals from within the tear film, while the

Table 2

Average concentration of the principal tear proteins

Protein component Total protein Lysozyme Albumin Tear specific prealbumin Lactoferrin Immunoglobulins (IgA, IgG, IgM, and IgE)

Average concentration (mg ml 1) 7.51 2.36 1.30 1.23 1.84 0.43

Adapted from Sariri R. and Ghafoori, H. (2008). Tear proteins in health, disease, and contact lens wear. Biochemistry (Moscow) 73(4): 381–392, with permission.

abundance of growth factors facilitates constant epithelial regeneration and promotes wound healing. Alterations in tear composition or inflammatory changes within the conjunctival vascular endothelia can act as the stimulus to ocular surface inflammation in which both cellular and soluble mediators play a significant role. The numbers of T lymphocytes and the relative proportions of activated T cells are increased in dry eye. The ocular surface epithelial cells are directly involved in such ocular surface inflammation with the release of a number of pro-inflammatory cytokines such as interleukin (IL)-1a, IL-1b, IL6, IL8, transforming growth factor beta 1 (TGF-b1) and tumor necrosis factor alpha (TNFa), and increased expression of immune activation molecules such as CD54 and HLA-DR. Increased proteolytic enzyme levels and activity have been observed in dry eye with, in particular, high levels of matrix metalloproteinase 9 (MMP9), which are not present on the normal ocular surface. The inflammatory markers described as precipitating dry eye are also recognized to perpetuate ocular surface inflammation, triggering an escalating cycle of ocular irritation, inflammation, apoptosis, and tear film dysfunction and instability, epithelial cell disease, and disruption of corneal epithelial barrier function.

Tear Production Traditional methods of measuring tear production rates are based on absorption of tears by Schirmer strips or cotton threads; however, both tests have been found to be poor quantifiers of tear production; the Schirmer test is marred by low specificity and sensitivity and the exact parameter measured with the cotton thread test has been questioned. As a result, a number of tests have been devised to measure the rate of disappearance of a dye marker placed in the tear film, as new tears are produced and the waste eliminated. In most studies in recent years, the rate of disappearance of instilled sodium fluorescein dye has been used to determine tear turnover (TTR) by the technique of fluorophotometry (Figure 3).

Tear Film

4.2

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C0

FCE (ng ml−1)

4.0 3.8 3.6 3.4 3.2 3.0

0

2

4

6

8

10

12

14

16

Time (min) Figure 3 Commercial fluorophotometer (Fluorotron Master, Coherent Radiation Inc, CA, USA) shown with a typical trace of ocular surface fluorescence decay following instillation of fluorescein sodium into the eye. A biphasic curve of fluorescence is observed with initial rapid decay (due to reflex tearing) followed by a more gradual decay (due to basal tear turnover). Adapted from The Ocular Surface (www.theocularsurface.com), with permission.

The values reported for tear turnover (%min–1) and tear flow (ml min–1) in the major studies in the literature for normal and dry eye subjects of studies using the commercial fluorophotometer have recently been collated. The data reported for normals in the majority of studies ranges from 10% to 20% min–1, which equates to an average basal tear flow rate of 1.03 0.39 ml min–1 (16.19 5.10% min–1). For dry eye, in all its forms, it averages 0.58 0.28 ml min–1 (9.36 5.68% min–1) and, within the dry eye subtypes, averages 0.40 0.10 ml min–1 (7.71 1.02% min–1) and 0.71 0.25 ml min–1 (11.95 4.25% min–1) for ADDE and for EDE, respectively. These are the rates of tear production under nonstimulated conditions in normal and dry eyes. However, the eye is capable of producing copious reflex tears under provocative conditions, providing the lacrimal gland has the ability to function at the required capacity. Reflex rates have been quoted as approximately 100-fold those under basal conditions.

The Mucin Layer The innermost, mucin layer of the tear film lies adjacent to the hydrophobic epithelial cells of the ocular surface. The layer consists of soluble, gel-forming mucins, which are capable of retaining large quantities of water, and corneal and conjunctival epithelial mucins (principally MUC1, 2, 4, and 16), which form the glycocalyx. The glycocalyx functions, through the membrane-spanning domain of MUC1, to anchor the soluble mucin layer to the plasma membrane of the corneal and conjunctival epithelial cells, while the soluble mucins interact with these transmembrane mucins and with the overlying aqueous layer, to form a water-retaining gel. The most significant soluble mucin for the ocular surface is MUC5AC, secreted by the goblet cells of the conjunctiva. The high-molecular-weight glycoproteins, with additional proteins, electrolytes, and cellular material that

contribute to the mucous layer, enable fulfilment of several important functions in the maintenance of a healthy ocular surface. In addition to providing a hydrophilic surface upon which to support a stable aqueous layer, the mucous layer offers protection against the shear force of blinking and environmental insult, and facilitates maintenance of a smooth ocular surface for optical clarity. The constituents are also believed to protect the ocular surface by inhibiting inflammatory cell adhesion.

Tear Distribution and Stability The distribution of tear fluid on the ocular surface is highly dependent on the blink. Lid closure during a blink progresses from the temporal to the nasal side of the eye spreading tears across the ocular surface and facilitating tear drainage through the lacrimal puncta. The inter-blink period in normal individuals averages 4.0 2.0 s and is significantly decreased in patients with dry eye (to 1.5 0.9 s); a high blink rate in dry eye patients maximizes the tear supply to the ocular surface. In detailed reading tasks, requiring concentration, the blink rate drops to about a half (from 22.4 8.9 to 10.5 6.5 min–1). In the clinical setting, tear film stability has traditionally been measured following the instillation of fluorescein sodium solution into the tear film, to improve visualization of the film. Tear breakup time has been defined as the time taken for the tear film to form a dark spot or streak, following a blink. However, subsequent awareness of the disruptive effect of fluorescein instillation on the tear film has encouraged use of noninvasive techniques where tear film stability is determined by observing mires reflected from the tear film surface, for signs of disruption or distortion following a blink. A tear breakup time of greater than 10 s is considered normal while values less than 5 s are suggestive of dry eye. Values between 5 and 10 s are

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generally considered to correspond to borderline dry eye, although it should be noted that this reported range was originally established for Caucasian eyes, and Asian eyes may exhibit significantly shorter tear film stabilities. In noninvasive techniques, without instillation of fluorescein, the reported cut-off values are longer with mean values around double those of the traditional fluorescein breakup test. The distribution of the tear film can further be observed in vivo using thin film interferometry. Interference fringes are produced by light reflected at the air-lipid and at the lipid-aqueous boundaries of the tear film due to the changes in refractive index. Specular reflection from the lipid layer precludes a clear view of the aqueous layer of the precorneal tear film although where the lipid layer is very thin or absent, aqueous fringes may be observed. Based on this optical principle, a number of clinical instruments, together with qualitative grading systems have been developed. These are useful for observing the structure of the tear film and offer some insight into its stability. Significant differences in appearance (and grade)

have been observed in dry eye conditions, with the partial or complete absence of the lipid layer being a feature. Recent work in this field has concentrated on developing quantitative analyses of interferometric images from the tear film of normal and dry eye patients (Figures 4 and 5, respectively). With the use of kinetic analysis of sequential interference images, it has been possible to quantify the lipid-spread time of tears in normal and dry eye patients. This spread time, defined as the time taken for the lipid film to reach a stable interference image, is significantly slower in ADDE, at 2.17 1.09 s, than it is in normal eyes (0.36 0.22 s). Because of this slower spread time, the resultant lipid film has been found to be thicker on the inferior cornea than the superior cornea, with the thickness being measured from a color reference chart created from the reflectance images of thin film interference generated by a white light source. Almost 90% of the patients with aqueous tear deficiency exhibit an interferometric pattern with vertical streaking, rather than the horizontal propagation typically observed in the superior corneal region.

Figure 4 Series of images obtained by dynamic thin film interferometry in a normal, asymptomatic subject. The images are obtained at 1 s intervals, following a blink. The lipid layer of the normal tear film reaches a relatively stable pattern within the first second after the blink. This pattern is then stable for about 6 s. Reprinted from The Ocular Surface (www.theocularsurface.com), with permission.

Tear Film

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Figure 5 Series of thin film interferometry images obtained from a patient with severe dry eye. The patient had primary Sjo¨gren’s syndrome with a tear turnover rate of 4% min–1, evaporation of 25.5 g cm 2 s 1, volume of 3.9 ml and osmolarity of 337.6 mOsm ml–1. The images are obtained at 1 s intervals following a blink. The lipid layer of the tear film is incomplete and variable in thickness, exhibiting color fringe patterns. A stable pattern is reached in 2–3 s after the blink, but this pattern begins to be disrupted within the next 3 s. Reprinted from The Ocular Surface (www.theocularsurface.com), with permission.

Evaluation of tear film particle movement can also provide an indication of the time necessary to obtain stability of the tear film after the blink. The observed particles are thought to be accumulations of newly secreted lipid from the meibomian glands. Measuring the displacement of these tear film particles immediately after a blink has shown that the time necessary to reach zero velocity (tear stabilization time) is 1.05 0.3 s. A commercial thin film interferometer has been developed, which enables the specular reflection from the tear surface to be monitored digitally and the tear film interference patterns classified. Research with this apparatus has shown that thicker lipid layers are associated with greater tear film stability. A number of grading systems have been developed mostly assessing the uniformity of the interference fringe pattern. A change in color and loss of uniformity in distribution indicates tear film instability. Such patterns are found more commonly in dry eyes in association with thin lipid layers and reduced stability. Assessment of the reflected images from the cornea and tear film has been used to evaluate tear film quality and stabilization following the blink. High-speed videokeratoscopy assesses the regularity indices, such as surface regularity index (SRI) and surface asymmetry index (SAI), in the time interval following a blink. These indices

have been found to correlate significantly with the results of standard diagnostic tests for dry eye, such as symptoms, tear breakup time, Schirmer test, fluorescein staining score, and best corrected visual acuity.

Tear Film Osmolarity Adequate production, retention, distribution, and balanced elimination of tears are necessary for ocular surface health and normal function. Any imbalance of these components can lead to the condition of dry eye. A single biophysical measurement that captures the balance of inputs and outputs from the tear film dynamics is tear osmolarity, the end-product of variations in tear dynamics. Normal homeostasis requires regulated tear flow, the primary driver of which is osmolarity. Hyperosmolarity is thus an important biomarker for dry eye disease. Tear hyperosmolarity has been found to be the primary cause of discomfort, ocular surface damage, and inflammation in dry eye. In studies of rabbit eyes, tear osmolarity has been found to be a function of tear flow rate and evaporation. In rabbit conjunctival cell cultures, hyperosmolarity has been demonstrated to decrease the density of goblet cells and, in humans, a 17% decrease in

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goblet cells density for subjects with dry eye has been reported. Granulocyte survival is significantly decreased with increases in solute concentration. Rabbit cells cultured in hyperosmolar states, above 330 mOs ml–1, show significant morphological changes, similar to those seen in subjects with dry eye. Hyperosmolarity-induced changes in surface cells in dry eye can be correlated with the degree and distribution of rose bengal staining. Measuring tear osmolarity is of benefit in the diagnosis of conditions such as dry eye. In a meta-analysis of human tear osmolarity values recorded in studies between 1978 and 2004 with freezing point depression (FPD) and vapor pressure (VP) osmolarity tests, normal values averaged 302.0 9.7 compared with 326.9 22.1 mOs ml–1 for patients with dry eye disease.

Drainage of Tears A principal means of elimination of tears from the eye is by drainage through the puncta of the eye. Tears then pass through the canaliculi, the lacrimal sac, and finally the nasolacrimal duct before reaching the nose. A technique for measuring tear turnover, which allows direct observation of tear drainage, involves instilling a radioactive dye into the tear film. In the technique of lacrimal scintigraphy a small quantity (0.013 mls) radioactive tracer such as technetium 99 (99M Tc), is introduced into the lower marginal tear strip. The distribution of the tracer is imaged serially by a gamma camera as it passes down the lacrimal drainage system (Figure 6 a–c). Images are typically taken at 10-s intervals for 1 min and then at less frequent intervals until all of the tracer has drained into the nasal cavity. The technique has been used to quantify tear turnover from the eye and drainage through

the lacrimal system. The drainage through this system is not linear, as a significant number of naso-lacrimal folds and ducts offer physiological obstruction to normal tear flow, and variable tear flow has been shown to be a typical feature of the drainage facility in asymptomatic individuals (Figure 6(d) and (e)). Therefore, most models of lacrimal drainage favor compartmental analysis to evaluate tear flow through the system, with separate components for the conjunctival sac, lacrimal sac, the nasolacrimal duct, and the nasal cavity. Although most quantitative lacrimal scintigraphy measurements describe the transit time of the radioactive tracer through the system, the compartmental model has be used to estimate tear flow rates. Depending on the number of compartments considered, basal flow rates have been estimated to fall between 0.45 and 8 ml min–1. Using a single compartment model for decay of the radioactive tracer on the conjunctival surface, mean values of reflex and basal turnover of 3.33 1.95 ml min–1 and 0.56 0.32 ml min–1, respectively, have been recorded by gamma scintigraphy. The mechanism of lacrimal drainage and the influence of blinking on the mechanics of the system have been observed by high-speed photography and by intracanalicular pressure measurements. Taking an anatomical approach and observing the lacrimal systems of human cadavers has shown that the surrounding vascular plexus of the lacrimal sac and the nasolacrimal duct is comparable to a cavernous body. While regulating the blood flow, the specialized blood vessels of this body permit opening and closing of the lumen of the lacrimal passage, which is effected by the bulging and subsiding of the cavernous body, thereby regulating tear outflow from the eye. Attempts have been made to quantify the regulation of tear outflow by measurement of the transit time of a fluorescein drop from the conjunctival sac into the inferior meatus

(a)

(b)

(c)

(d)

(e)

Figure 6 Gamma camera (a–c) used in the recording of intensity of a radioactive dye at various stages as it passes through the lacrimal system (d). In many cases of normal systems, the tracer does not proceed beyond the lacrimal sac (e). Reprinted from The Ocular Surface (www.theocularsurface.com), with permission.

Tear Film

of the nose. Application of a decongestant drug or placement of a foreign body on the ocular surface have both been found to significantly prolong the dye transit time, indicated restricted drainage through the lacrimal system in these conditions. It has therefore been concluded that the cavernous body of the lacrimal sac and naso-lacrimal duct plays an important role in the physiology of tear outflow regulation; it is subject to autonomic control and is integrated into a complex neural reflex feedback mechanism between the blood vessels, the cavernous body, and the ocular surface.

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interdependent and have a close relationship with those of the adjacent ocular tissues such that failure of any one of aspect of the tear film or lacrimal system can cause imbalance and result in dry eye. See also: Conjunctival Goblet Cells; Contact Lenses; Defense Mechanisms of Tears and Ocular Surface; Dry Eye: An Immune-Based Inflammation; Eyelid Anatomy and the Pathophysiology of Blinking; Inflammation of the Conjunctiva; Lacrimal Gland Overview; Lids: Anatomy, Pathophysiology, Mucocutaneous Junction; Meibomian Glands and Lipid Layer; Tear Drainage.

Absorption of Tears by the Ocular Surface Another method by which tears can be eliminated from the eye is by absorption into the tissues of the ocular surface and the drainage system. The possibility has been suggested that the epithelial lining of the drainage system absorbs tear fluid before it reaches the nose. It has been shown in an animal model that lipophilic substances are absorbed from the tear fluid by the epithelium of the naso-lacrimal duct and that the cavernous body surrounding this duct may play a role in drainage of absorbed fluid. No quantification of fluid volume eliminated by this route has been reported. However, tears absorbed in the blood vessels of the cavernous body may, because these vessels connect to the blood vessels of the outer eye, have a role in a biofeedback mechanism for tear production. Observations of the absorption of tear film onto the anterior ocular surface have been made in studies of corneal permeability. The proportion absorbed, in the absence of compromised corneal function, appears to be small at 0.24 0.13% of the dye instilled in the eye. The lacrimal system of the human eye is, in the vast majority of individuals, a robust system, which allows the ocular surface to maintain its health and normal function throughout life, and under modest provocation. It is only in a relatively small proportion (15%) that the imbalance between evaporative loss and tear production results in dry eye. Recent research has confirmed that an increase in this ratio of approximately 2–3 times, as most often occurs in older individuals, appears to lead the condition of dry eye. The tears covering the anterior ocular surface, form a dynamic structure with a complex nature and a number of important functions. The tear film components are

Further Reading Bron, A. J., Yokoi, N., Gaffney, E., and Tiffany, J. M. (2009). Predicted phenotypes of dry eye: Proposed consequences of its natural history. Ocular Surface 7(2): 78–92. Craig, J. P. (2002). Structure and function of the preocular tear film. In: Korb, D. R. (ed.) The Tear Film: Structure, Function and Clinical Examination, pp. 18–50. London: Elsevier Health Sciences. Dartt, D. A. (2004). Dysfunctional neural regulation of lacrimal gland secretion and its role in the pathogenesis of dry eye syndromes. Ocular Surface 2(2): 76–91. Doane, M. G. (1994). Abnormalities of the structure of the superficial lipid layer on the in vivo dry-eye tear film. Advances in Experimental Medicine and Biology 350: 489–493. Gilbard, J. P. (1985). Tear film osmolarity and keratoconjunctivitis sicca. Contact Lens Association of Ophthalmologists Journal 11(3): 243–250. Gipson, I. K., Hori, Y., and Arguëso, P. (2004). Character of ocular surface mucins and their alteration in dry eye disease. Ocular Surface 2(2): 131–148. King-Smith, P. E., Fink, B. A., Fogt, N., et al. (2000). The thickness of the human precorneal tear film: Evidence from reflection spectra. Investigative Ophthalmology and Visual Science 41(11): 3348–3359. Mathers, W. D. and Choi, D. (2004). Cluster analysis of patients with ocular surface disease, blepharitis, and dry eye. Archives of Ophthalmology 122(11): 1700–1704. McCulley, J. P. and Shine, W. E. (2003). Meibomian gland function and the tear lipid layer. Ocular Surface 1(3): 97–106. Sariri, R. and Ghafoori, H. (2008). Tear proteins in health, disease, and contact lens wear. Biochemistry (Moscow) 73(4): 381–392. Stern, M. E., Beuerman, R. W., and Pflugfelder, S. (2004). Dry Eye and Ocular Surface Disorders; the Normal Tear Film and Ocular Surface. New York: Marcel Dekker. Tiffany, J. M. (2008). The normal tear film. Developments in Ophthalmology 41: 1–20. Tomlinson, A. and Khanal, S. (2005). Assessment of tear film dynamics: Quantification approach. Ocular Surface 3(2): 81–95. van Best, J. A., Benitez del Castillo, J. M., and Coulangeon, L. M. (1995). Measurement of basal tear turnover using a standardized protocol. European concerted action on ocular fluorometry. Graefes Archive for Clinical and Experimental Ophthalmology 233(1): 1–7.

Meibomian Glands and Lipid Layer T J Millar, P Mudgil, and S Khanal, University of Western Sydney, NSW, Australia ã 2010 Elsevier Ltd. All rights reserved.

Glossary Acinus – A gland that is shaped like a hollow sphere with the gland cells lining the sphere and secreting into the center of the sphere. The secretions are removed from the center of the sphere by a duct. HMG-CoA – The 3-hydroxy-3-methyl-glutarylcoenzyme A is a precursor molecule for lipid synthesis with the small precursor molecule attached to a carrier molecule, coenzyme A. Holocrine – A mechanism of secretion by a gland wherein the whole gland cell is secreted. Hydrophilic – A substance that dissolves readily in water (water loving). Hydrophobic – A substance that does not like water. Fats, lipids, and oils are common hydrophobic substances. mN m 1(millinewtons per meter) – A unit used for measuring surface pressure relative to that of water which is regarded as 0 mN m 1. Osmolarity – A measure of the number of individual molecules dissolved in water. It is important to cells because water can pass readily through a cell membrane, but the dissolved chemicals in the cytoplasm cannot. Hence, a cell will either take on water or release water depending upon whether its osmolarity is more (hyperosmolar) or less (hypoosmolar) than its environment, respectively. Refractive index – When light travels from one medium to another, for example, from air into water, it is bent. The refractive index is a measure of the extent to which the light is bent and is a constant for a particular substance. Tarsal plate – A sheet of fibrous cartilage in the eyelids of mammals that gives the eyelids their stiffness and shape.

Overview Meibomian glands are a series of fat (lipid)-producing glands found in the upper and lower eyelids of mammals, named after a German anatomist, Heinrich Meibom (1638–1700), who recorded their presence in De Vasis Palpebrarum Novis Epistola (1666). In humans, there are 30–40 evenly spread glands in the upper lid and 20–30 in the lower lid (Figure 1). Each gland is aligned vertically in

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the eyelid and located within the tarsal plate (a sheet of fibrous cartilage that gives the eyelids their stiffness and shape) which lies closer to the ocular surface than the dermal surface. Structurally, meibomian glands have a central tubular duct surrounded by grape-like acini (glands). The duct is blind at one end and the other end opens onto the eyelid margin. By everting the lower eyelid, the openings can be readily seen as a row of small dots behind the eyelashes (Figure 2). Phylogenetically, meibomian glands are present in marsupials, but are absent in the two monotremes (echidna, Tachyglossus aculeatus; platypus, Ornithorhynchus anatinus) that we have studied. They are not present in reptiles or birds. It is believed that many of these animals use a different gland, the harderian gland, to secrete lipids onto the ocular surface. While the distribution and appearance of meibomian glands in other mammals are generally similar to those of humans, this is not always the case. Some species of voles and musk rats have few glands, for example, Microtus pinetorum, which has only two large glands in the upper eyelid – one at the medial canthus and the other at the lateral canthus. Whales have neither meibomian glands nor a tarsal plate in the eyelid. Dolphins and sea lions have a very oily secretion in their tears, but this is thought to originate from the harderian gland. Currently, the literature is not clear about the presence or absence of meibomian glands in sea-dwelling mammals. However, it is of interest to note that dolphins, sea lions, and sea otters have no eyelashes (or eyebrows), which means that they also lack the other major sebaceous glands in the eyelid, for example, the glands of Zeis which are the sebaceous glands of the eyelashes. The major function of the meibomian gland is to supply the main components of the outer layer of the tear film. The tear film is a thin (7–10 mm thick), watery fluid that covers the exposed surface of our eyes (Figure 3). Lipids from the meibomian glands are secreted onto the inner margin of the eyelids where they contact and then spread over the aqueous part of the tear film to form a covering layer (90 nm thick) in contact with the air. This layer is referred to as the lipid layer of the tear film. It is believed to decrease evaporation from the tear film and hence prevent dry eyes. However, this role for the meibomian lipids is by no means certain, and it is likely that it has other roles such as preventing tears from flowing onto the skin and skin lipids from flowing onto the ocular surface, assisting the spread of the tear film over the eye by lowering the surface tension, and forming a watertight seal when the lids are closed. The Meibomian lipids provide a smooth and

Meibomian Glands and Lipid Layer

Figure 1 Arrangement of the meibomian glands in the upper and lower eyelids.

Figure 2 Meibomian gland orifices (arrows) in the lower lid margin.

highly refractive (1.4766 at 589 nm and 35 C) surface. Clinically, a mechanism for measuring tear breakup time (TBUT) is to observe changes to interference colors of the surface layer of the tear film. This can be used as one measure of the tear film performance.

The Lipid Layer of the Tear Film The lipid layer of the tear film provides an optically smooth surface at the interface between the air and the aqueous part of the tear film. Although the structure of this layer has not been determined, the meibomian lipids form a major component (Figure 3). A useful model of the lipid layer, based on the idea that only lipids are present, was developed by McCulley and Shine. The model is presented as a crystalline array, and, while very useful

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for developing understanding, the lipid layer is unlikely to be crystalline in practice. This model proposed that the wax esters, steryl esters, triglycerides, and hydrocarbons (hydrophobic lipids) from the meibomian glands reside in the outermost layers and are linked to the aqueous subphase by polar lipids (phospholipids, free cholesterol, and free fatty acids). Some of these polar lipids may arise from the aqueous layer of the tear film and others from meibomian lipids and hydrolysis (cleavage) of the wax esters, steryl esters, and triglycerides. These polar lipids have a hydrophobic part which interacts with the hydrophobic lipids and a hydrophilic part that is able to interact with water (the aqueous layer). Assuming that the outer layer of the tear film is entirely made from lipids implies that the interfacial molecules (surfactants) must be polar lipids. An alternate model proposing that proteins and mucins also contribute to this layer is more realistic. Strong evidence comes from surface tension measurements. It has been found that the surface tension of tears is 42–46 mN m 1 and this can only be achieved by a mixture of lipids and proteins and not with meibomian lipids alone. The interaction of meibomian lipids with tear proteins is often cautiously presented as lipids occupying the outer surface of the lipid layer, and the inner surface interacting with proteins from the aqueous layer. A more recent model includes proteins and mucins as integrated parts of this layer (Figure 3). Although there has been a focus on lipocalin, an abundant lipid-binding protein in tears, being the main tear protein interacting with the meibomian lipids, lysozyme and lactoferrin may be more involved. Lipocalin is thought to scavenge lipids that have adhered to the epithelial cells of the ocular surface and lipids that are in the aqueous layer. Although it has been claimed that these are then transported to the outer lipid layer, this may not be the case. Once a lipid is bound into the central pocket of lipocalin, lipocalin is in a low-energy state and unlikely to interact with the meibomian lipids at the outer surface of the tear film. The presence of proteins in the lipid layer has important conceptual implications because they are large molecules with complex mixtures of hydrophobic, hydrophilic, and distinctly charged components. These properties mean that they can unfold and form a range of shapes according to their local molecular environment. Due to this unfolding, it is possible for them to extend across the lipid layer and interact with the hydrophobic lipids and other proteins. As the model suggests, this means that the layer comprises a complex mixture of islands of proteins, islands of lipids, islands of mucins, and various mixtures of these (Figure 3). This model is more akin to models for cell membranes and for lung surfactant. Some advantages of this model are that: the outer layer would be a noncollapsible viscoelastic gel; it would allow for the lowest free energy states of the proteins in contact with lipids; and the changes in salt concentrations in the tears

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Key for molecules shown on the right side

L

Lipocalin

Cholesterol ester

Lysozyme

Wax ester

Lactoferrin

Phospholipid

IgA

Ganglioside

Mucins

Fatty acid

Lipid layer

Aqueous/mucin layer

Corneal epithelium Figure 3 Tear film comprising an outer lipid layer covering an aqueous/mucin layer. The exploded view of the lipid layer shows a mixture of different lipids and denatured proteins (see key).

(e.g., hyperosmolarity) would affect the ability of the proteins to unfold and interact with lipids in the outer layer. Taking these one at a time, the lipid-only model needs some mechanism to spread the lipids over the aqueous surface after a blink. Both phospholipids and mucins have been proposed as enabling this by spreading slightly ahead of the hydrophobic lipids, and this concept also means that a new lipid layer is formed at each blink. Lack of change over a number of blinks to the interference patterns formed by the outer layer of the tear film does not support this concept. However, a noncollapsible viscoelastic gel that would be formed with proteins and mucins in the layer obviates the need for spreading and would account for the consistent interference pattern over a number of blinks. Furthermore, in other fields of study, mixtures of molecules in a liquid environment autoassemble into states of lowest free energy, which means that when lipids and proteins are placed together, they will mix at the molecular level rather than remain separate as the lipid-only model suggests. Some of the proteins can act as the surfactants, meaning that while phospholipids may co-jointly serve as surfactants, they are not absolutely necessary. It has also been found that while lipids alone are not capable of lowering the surface tension to the levels found in tears, mixtures of tear proteins and tear lipids possess that ability. Hyperosmolarity of the tears has the strongest correlation with a dry eye. Salt concentrations have strong effects on how proteins fold; therefore, it is possible that high salt concentrations would affect the folding of proteins and hence their ability to interact with the lipid layer to form a stable outer layer.

In turn, this could affect the surface tension and spreading of the outer layer and, consequently, its protective function.

Meibomian Glands Anatomy and Histology There are no anatomical differences in the meibomian glands of human males and females. The ducts in humans are approximately 1.6 mm long with the central ducts being slightly longer than the nasal and temporal ducts, and are surrounded by a dense banding of elastic fibers. The ducts are lined by keratinized epithelium and lie nearly 780 mm from the dermal surface of the eyelid. A horny cell layer overlies one or two layers of intermediate cells that rest on cuboidal basal cells connected to a basement membrane, and there is no difference in this appearance between the proximal and distal portions of the duct (Figure 4). Acini are arranged circularly around the central duct and are connected to it by short ductules. The acinar cells are distinct from the ductal cells with no keratohyaline granules or lamellar bodies in the acinar cells, and no lipid vesicles in the ductal cells. The acinar cells continually differentiate into holocrine-secreting cells from basal acinar cells. They contain an abundance of smooth endoplasmic reticulum that surrounds the lipid vesicles. These cells also develop from the division of basal cells and move toward the center of the acinus (migration rate of 0.62 mm d 1 in rats), and slowly increase in neutral lipid content and in the size of the lipid-containing vesicles. The acinar cells die and gradually breakdown, leaving a


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central epithelial cells. At 15 weeks (100 mm), cuboidal secretory cells line the ducts. The upper and lower eyelids are fused during this whole process. At 7.5 months (250 mm), after the eyelids have separated, acini are present, epithelial cells plugging the meibomian gland orifices disintegrate, and secretion begins just prior to birth. A similar process takes place in the mouse; however, in this case, the eyelids are still fused at birth, which is also the time when the first signs of meibomian gland development show, again from the eyelid margin. In contrast to meibomian gland development, eyelashes in humans begin development earlier at 8 weeks (35 mm) as solid epithelial invaginations from the external face of the eyelid margin. Figure 4 An illustration of meibomian glands and duct. A: Ghosts of acini not filled in with detail. B: Acini showing nucleated basal cells around the periphery which gradually lose their nuclei as they mature and move to the center of the acinus. C: Secondary duct surrounded by ductal cells (stratified epithelium) containing meibomian secretion and cell remnants. D: Main duct surrounded by ductal cells containing meibomian secretion and cell remnants. The arrow shows a bundle of nerve endings close to the acinus but separated by collagen fibers.

lipid mass that is secreted via the duct. In rats, it takes close to 9 days for a cell to migrate to the center of the acinus, and it probably divides twice during this time. Surrounding the glands is a distinct extracellular matrix comprising collagen types I, II, and IV, aggrecan, dermatan sulfate, and chondroitin-6-sulfate. Close to the acini are numerous unmyelinated varicose nerve fibers with boutons in contact with collagen fibers within the basal lamina of the basal acinar cells. These are mainly parasympathetic fibers which contain the neurotransmitter, acetylcholine, and the neuropeptide vasoactive intestinal polypeptide. The cell bodies for these fibers lie in the pterygopalatine ganglion and their fibers reach the eyelid by the greater petrosal nerve. Preganglionic neurons are ipsilateral and cholinergic, and lie in the superior salivary nucleus located lateral, dorsal, and caudal to the superior olive and lateral, dorsal, and rostral to the facial nucleus. Sympathetic innervation is sparse and mainly associated with blood vessels. Sensory nerve fibers are also sparsely distributed close to the basal region of acini, and these are immunoreactive for the neuropeptides substance P and calcitonin gene-related peptide. Development Meibomian glands in humans develop as small, solid epithelial invaginations from the ocular face of the eyelid margin at approximately 9 weeks of development (crownrump length of 40 mm). By 12 weeks (60 mm), the epithelial growth has extended the depth of the tarsal plate and has a central tube that was formed from apoptosis of the

Composition of Meibomian Lipids The meibomian gland is often referred to as a modified sebaceous gland. In this case, the term sebaceous means lipid producing rather than sebum producing because the composition of the lipids differs from those produced by the sebaceous glands of hair follicles. The main lipid types produced by meibomian glands are wax and steryl esters (60–70%) which are very hydrophobic (dislike water). Wax esters are formed by linking a long-chain carboxylic acid (fatty acid) to a long-chain alcohol (fatty alcohol). Since different fatty acids and fatty alcohols are linked together, the wax esters are a complex family of lipids and their detailed structure varies between species. In humans, oleic acid is the most prevalent fatty acid found in these waxes. Similarly, the steryl esters are mainly cholesterol esters which are formed by linking cholesterol to a longchain fatty acid. These fatty acids are generally longer than those found in the wax esters. Small amounts of other lipids (mainly polar) – such as mono-, di-, and triglycerides; fatty acids; fatty alcohols; free cholesterol; and phospholipids – make up the remainder. While phospholipids are readily detected in meibomian lipids of rabbits, there is contention as to whether they are a component of human meibomian lipids. This is important because in models of the lipid layer of the tear film, phospholipids are crucial as a link between the hydrophobic molecules (wax and cholesterol esters) and the aqueous layer. If they are not present in the meibomian lipids, then they must be derived from elsewhere, such as the aqueous layer of the tear film, or alternative surfactants need to be present in the model. The nature and mixture of the lipids give them a melting range of 19–33 C, which means that they are fluid on the ocular surface. Meibomian Lipid Turnover and Synthesis Since the meibomian gland is a holocrine gland, lipid turnover is related to the cell turnover rate and the lipids are synthesized by the glands rather than being adsorbed from the bloodstream. For instance, the levels of

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cholesterol found in meibomian lipids are independent of cholesterol levels in the blood. Synthesis of the straightchain fatty acids is typical of elsewhere in the body, occurring in the cytoplasm using acetyl-coenzyme A (CoA) and malonyl-CoA as the starting molecules and fatty acid synthase as the major enzyme. Some of the fatty acids have branching and, at least in rabbits, the branched carbon chains are derived mainly from the amino acids valine and isoleucine. The acyltransferases seem to be nonselective and will connect any fatty acid to a fatty alcohol. Similarly, cholesterol synthesis is typical of other tissues converting b-hydroxy-b-methylglutarylCoA to mevalonate, isoprene, squalene, and cholesterol. The acylcholesterol transferase, involved with cholesterol ester synthesis, appears to be selective for longer-chain fatty acids based on the predominance of long-chain fatty acids in cholesterol esters. The meibomian acinar cells have nuclear androgen receptors and the level of androgens or, more likely, the ratio between androgens and estrogens is critical for controlling lipid synthesis in the meibomian glands. Stimulating androgen receptors increases gene transcription in enzymes associated with fatty acid and cholesterol synthetic pathways (adenosine triphosphate (ATP)-citrate lyase, acetyl-CoA synthase, acetoacetyl-CoA synthase, 3-hydroxy-3-methylglutary (HMG)-CoA synthase 1, HMG-CoA reductase, acetyl-CoA carboxylase, glyceraldehyde-3-phosphate dehydrogenase, and sterol regulatory element-binding protein 1 and 2) and hence stimulates lipid production. Androgen deficiency has been associated with meibomian gland dysfunction (MGD) and dry eye. P2Y2 receptor gene expression has also been detected in meibomian gland acini. This suggests that extracellular ATP or UTP might influence lipid synthesis or composition through activation of G proteins. Specific changes to lipid composition through this pathway have not been investigated. The total amount of meibomian lipids on the lid margins has been estimated and children under 14 years of age showed the lowest levels (1.5 mg mm 2 lid margin). After puberty, there is a steady increment with age until the late 60s (3.26 mg mm 2) and throughout this period, males have nearly 10% greater levels than females. Given that one of the reasons for dry eye is insufficient meibomian lipid secretion and that there is an increase in the incidence of dry eye with age, it is surprising that the lowest levels of meibomian lipids have been found in children. Morning and afternoon basal levels are the same and there is no correlation between lid temperature (30–34 C) and basal levels of meibomian lipids. However, deliberately increasing the eyelid temperature from 33 to 37 C increases the lipid values on the lid margin by close to 25%. Despite the presence of parasympathetic nerves around the acini, lipid secretion is most likely due to the mechanical force of blinking which causes compression of the territorial fibrocartilaginous matrix that surrounds the

meibomian glands. This has been shown by measuring the reappearance of lipids on the eyelid margin after cleaning with an organic solvent. No lipids appear until a blink occurs (3 min was the longest). After approximately 10 blinks, the levels return to nearly a third of their basal levels. It is estimated that close to 10 mg of lipids are delivered per blink and that there is approximately 20–40 times excess basal amount of lipids available on the eyelid margin than what is required for forming a complete lipid layer on the tear film. How the lipids are removed from the eye is uncertain. It is believed that most of them flow over the eyelid margin onto the skin and eyelashes. This constant flow prevents the skin lipids from contaminating the tear film. It has been shown that skin lipids disrupt the tear film. Some lipids are likely to bind to proteins of the aqueous layer, particularly lipocalin, and are removed with the aqueous layer through the lacrimal ducts. The crusty buildup that collects in the corner of the eye during sleep is primarily a mixture of lipids and mucins and thus another mechanism for removing lipids from the ocular surface.

Pathology of the Meibomian Gland Disorders of the meibomian glands are manifest by the obstruction of the gland orifices, inflammation, or loss of the glands. This is often associated with one or more of the following: thickening of the lid margin, exaggerated vascularization around the gland orifices, and hyperkeratinization. Meibomian gland diseases are usually more uncomfortable rather than painful and, when chronic, are associated with dry eye which can be very painful. Absence or deficiency of meibomian glands is often congenital. Clinically, the state of meibomian glands is determined by examining their morphology and function. In eye clinics, the orifices of the glands lining both the upper and lower eyelids are observed through a slit lamp biomicroscope. If a gland is blocked, the orifice appears swollen on the lid margin. Some practitioners also squeeze the lower eyelid gently to expel meibomian lipids. A clear fluid is considered to be normal, whereas a thick, yellowy secretion is an indication of meibomian gland disorder. If excessive pressure is applied, a thick pasty expression can be obtained from people with normal meibomian gland function (Figure 5). This technique is used for obtaining meibomian lipids for experimental purposes or for analysis. In research settings, more specific tests are performed to assess meibomian gland function. Transillumination of the lower eyelids is widely used to evaluate the morphology of the glands. In particular, shorter-than-normal meibomian glands and meibomian gland dropout are strong indications of MGD (Figure 6). Changes to the shape and form of the glands do not occur with aging,


although expression of secretion is commonly more difficult. A clear, noninvasive view of meibomian glands can be achieved by using infrared light, but the need for specialized equipment means that this technique is only used in a few research laboratories. Rather than examine the meibomian glands themselves, observation of the lipid layer of the tear film is regarded as an indirect measure of both the quantity and the quality of meibomian gland secretions. Using specialized interferometry, the lipid layer stability, distribution, dynamics, and thickness can be assessed. Generally, a thick amorphous layer is an indication of high-quality meibomian oil secretions and, hence, excellent meibomian gland function. Such a layer is also commonly associated with longer TBUTs, presumably because of the lowered surface tension. The

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results obtained from tear breakup tests have been shown to be comparable to interferometry findings, although a cause–effect relationship between the two is yet to be established. Another method for evaluating meibomian gland function indirectly is to measure evaporation from the ocular surface. This is a difficult technique and is normally confined to research settings. Evaporimetry is based on the theory that evaporation from the ocular surface is normally minimal due to the well-spread lipid layer acting as a blanket, and that an inadequate lipid layer or disruption to this lipid layer causes tear evaporation to increase. It is thought to be the cause of evaporative dry eye. However, this idea has been difficult to substantiate and a wide variability in tear evaporation rates has been reported irrespective of the presence or absence of dry eye and, more importantly, the appearance of the lipid layer. Further, a large quantity of lipid on the tear surface does not necessarily correlate with an adequate barrier to evaporation. Evaporative dry eye can occur with an excessively thick lipid layer. Current areas of research center on whether the biochemical composition of the meibomian lipids can influence their surface activity and ability to diminish evaporation, but clear outcomes are still in the future.

Chronic Blepharitis

Figure 5 Hard squeezing of meibomian glands. The secretions are indicated by arrows.

The term blepharitis has different meanings depending upon the user. Acute blepharitis (normally just called blepharitis) is an infection of the anterior eyelid and Staphylococcus epidermidis or Staphylococcus aureus are the most likely cause. Chronic blepharitis is caused by dysfunction of the meibomian glands and is synonymous with

Region of meibomian gland dropout Meibomian gland in eyelid

Figure 6 The transillumination of eyelid showing the dark meibomian gland acini. There is meibomian gland dropout in the middle region of the lid. Courtesy of Jerry Paugh, Southern California College of Optometry.

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Chalazion

Stye

Figure 7 A chalazion appears as a swelling deep within the eyelid. A stye is associated with the eyelashes (infection of the eyelash follicle). Courtesy of Rob Terry, Institute of Eye Research, University of New South Wales.

MGD and posterior blepharitis. Many studies have looked at the association between blepharitis or chronic blepharitis and dry eye. However, the conclusions drawn about the association depend on the definitions being used in the study. Although MGD occurs in nearly three-quarters of patients with chronic blepharitis, it also occurs in approximately 20% of people with normal tear function. In MGD, the orifices of the meibomian glands are blocked, reducing the secretion of meibomian lipids onto the ocular surface. Regular warm compresses help to open the orifices and allow normal lipid secretion. Some patients with chronic blepharitis have similar symptoms to those of dry eye and are prescribed tear lubricants for palliative purposes, that is, they do not resolve the blepharitis. Chalazion The blockage of the meibomian glands can lead to formation of a chalazion (Figure 7). This is a cyst on the eyelid that is normally sterile and composed of a lipid granuloma. It looks similar to a stye, which is caused by an infected sebaceous gland of the eyelash, but can be easily distinguished clinically because a chalazion is painless and develops gradually, whereas a stye is always painful and forms over a few days. For both conditions, warm compresses are recommended. In extreme cases of chalazion, it is surgically incised and the granulomatous material is removed by curettage. Antibiotics are often prescribed to treat a stye. Surgical Damage Treatment of trachoma is a common cause for surgical damage to the meibomian gland. Trachoma, a leading cause of blindness, is an infectious disease of the palpebral conjunctiva that leads to the eyelids folding inward (entropion), causing the lashes to rub against the cornea. The lids, and hence the meibomian glands and ducts, are cut to relieve this condition. It is yet to be determined whether this compromises the functionality of the outer lipid layer of the tear film. Other surgical procedures such

as correction of lid malpositioning, particularly ptosis (drooping of the upper eyelid) and genetic entropion, can also sometimes require the cutting of the meibomian glands.

Contact Lenses and the Lipid Layer Anomalies of the lipid layer, in themselves, are not a deterrent for contact lens wear. Lipids or proteins or both are deposited on contact lenses during wear. These deposits can block the small pores of the contact lenses, which are essential for the passage of air to the cornea for its metabolism. It is impossible to ascertain beforehand how long a contact lens needs to be worn for it to be unsuitable for an individual as the amount and pattern of lipid deposition depends on the composition of the ocular lipids, which can vary between people, and the specific material the lens is made from. Contact lens cleaning agents are designed for the specific type of contact lens and normally contain a surface active agent that removes lipid deposits. See also: Dry Eye: An Immune-Based Inflammation; Ocular Mucins; Tear Film.

Further Reading Bron, A. J., Benjamin, L., and Snibson, G. R. (1991). Meibomian gland disease, classification and grading of lid changes. Eye 5: 395–411. Bron, A. J., Tiffany, J. M., Gouveia, S. M., Yokoi, N., and Voon, L. W. (2004). Functional aspects of the tear film lipid layer. Experimental Eye Research 78: 347–360. Butovich, I. A., Millar, T. J., and Ham, B. M. (2008). Understanding and analysing Meibomian lipids – a review. Current Eye Research 33: 405–420. Glasgow, B. J., Marshall, G., Gasymov, O. K., et al. (1999). Tear lipocalins: Potential scavengers for the corneal surface. Investigative Ophthalmology and Visual Science 40: 3100–3107. Goto, E. and Tseng, S. C. G. (2005). Kinetic analysis of tear interference images in aqueous tear deficiency dry eye before and after punctual occlusion. Investigative Ophthalmology and Visual Science 44: 1897–1905. Gouveia, S. M. and Tiffany, J. M. (2005). Human tear viscosity: An interactive role for proteins and lipids. Biochimica et Biophysica Acta 1753: 155–163.

Meibomian Glands and Lipid Layer Holly, F. J. (1973). Formation and rupture of the tear film. Experimental Eye Research 15: 515–525. Hykin, P. G. and Bron, A. J. (1992). Age related morphological changes in lid margin and Meibomian gland anatomy. Cornea 11: 334–342. Jester, J. V., Nicolaides, N., and Smith, R. E. (1981). Meibomian gland studies: Histologic and ultrastructural investigations. Investigative Ophthalmology and Visual Science 20: 537–547. Mathers, W. (2004). Evaporation from the ocular surface. Experimental Eye Research 78: 389–394. McCulley, J. P. and Shine, W. (1997). A compositional based model for the tear film lipid layer. Transactions of the American Ophthalmological Society 55: 79–93.

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Millar, T. J., Tragoulias, S. T., Anderton, P. J., et al. (2006). The surface activity of purified ocular mucin at the air–liquid interface and interactions with meibomian lipids. Cornea 25: 91–100. Nagyova, B. and Tiffany, J. M. (1999). Components responsible for the surface tension of human tears. Current Eye Research 19: 4–11. Sullivan, D. A., Sullivan, B. D., Evans, J. E., et al. (2002). Androgen deficiency, Meibomian gland dysfunction, and evaporative dry eye. Annals of the New York Academy of Science 966: 211–222. Tiffany, J. M. (1995). Physiological functions of the Meibomian glands. Progress in Retinal and Eye Research 14: 47–74.

Lacrimal Gland Overview M C Edman, R R Marchelletta, and S F Hamm-Alvarez, University of Southern California School of Pharmacy, Los Angeles, CA, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Acinus – Originating from the Latin word grape, it refers to the sac-like ending of a secretory exocrine gland. Endocytosis – The process of internalization of plasma membrane as well as membrane-bound constituents and extracellular fluid by invagination of the plasma membrane, budding of the membrane vesicle, and its movement to the interior. Different types of endocytosis are known, including clathrin-mediated and caveolar endocytosis. Exocytosis – The process by which a cell releases the contents of secretory vesicles to the extracellular environment by fusion of secretory vesicles with a plasma membrane domain. Motor proteins – Mechanochemical proteins that utilize the energy of ATP hydrolysis to generate motive force along a polar surface, typically an actin filament or a microtubule. Rab proteins – Small GTP-binding proteins that utilize the GTP binding and hydrolysis cycle to trigger protein on and off states, and which serve as molecular zip codes to specify the accurate sorting and targeting of membranes. SNARE proteins – Proteins associated with donor and acceptor membranes which associate to form a fusion pore, allowing the contents of opposing membrane vesicles to intermingle, or allowing the extrusion of membrane-encapsulated contents to the cell exterior. Transcytosis – The process by which macromolecules are transported through a polarized cell. trans-Golgi network – A post-Golgi processing compartment responsible for the accurate segregation of contents into membrane vesicles destined for regulated exocytosis, constitutive exocytosis, or for targeting to intracellular membrane compartments.

Anatomy of the Main Lacrimal Gland The human main lacrimal gland, located laterally above the eye, measures approximately 20 12 5 mm and has an almond-like shape. The major part of the gland, designated as the orbital portion, or the intraorbital gland,

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is located in the shallow lacrimal fossa of the frontal bone, while the smaller palpebral, or extraorbital portion, which is separated from the orbital portion by the lateral horn of the levator palpebrae muscle, is located above the temporal segment of the superior conjunctival fornix. In contrast, the mouse and rat have two pairs of lacrimal glands including a small orbital gland which is located laterally beneath the upper lid and a larger extraorbital gland which is located ventral and anterior to the eye. The rabbit lacrimal gland is unusually large and is comprised of a larger portion (4 cm) located below the eye and a smaller portion (0.5 cm) located above the eye. The lacrimal gland is constituted largely (80%) of acinar epithelial cells organized within the tubuloacinar units that are arranged into multiple globuli surrounded by fibrovascular septa. The remaining 20% of the mass of the lacrimal gland is composed of ducts, nerves, myoepithelial cells, leukocytes, and connective tissue. A schematic diagram showing the positioning of the gland relative to the ocular surface and the organization of several of the cell types within the gland is shown in Figure 1.

Cell Types within the Lacrimal Gland Acinar Cells The acinar epithelial cells within the lacrimal gland are triangular-shaped cuboidal cells organized in single cell layers in clusters with a narrow microvillus-covered apical domain oriented toward a central lumen and a more extensive basolateral domain which faces the tissue interstitium. Tight junctions near the apices segregate these two domains and result in polarization of the cells, which also are cytoplasmically coupled through gap junctions, thus making the acinus a single functional unit. The apical side of the cell is enriched in numerous large (1–2 mm) secretory granules or vesicles, containing proteins released upon cell stimulation, while the Golgi apparatus and endoplasmatic reticulum compartments are located more toward the basolateral side adjacent to the basolateral nucleus. Figure 2 shows the characteristic distribution of secretory vesicles and other organelles within an acinar cell from mouse lacrimal gland. Ductal Cells The lumena of several acini come together to form a duct; each duct merges with others into gradually larger ducts

Lacrimal Gland Overview

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Myoepithelial Cells L D

CNS

N

BM AM

LG

SV

ME

OS

NE

Figure 1 Schematic diagram showing the positioning of the human main lacrimal gland relative to the ocular surface and the organization of several of the cell types within the gland. AM, apical membrane; BM, basolateral membrane; CNS, central nervous system; D, duct; L, lumen; LG, lacrimal gland; ME, myoepithelial cell; N, nucleus; NE, nerve ending; OS, ocular surface; SV, secretory vesicle.

N

The acini are surrounded by stellate-shaped myoepithelial cells with long slender processes. The myoepithelial cells not only exhibit characteristics of other epithelial cells, such as expression of cytokeratin, but also exhibit properties of smooth muscle cells such as expression of a-smooth muscle actin. The exact role of the myoepithelial cells in the regulation and maintenance of the lacrimal gland remains unclear, but it has been shown that they express receptors for neurotransmitters, suggesting that they play a role in facilitating the secretion from the lacrimal gland. It is also likely that an important role is to support and maintain the structure of the lacrimal gland. Bone-marrow-derived Cell Population The lacrimal gland is a part of the mucosal-associated lymphoid tissue (MALT). The bone-marrow-derived cells in the lacrimal gland are mainly immunoglobulin A (IgA)-producing plasma cells and T and B lymphocytes, but macrophages and mast cells are also present. The bone-marrow-derived cells are clustered into lymphoid follicles scattered in the stroma surrounding the acini.

Innervation of the Lacrimal Gland SV

L 2000 nm

Figure 2 Transmission electron micrograph of mouse lacrimal gland. N, nucleus; L, lumen; SV, secretory vesicle.

that finally, in humans, drain into 6–12 major ducts with openings in the upper lateral fornix. In rat and mice, the ducts from the extraorbital gland join to a single major duct that then joins the duct from the intraorbital gland before it empties onto the conjunctiva in the lateral canthus of the eye. In rabbit, a single duct each forms from the upper and lower portions of the gland, which empty onto the conjunctiva of the upper and lower lids, respectively, near the temporal angle. The ducts are formed by one to two layers of cuboidal epithelial cells. Similar to the acinar cell, the ductal epithelial cells of small ducts are polarized by tight junctions in the apical area. However, in these cells, the Golgi and endoplasmic reticulum are located more apically, and secretory vesicle content is lower. Interlobular ducts are embedded with and supported by perivasculoductal connective tissue containing associated structures such as nerve fibers, capillaries, and mast cells and a dense population of fibroblast-associated collagen fibrils.

The lacrimal gland is innervated by parasympathetic, sympathetic, and sensory nerves. Parasympathetic nerves originate in the lacrimal nucleus of the pons and travel along the nervus intermedius, the deep and superficial petrosal nerves, and the vividian nerve before they synapse in the pterygopalatine ganglion. The postganglionic parasympathetic fibers can take different routes to the lacrimal gland. They leave the ganglion through the pterygopalatine nerves but can also reach the lacrimal gland via the maxillary portion of the trigeminal, the zygomatic, or the lacrimal nerves. Parasympathetic fibers can also travel along a branch of the middle meningeal artery to join the ophthalmic or lacrimal artery en route to the lacrimal gland. Sympathetic nerves originate from the superior cervical ganglion and travel along with the parasympathetic nerves through the pterygopalatine ganglion, reaching the lacrimal gland through the lacrimal branch of the zygomatic branch of the maxillary trigeminal nerve that joins the ophthalmic branch of the trigeminal nerve. The sensory nerves innervating the lacrimal gland carry sensory information from the gland through the ophthalmic branch of the trigeminal nerve to the trigeminal ganglion. The parasympathetic nerves, being the most abundant, regulate the lacrimal gland mainly through the release of neurotransmitters, acetylcholine and vasoactive intestinal

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peptide (VIP), with a possible co-secretion of nitric oxide (NO). Acetylcholine activates M3 muscarinic receptors located in the basolateral membrane of the lacrimal cells, while VIP binds to VIP receptors that are similarly located. The sympathetic nerves exert their effects on the lacrimal gland through release of norepinephrine that binds to a- and b-adrenergic receptors, and possibly through neuropeptide Y receptors also located at the acinar cell basolateral membranes. The sensory nerves release substance P and calcitonin gene-related peptide. Not every individual acinar cell is independently innervated; rather cells that are not directly innervated can respond to stimulation of neighboring cells due to the intercellular gap junctions that connect the cells. The density of synapses within each acinus varies according to the species: in the rat, orbital glands fewer than 15% of acinar cells have an adjoining nerve fiber in contrast to the mouse orbital glands where close to 100% of the cells have an adjoining nerve fiber.

Blood Supply The main blood supply to the lacrimal gland is not only through the lacrimal artery, a branch of the ophthalmic artery, but it also receives minor contributions from the infraorbital and the meningeal arteries. The veins mainly follow the same pathways as the arteries inside the orbit and drain into the superior ophthalmic vein.

Contents of Lacrimal Fluid The tear film consists of three layers: a mucus layer located directly above the ocular surface epithelium, an aqueous layer, and a thin external lipid layer. The lacrimal fluid produced by the main lacrimal gland constitutes the major part of the aqueous layer of the tear film, to which the accessory glands of Krause and Wolfring and the ocular surface epithelium also contribute, in a minor fashion. Although the major part of the aqueous layer is water, it also contains electrolytes and a high concentration of proteins. Human tear fluid, for instance, has a protein concentration of about 8 mg ml1. Although the three layers of the tear film largely originate from different sources, these sources can contribute in part to each layer; therefore, it is hard to determine the origin of a specific protein. Recently, researchers identified 419 different proteins in human tear fluid; however, many of these may not have an active function in the tear fluid but are simply debris shed from epithelial cells. Three proteins constitute 80% of the total protein within the aqueous tear film, that is, lipocalin, lysozyme C, and lactoferrin. The functions of the different proteins in the tear fluid are varied. For instance, many proteins contribute

to the antimicrobial properties of the tear fluid. Secretory IgA and cytokines are involved in immune responses, while others such as lysozyme C and lactoferrin provide a more direct defense against bacteria. The novel protein, lacritin, acts as a mitogen in corneal regeneration. Other proteins in the lacrimal fluid are involved in diverse activities in wound healing, blood coagulation, and oxidative stress reduction – all functions essential to maintain a healthy ocular surface. The protein pattern of both active and inactive proteins in the tear fluid can reflect disease states, including diabetes, dry eye, and cystic fibrosis.

Mechanisms of Protein Secretion in the Lacrimal Gland The acinar cells of the lacrimal gland are professional excretory cells that engage in several types of secretion that collectively contribute to the lacrimal fluid. Protein secretion at the apical membrane into lacrimal fluid can be subdivided into several types: regulated exocytosis, constitutive exocytosis, and transcytosis. Regulated exocytosis is a process in which proteins destined for a particular plasma membrane domain are sorted into secretory vesicles after their biosynthesis within a post-Golgi sorting compartment called the trans-Golgi network. These secretory vesicles mature and migrate toward the site of release, where they are stored until the appropriate signal triggers their movement and fusion with the acceptor membrane domain. Examples of content proteins thought to be released from the lacrimal acini in animal model systems through regulated exocytosis at the apical plasma membrane include peroxidase and b-hexosaminidase. Constitutive exocytosis occurs, similarly, as components for release to the exterior of the cell are sequestered into vesicles at the trans-Golgi network that are immediately targeted to the acceptor membrane. Unlike regulated exocytosis, constitutive exocytosis is not reliant on extracellular activation of receptors by a ligand (such as a hormone or a neurotransmitter) to elicit the event. Although both forms of exocytosis have been observed in lacrimal acini, most studies have focused on the role of regulated exocytosis in the release of proteins at the apical plasma membrane of the acinar cell into lacrimal fluid. The secretory vesicles in the lacrimal gland acinar cells are generally larger (1–2 mm) and considerably more heterogeneous in both size and content compared to vesicles in other exocrine glands such as exocrine pancreas and the salivary gland. The spectrum of proteins secreted from the lacrimal acinar secretory vesicles appear to span a greater functional range than the spectrum release from other exocrine tissues as well, including nutrient and protective factors as well as factors that protect the mucosal surface from pathogens. This is an area of very active research since there are an unusually large number of

Lacrimal Gland Overview

proteins of unknown function in the lacrimal gland secretory proteome. Transcytosis is a process in which material internalized at the basolateral membrane is recruited into vesicles by endocytosis, followed by the movement of these vesicles to apically located compartments, and ultimately their targeting to the apical plasma membrane for release. Major cargo known to be carried through this pathway includes dimeric IgA, through association with the polymeric IgA receptor. Although not specifically characterized in lacrimal acini, other abundant tear proteins, including albumin and transferrin, are known to be transported through transcytotic pathways in other epithelial cells, suggesting that these proteins may be comparably transcytosed into lacrimal fluid by lacrimal acini. Regulated exocytosis and transcytosis utilize a number of different processes, globally referred to as membrane trafficking, to achieve the unidirectional transport of cargo-laden vesicles over short and long distances followed by their targeted release. Several of these processes have been studied in acinar cells, including cytoskeleton and motor proteins, rab proteins, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors or SNARE proteins. All mammalian cells contain filamentous structures collectively referred to as the ‘cytoskeleton’, which includes actin filaments, intermediate filaments, and microtubules. Each of these structures is formed from individual subunit proteins that exist in equilibrium with polymeric assemblies. Cytoskeletal filaments are critical in maintaining the integrity of cell shape as well as conferring cellular polarity or asymmetry, a function critical in aiding the movement of materials to different membrane domains in a polarized cell. Filamentous actin and microtubules, in particular, participate in several capacities in the movement of membrane vesicles to the apical plasma membrane, where the release of proteins into lacrimal fluid takes place. Microtubule- and actindependent membrane transport events can be facilitated either by the use of compressive force associated with cytoskeletal assembly to physically compress or direct membrane traffic, and/or by the use of these polymers as tracks which support the movement of motor proteins which carry membrane vesicles to specific destinations. In lacrimal acini, actin filaments are localized in a dense network below the apical membrane called the subapical actin network, and this network is also present to a lesser extent below the basolateral membrane. Beneath the subapical actin, the ends of microtubules are anchored. Microtubules extend from the subapical region to the basolateral membrane. Both actin filaments and microtubules sustain aspects of protein secretion in lacrimal acinar cells. When microtubules are disrupted in acinar cells using the agent, nocodazole, stimulated protein secretion is reduced because the microtubule scaffolding required for vesicle motility has been disrupted. Other studies have

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suggested that a particular motor protein, cytoplasmic dynein, is required for the movement of membrane vesicles involved in secretory vesicle maturation and, possibly, transcytotic vesicle transport, to the subapical cytoplasm. The subapical actin cytoskeleton plays complex roles in lacrimal acinar secretion. With its location immediately beneath the apical plasma membrane in a dense network, it poses an intracellular barrier for vesicle fusion to the apical membrane. For fusion to occur, this actin barrier must be disassembled to allow access of large secretory vesicle to the apical plasma membrane. Recent work has shown in fact that regions of the actin cytoskeleton located immediately beneath the regions of apical plasma membrane do disassemble, but that actin filaments also reassemble and contract around the base of multiple fusing secretory vesicles. The force generated through compression and retraction of actin filaments toward the apical membrane aids in compound fusion and content extrusion from the fusing vesicles. Regulated exocytosis can therefore be further characterized in lacrimal acini into a type known as multivesicular exocytosis. Two specific actin-dependent motors have been implicated so far in this actin remodeling and compound fusion of activated secretory vesicles, a conventional myosin motor known as nonmuscle myosin 2 and an unconventional myosin motor known as myosin 5c, with the possibility that other members of the myosin motor superfamily may also participate in this complex process. Other major membrane trafficking effectors that have been implicated in acinar cell protein secretion include rab proteins. Rabs are major effectors of all intracellular steps of membrane trafficking and fusion in the eukaryotic cell, serving as the molecular address code on donor membrane vesicles which specify the acceptor compartment destination. GTP binding and hydrolysis serves as the on/off switch that activates these proteins. Specific rabs are localized to distinct compartments, conferring identity to these compartments. For instance, rab3D is enriched in secretory vesicles in lacrimal acinar cells and appears to regulate compound fusion of these vesicles. Other data suggest that rab27 isoforms also participate in the maturation and fusion of secretory vesicles during regulated exocytosis in lacrimal acini. By analogy with other systems, rab4 and rab5 isoforms are likely to participate in early events in acinar transcytosis, specifically basolateral endocytosis and sorting, while rab 11 isoforms are enriched in apical endosomes and may facilitate terminal transcytotic traffic of materials destined for the lacrimal fluid. Specific types of SNARE proteins are located on donor and acceptor membranes and interact to form fusion pores which allow membrane contents to mingle or secretory vesicle contents to be extruded to the cell exterior. Several types of SNARE proteins have been demonstrated in lacrimal acini. Regulated exocytosis of secretory vesicles in acini is thought to use both vesicle-associated

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Resting

AM

TGN

membrane protein 2 (VAMP 2) and VAMP 8 on donor membranes, and syntaxin 2 and SNAP23 on acceptor membranes. Figure 3 shows the organization of the two major protein secretory pathways that contribute proteins to lacrimal fluid: regulated exocytosis and transcytosis, as well as some of the effectors within each pathway. Morphological analysis of ductal epithelial cells has revealed the presence of large secretory vesicles, presumably containing additional constituents destined for release into the lacrimal fluid. However, due to limitations in the ability to isolate these individual cells and conduct cellular investigations into the membrane trafficking mechanisms, little is known about the precise mechanisms involved in ductal cell exocytosis and transcytosis.

BM

Mechanisms and Regulation of Electrolyte and Water Secretion by the Lacrimal Gland

(a) AM

Stimulated

Acinar Cells

TGN

(b)

BM Actin

Microtubules

Snares

Rabs

Unconventional myosin

Conventional myosin

Microtubule motor Figure 3 Protein secretion in lacrimal acinar cells at the apical membrane. (a) Depicts vesicles participating in the transcytotic pathway from the basolateral membrane (BM) to the apical membrane (AM) as shown in blue vesicles. Depicted as well is the maturation and movement to the AM of secretory vesicles after budding from the trans-Golgi network (TGN) as shown in red vesicles. Initially both transcytosis from the BM and movement from the TGN are reliant on microtubule-based motor proteins. As the vesicles move to the actin-rich subapical region, unconventional myosin motors become important in actin-based movement through the subapical actin network. (b) Depicts multivesicular exocytosis after stimulation with secretogogs such as carbachol. Unconventional myosins such as myosin 5c have been shown to have an important role in the reorganization of actin filaments around clusters of secretory vesicles primed for fusion. Actin and conventional myosins then work together to compress the fusing secretory vesicles and to promote content extrusion. Rab and SNARE

The lacrimal fluid is hypertonic due to a high Cl and Kþ 2þ content, whereas the levels of Naþ, HCO are 3 , and Ca similar to the plasma levels. The electrolyte concentration of the lacrimal fluid is however not static, but varies with the flow rate to become more isotonic with an accelerated flow rate. Fluid secretion is an osmotic process driven by ion movement through the membrane of the acinar cells. Parasympathetic stimulation of the acinar cells triggers an acute increase of cytosolic Ca2þ and adenosine 30 ,50 cyclic monophosphate (cAMP) which opens Cl channels in the apical membrane, resulting in a movement of Cl into the lumen. The increase in cytosolic Ca2þ also activates Kþ channels in the apical as well as the basolateral membrane, causing Kþ to move out of the cell. Naþ follows the flux of Cl and Kþ, moving from the basolateral side toward the lumen traveling through paracellular channels between the cells. To maintain an isotonic secretion, water exits the cell through water-channel proteins called aquaporins. The movement of Cl and Kþ out of the cell is dependent on their electrochemical gradient, that is, the intracellular concentration of these ions must be higher than in the extracellular fluid. This is made possible by ion pumps and co-transporters located in the basolateral membrane. Naþ/Kþ-ATPase transports Kþ into the cell and Naþ out of the cell; the Naþ/Kþ/2Cl co-transporter (NKCC1) moves all three ion types into þ þ the cell; and Cl/HCO 3 and Na /H anti-porters trans þ port Cl and Na into the cell and Hþ and HCO 3 out of the cell. Figure 4 illustrates the ion channels and transporters present in the lacrimal acinar cell. proteins participate in the targeting and fusion events in each pathway. It should be noted that the rabs and the SNAREs participating in transcytosis and exocytosis are different for each pathway.

Lacrimal Gland Overview K+

Cl– Lumen

Ca2+

cAMP

K+ Cl–

Na+ ATP ADP

BM Na K+ 2Cl–

K+ HCO3

Na+

H+

Figure 4 Schematic diagram showing the major ion transporters active in the electrolyte and water release from the lacrimal gland acinar cell. Increases in cytosolic Ca2þ and cAMP following neural stimulation opens Kþ and C channels, resulting in an outward flux of these ions. Naþ/Kþ-ATPase transports Kþ into the cell and Naþ out of the cell, the Naþ/Kþ/2Cl co-transporter moves all three ion types into the cell, and þ þ þ Cl/HCO 3 and Na /H anti-porters transport Cl and Na into þ the cell and H and HCO3 out of the cell.

Ductal Cells Due to difficulties in specifically isolating the ductal cells, the ion transport mechanisms have not been extensively studied. However, it has been hypothesized that the water and ion transport events continue as the lacrimal fluid travels through the ductal system in a pattern comparable to that in the acinar cells. Recently, several approaches to study the ductal cells have been developed, including microdissection and culturing of individual ducts and laser capture microdissection of individual ductal cells. In these studies, some of the most common acid/base transporters were characterized in the ductal cells. The same work also showed that the ion transport in the ductal cells can be regulated by parasympathetic neurotransmitters. Furthermore, studies showed that the lacrimal fluid released from the acinar cells is isotonic, leading to the hypothesis that the ductal cells are responsible for the high Kþ levels in tears. The finding that NaþKþ-ATPase and NKCC1 are expressed at higher levels in the ductal cells than in the acinar cells supports this hypothesis.

Conclusion Previous and ongoing studies have established many of the functions of the principal cells of the lacrimal gland. The major cell type, the acinar cell, is responsible for the regulated release of proteins, fluid, and electrolytes into

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the lacrimal fluid, while ductal cells appear to further modify the electrolyte composition and likely also contribute additional proteins to the aqueous tear film. Some of the signaling pathways have been elucidated that stimulate the production of lacrimal fluid, while some of the molecular mechanisms involved in the fundamental exocytotic and transcytotic events have likewise been elucidated. However, the complexity of the signaling and membrane trafficking events even in lacrimal acinar cells, the best-studied cell type in this complex organ, means that considerable work remains to be done. Although some insights regarding changes in signaling and membrane trafficking pathways that result in altered production of lacrimal fluid have been obtained in dry eye disorders, considerable additional work is required in order to truly understand the etiology of these disorders. In some studies, changes in tear protein composition have been associated with dry eye disorders, so future challenges also include the identification of tear biomarkers that can be used to diagnose different types of dry eye disorders to aid in determination of the appropriate course of treatment. See also: Dry Eye: An Immune-Based Inflammation; Innate Immune System and the Eye; Lacrimal Gland Hormone Regulation; Lacrimal Gland Signaling: Neural; Meibomian Glands and Lipid Layer.

Further Reading Cohen, A. J., Mercandetti, M., and Brazzo, B. G. (eds.) (2006). The Lacrimal System, Diagnosis, Management and Surgery. New York: Springer. Hodges, R. R. and Dartt, D. A. (2003). Regulatory pathways in lacrimal gland epithelium. International Review of Cytology 231: 129–196. Jerdeva, G., Wu, K., Yarber, F. A., et al. (2005). Actin and non-muscle myosin II facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells. Journal of Cell Science 118: 4797–4812. Marchelletta, R. R., Jacobs, D., Schechter, J. E., Cheney, R., and Hamm-Alvarez, S. F. (2008). Myosin Vc facilitates actin filament remodeling and compound fusion of mature secretory vesicles during exocytosis in lacrimal acini. American Journal of Physiology (Cell Physiology) 295: C13–C28. Pflugfelder, S. C., Beuerman, R. W., and Stern, M. E. (eds.) (2004). Dry Eye and Ocular Surface Disorders. New York: Marcel Dekker, Inc. Ubels, J. L., Hoffman, H. M., Srikanth, S., Resau, J. S., and Webb, C. P. (2006). Gene expression in rat lacrimal gland duct cells collected using laser capture microdissection: Evidence for Kþ secretion by duct cells. Investigative Ophthalmology and Visiual Science 47: 1876–1885. Walcott, B., Moore, L., Birzgalis, A., Claros, N., and Brink, P. R. (2002). A model of fluid secretion by the acinar cells of the mouse lacrimal gland. Advances in Experimental Medicine and Biology 506(Pt. A): 191–197. Wu, K., da Costa, S. R., Jerdeva, G., et al. (2006). Mechanisms of exocytosis in lacrimal gland. Experimental Eye Research 83: 84–96. Zierhut, M., Stern, M. E., and Sullivan, D. A. (eds.) (2005). Immunology of the Lacrimal Gland, Tear Film and Ocular Surface. New York: Taylor and Francis.

Lacrimal Gland Hormone Regulation A K Mircheff, D W Warren, and J E Schechter, University of Southern California, Los Angeles, CA, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary CD86 – A co-receptor expressed on the surfaces of antigen-presenting cells. When engaging either of its cognate receptors – CD28 and CTLA-4 – on the surface of T cells, it generates signals essential for T-cell activation and activates signaling cascades within the antigen-presenting cells. Chemokines – The proteins that promote recruitment of lymphocytes and leukocytes to inflamed tissues and to lymphoid tissues. Hypophysectomy – The surgical removal of the pituitary gland. Interferon gamma (IFN-g) – A cytokine released primarily by T cells and natural killer cells that induces T cells to express the TH1 phenotype, activates macrophage to express microbicidal functions, and induces B cells to switch from immunoglobulin M (IgM) to complement-fixing IgG isotypes. Interleukin 1 alpha and 1 beta (IL-1a, IL-1b) – The related cytokines released primarily from macrophages, endothelial cells, and epithelial cells that induce inflammatory responses. Interleukin 6 (IL-6) – A cytokine that promotes inflammatory responses and supports survival of T and B cells. Interleukin 12a (IL-12a) – A cytokine released in innate immune responses that induces expression of IFN-g and, thereby, promotes the evolution of adaptive responses mediated by TH1 cells. Lactation – The production and secretion of milk. Lactogenesis – The secretory differentiation of the mammary epithelium. Orchiectomy – The surgical removal of the testes. Sex hormone-binding globulin (SHBG) – A protein which binds estrogens and androgens. It is produced by the liver and secreted into the blood. Estrogens stimulate its production and androgens suppress its production. Sodium-potassium-dependent ATPase (Na,K-ATPase) – The sodium–potassium pump enzyme; it generates the chemiosmotic energy essential for lacrimal fluid production by pumping Na+ out of, and K+ into, the cytosol. Transforming growth factor-beta (TGF-b) – A cytokine released by T cells and macrophages, as well as by some epithelial cells and mesenchymal cells. Its actions on immune cells include: inhibiting T-cell proliferation and expression of effector

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functions; inhibiting B cells from proliferating and inducing them to undergo IgM-to-IgA isotype classswitch recombination; and suppressing macrophage activation. It often exerts antiproliferative or proapoptotic influences on epithelial cells.

Gender-Related Dimorphisms The lacrimal glands produce most of the aqueous fluid that comprises the milieu exte´rieur sustaining the live, metabolically active cells of the superficial layers of the cornea and conjunctiva, and insufficient production of this fluid and alterations of its composition are associated with dry eye disease. Because dry eye disease is considerably more prevalent among women, it has seemed intriguing that morphological differences can be readily discerned between the acini – that is, the primary secretory structures of the lacrimal glands – of male and female rats. The structural dimorphisms were, some years ago, found to be accompanied by equally striking biochemical and functional dimorphisms. Classic work by Sullivan and colleagues showed that many of the evident dimorphisms are supported by the higher levels of androgens characteristic of males. However, it is taking much longer to learn how, in mechanistic terms, the gender-related dimorphisms might relate to females’ greater predilection for lacrimal dysfunction. Indeed, one of the first functional dimorphisms to be documented appeared paradoxical: basal precorneal tear volume is smaller in intact male rats than in females, and it increases in males after they are castrated – a surgical maneuver that causes the size of the acini to decrease, that is, to become more female like. One of the products that the lacrimal glands contribute to the ocular surface fluid is secretory immunoglobulin A (sIgA), which is the effector of the adaptive mucosal immune defense against microbial infection. The lacrimal glands of rats exhibit several readily quantified dimorphisms relating to the production and secretion of dimeric IgA (dIgA). The stromal spaces of the lacrimal glands of male rats are populated by larger numbers of dimeric IgA-secreting plasmacytes. Whole gland extracts contain greater masses of dIgA. Glandular epithelial cells express higher levels of the polymeric Ig receptor (pIgR), which mediates uptake of dimeric dIgA at the stromal-facing surface of the lacrimal epithelium, chaperones it through

Lacrimal Gland Hormone Regulation

the cells’ transcytotic apparatus, and provides the secretory component (SC) portion of secretory IgA (sIgA). Lacrimal gland fluid from sexually mature male rats contains both more sIgA and SC.

Sex Steroids Androgens As noted above, it was established early on that the androgens support the gender-related dimorphisms of acinar size and of precorneal tear volume, which is presumably related to basal rates of lacrimal fluid production. It was also found that the androgens also support epithelial cell expression of pIgR and secretion of SC. However, androgen effects on the numbers of IgAþ-plasmacytes populating the glands’ stromal spaces varied considerably among individual animals. Although different laboratories have reported discrepant findings, there is some evidence for the theoretical paradigm that the androgens exert general trophic influences on the glandular epithelium. Hypophysectomizing rats decreases circulating levels of gonadal and adrenal steroids, as well as pituitary hormones (see Box 1). This maneuver reduces the lacrimal glands’ gross weight and their weight as a fraction of total body weight. In some studies, administration of dihydrotestosterone (DHT) did not change lacrimal gland weight appreciably. In other studies, administration of DHT partially reversed hypophysectomy-induced decreases in the total amounts of protein and Na,K-ATPase catalytic activity measured in lacrimal gland lysates. Ovariectomizing female rabbits decreases serum sex steroid levels. This maneuver decreased the total protein and DNA contents of lacrimal gland lysates. Administration of DHT prevented the ovariectomy-induced decreases, and it increased the Na,K-ATPase catalytic activity measured in lacrimal gland lysates. Surprisingly, in view of the role Na,K-ATPase plays in lacrimal fluid production, DHT did not increase the basal rate of lacrimal fluid production. However, it significantly increased the volume of fluid intact glands produced when they were stimulated with cholinergic agonists. These findings make it clear that several independent layers of regulation determine lacrimal fluid production: long-term regulation of the mass of cells comprising the glandular epithelium and of the levels at which the epithelial cells transcribe the genes specifying Na,K-ATPase and other iontransport proteins, and acute regulation – presumably neurally mediated – of the transport proteins’ functional states. A more complex paradigm, however, is needed to account for why the magnitude of the lacrimal gland regression caused by ovariectomy is small compared to the extent of atrophy that nuclear magnetic resonance (NMR) imaging studies have documented in the lacrimal glands of aging females. One possible paradigm is that in mature, but not aged, female rabbits the lacrimal glands compensate for ovariectomy-induced loss of testosterone

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by converting the weak androgen, dihydroepiandrosterone (DHEA) – which is produced by the adrenal cortex (see Box 1) – to testosterone and DHT. A second is that the loss of a small trophic influence must be compounded over time before its consequences become evident. A third is that androgens influence other parameters in addition to the cellular mass of the epithelium, and that it is the consequences of loss of those influences that are compounded over time. In ex vivo studies, DHT increased proliferation in models of acinar cells from rabbit lacrimal glands. Compared to the action of epidermal growth factor (EGF), however, the influence of DHT was relatively modest. When Azzarolo and colleagues tested the hypothesis that androgens support the number of cells in the epithelium by preventing apoptosis, as well as by promoting cell proliferation, they found that epithelial cell apoptosis is relatively rare, but plasmacytes in the glands’ stromal space began apoptosing within an hour following ovariectomy. Moreover, administration of DHT prevented ovariectomy-induced plasmacyte apoptosis. The finding that androgen withdrawal leads to a wave of apoptosis in the lacrimal gland plasmacyte population is one of several that accord with the paradigm that their higher levels of androgens protect men both from Sjo¨gren’s autoimmune dacryoadenitis and from the histopathophysiological syndrome commonly found in aging women by influencing immunophysiological processes within the gland. Indeed, it has been found that administration of DHT suppresses lacrimal gland disease in certain mouse models for Sjo¨gren’s syndrome, and preliminary clinical experiences suggest that androgen supplementation of hormone replacement therapy may improve symptoms and clinical signs in menopausal women with inflammatory autoimmune lacrimal diseases. In the models in which androgen administration is therapeutic, the responses are more pronounced in the lacrimal glands than in other affected organs. This finding led Sullivan and coworkers to propose that the androgens control the expression of critical immunoregulatory paracrine mediators by lacrimal gland epithelial cells. Recent microarray analyses indicate that androgens influence the expression of large numbers of gene transcripts in the lacrimal glands and corneas of mice. Some of the androgen actions might be expected to diminish inflammatory processes. For example, testosterone decreases expression of certain chemokines; interferon (IFN) response factors 4 and 7; caspase-1, which converts the inactive interleukin (IL)-1b precursor to active IL-1b; and the prolactin receptor, which – as discussed below – mediates mitogenic responses both in B and T cells and induces Tcells to express IFN-g. However, other testosterone actions might be expected to enhance inflammatory processes. For example, testosterone increases expression of IL-6; IL-12a; the chemokines CCL1, CCL8, CCL28, CCL5, and CXCL4; CD86; and interferon response factor 5.

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Box 1.

Regulation of reproductive hormone bioavailability

Figure 1 summarizes factors and feedback interactions that determine the bioavailabilities and local actions of the steroid reproductive hormones. Hypothalamic neurons release gonadotropin-releasing hormone (GNRH), which stimulates release of LH and FSH by the anterior pituitary. LH and FSH, in turn, stimulate synthesis of estradiol and progesterone in the ovaries, and synthesis of testosterone in the testes. Notably, testosterone is an obligatory precursor in the syntheses of estradiol and progesterone, and total serum testosterone levels increase as estradiol and progesterone levels increase. Progesterone can be converted to testosterone, but estradiol cannot. The anterior pituitary also produces adrenal corticotropic hormone (ACTH), which is induced by corticotrophin releasing hormone from hypothalamic neurons. ACTH, in turn, increases adrenal synthesis of the glucocorticoids and dihydroepiandrosterone (DHEA). DHEA can be converted to testosterone in peripheral tissues. Androgen target tissues frequently convert testosterone to dihydrotestosterone, a higher affinity ligand for androgen receptors. The liver plays an indirect but critical role in determining testosterone bioavailability, producing sex hormone binding globulin (SHBG) in response to increasing levels of estradiol and testosterone in the serum. In some cases SHBG sequesters the hormones, reducing their bioavailability. In other cases SHBG-sex steroid hormone complexes exert biological activities by interacting with nonclassical receptors at the surfaces of target cells, rather than with the classical receptors that traffic from the cytosol to the nucleus. After menopause, the ovarian stroma continues to produce some testosterone and androstenedione. During hormone replacement therapy, however, the exogenous estrogens suppress GnRH release, thus reducing ovarian testosterone production. Exogenous estrogens further decrease testosterone bioavailability by stimulating increased hepatic production of SHGB. Production of testosterone declines gradually as men age. Production of DHEA declines similarly in women and men. In women, reproductive steroid hormone and prolactin levels vary systematically during the menstrual cycle. They fluctuate, with more individual variation, during the perimenopause. They then remain consistently low postmenopausally, while prolactin levels are decreased only modestly. Estradiol, progesterone, and prolactin levels increase markedly over the course of a pregnancy. The steroid levels decrease abruptly at parturition; prolactin declines more gradually during a nonlactating puerperium, and it remains elevated during lactation. In contrast to positive regulation of LH, FSH, and ACTH by hypothalamic factors, the release of prolactin by the anterior pituitary is negatively regulated, that is, suppressed, by dopamine produced by hypothalamic neurons. Estradiol increases production of prolactin in the anterior pituitary, while prolactin suppresses estradiol production by acting upstream to suppress release of GNRH. As noted in the text, a number of peripheral tissues in addition to the pituitary produce prolactin.

Hypothalamus

Pituitary

CRH

Dopamine Gonads GNRH

ACTH LH, FSH Prolactin

Adrenal cortex

Testosterone

Cortisol

Gonads

DHEA Progesterone

Testosterone SHBG

Estradiol

DHT On Off

Liver

Figure 1 Production, regulation, and interactions of reproductive hormones. The pituitary produces the protein hormones, LH (luteinizing hormone), FSH (follicle stimulating hormone), ACTH (adrenalcorticotropic hormone), and prolactin. LH and FSH stimulate gonadal production of estradiol, progesterone, and testosterone. ACTH stimulates production of cortisol, as well as dihydroepiandrosterone (DHEA). Estradiol stimulates hepatic production of sex hormone binding globulins, which may either sequester steroids, particularly testosterone, reducing their bioavailability, or potentiate their actions by allowing them to bind to unconventional receptors at the surfaces of target cells. Estradiol also increases pituitary production of prolactin. Both prolactin and estrogen mediate negative feedback signals that decrease LH and FSH production.


Before considering tentative theoretical paradigms that might explain how the androgens might confer protection against dry eye disease, it is necessary to first review the influences other reproductive hormones exert on the lacrimal glands. Estradiol and Progesterone Whereas estradiol and testosterone often have opposing actions, Azzarolo and coworkers found that estradiol – like DHT – prevents ovariectomy-induced apoptosis of lacrimal gland plasmacytes. Estradiol does not appear to influence pIgR expression in rat lacrimal epithelial cells. However, tear lactoperoxidase levels vary during the estrus cycle in rats and the menstrual cycle in humans – correlating with the changes in estradiol and progesterone levels. Microarray analyses of mouse lacrimal gland extracts indicate that estradiol and progesterone influence the expression of numerous gene transcripts. They increase expression of the chemokines CCL2 and CXCL15 and decrease expression of FoxP3 – a central transcription factor in regulatory lymphocyte function; these influences might seem consistent with the greater risk for inflammatory lacrimal gland disease in females. However, estradiol and progesterone also decrease expression of CD86, IL-12, and the chemokines CCL6, CCL12, and CCL28 – influences which might be expected to diminish inflammatory responses.

Prolactin Prolactin is produced by the pituitary gland; as its name implies, its first discovered function was support of lactogenesis and lactation. However, prolactin has also been found to function as an autocrine/intracrine and paracrine mediator in a number of physiological systems. In the immune system, it acts as a mitogenic cytokine for Tcells and B cells, and as a differentiation factor for T cells – inducing them to express the prototypical TH1 cytokine, IFN-g. Administration of prolactin to hypophysectomized male rats increased Na, K-ATPase catalytic activity in lacrimal gland. However, a number of reports indicate that serum prolactin levels are elevated in women with Sjo¨gren’s syndrome and other autoimmune diseases. A study of reproductive hormone influences on lacrimal function revealed that increasing serum prolactin levels within the normal range of values for nonpregnant, nonlactating women were strongly correlated with decreasing lacrimal function – independently of menopausal status and use of estrogen replacement therapy. Exocrine Products and Autocrine/Intracrine and Paracrine Mediators While they respond to prolactin as a classic hormone, lacrimal gland epithelial cells also express prolactin, and

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they secrete it both as an exocrine secretory product and as a paracrine mediator. In the lacrimal glands of normal, nonpregnant female rabbits, immunopositivities for prolactin, as well as for transforming growth factor-beta (TGF-b), EGF, fibroblast growth factor (FGF)-2, are localized preferentially – but not exclusively – in ductal epithelial cells. In both acinar and ductal cells, the cytokine and growth factor immunopositivities are concentrated in the apical cytoplasm. Prolactin is localized in the regulated exocrine secretory vesicles; the mechanisms by which epithelial cells of the rabbit lacrimal gland secrete the other cytokines and growth factors have not been elucidated. Like prolactin and other secretory vesiclecontent proteins, TGF-b is released to the fluid forming within the lumen of the acinus-duct system in response to stimulation with cholinergic agonists. As illustrated in Figure 1, lacrimal epithelial cells use their apical recycling endosome and early basolateral endosome as a transcytotic secretory apparatus. It is this apparatus which secretes sIgA and some free SC into the lumena of the acinus-duct system. They also use the early and recycling endosomes as a paracrine secretory apparatus that delivers products to the underlying stromal space. Both transcytotic secretion and paracrine secretion occur constitutively; although they can be accelerated by stimulation with cholinergic agonists, the steady-state pools of secreted products in the endosomes are quite small compared to the pools of products stored in regulated exocrine secretory vesicles. Experiments with ex vivo acinar cell models showed that increasing the concentration of prolactin in the ambient medium induces increased transcription of prolactin messenger RNA (mRNA). Increasing epithelial cell prolactin expression or increasing the prolactin concentration in the ambient medium decreased the amount of secretory proteins stored in apical secretory vesicles, and it induced the cells to express a novel population of regulated secretory vesicles that accumulated in the basal cytoplasm and released their contents at the basolateral plasma membrane in response to acute cholinergic stimulation. Immunogold localization studies demonstrated that when acinar cells endocytose prolactin from their ambient medium, they traffic it dually to the endosomes that comprise their constitutive transcytotic-paracrine apparatus and to the secretory vesicles of the novel, induced paracrine apparatus. Thus, when acinar epithelial cells internalize prolactin secreted from the pituitary or from ductal epithelial cells, they may recycle it as a paracrine mediator. Serum prolactin levels do not differ greatly between normal men and normal, nonpregnant women. However, serum prolactin levels increase markedly during pregnancy, and, by the time a pregnancy reaches term, mean serum prolactin levels are 10- to 20-fold greater than the levels in nonpregnant, nonlactating females. Thus, the physiological hyperprolactinemia of pregnancy has

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Early endosome

Recycling endosome Multivesicular body

Stroma

Golgi ER Lumen Regulated paracrine secretory vesicles Late endosome

TGN Exocrine secretory vesicles

Storage lysosome Pre-lysosome Autolysosome

Prolactin

Constitutive traffic

Prolactin receptor

Induced by prolactin

Maturation

Endocytosed prolactin

Cholinergically induced exocytosis

Locally expressed prolactin

Figure 1 As in other exocrine glands, lacrimal gland epithelial cells use secretory vesicles to secrete proteins into the fluid being produced in the lumena of the acinus-duct system. They also use their early basolateral endosomes and apical recycling endosomes as a transcytotic apparatus to secrete SC and sIgA. Furthermore, the constitutive traffic of transport vesicles from the endosomes to the basolateral plasma membranes secreted paracrine mediators to underlying stromal space. Elevated levels of prolactin induce the cells to express a novel population of regulated paracrine secretory vesicles and decrease their population of exocrine secretory vesicles. The induced paracrine secretory vesicles allow ductal epithelial cells to secrete more prolactin and TGF-b to the stroma, and they allow acinar cells that have endocytosed prolactin from the stroma to recycle it as a paracrine secretory product.

seemed to offer a natural model in which to study prolactin’s influences on the lacrimal glands.

Influences of Prolactin, Estradiol, and Progesterone during Pregnancy The lacrimal glands of nonpregnant, sexually mature female rabbits normally contain small aggregates of

lymphocytes and plasmacytes, localized in the stromal spaces surrounding and spanning between venules and interlobular ducts. It might be noted that these are the same sites where the ectopic lymphoid tissues characteristic of Sjo¨gren’s dacryoadenitis develop. The immunoarchitecture undergoes a remarkable change during pregnancy, and it remains in the altered state throughout lactation and for some weeks following weaning. By the time a pregnancy reaches term, the aggregates have largely dissipated, and


lymphocytes and plasmacytes are primarily located in the thin stromal spaces surrounding acini. The immunoarchitectural change is associated with several notable cytophysiological changes and functional changes. The basal rate of lacrimal gland fluid production decreases, while the rate of fluid production under cholinergic stimulation increases. Immunopositivities for TGF-b and prolactin increase substantially, and their localizations shift from the apical cytoplasm to the basal cytoplasm. It now appears that these redistributions occur because the novel paracrine secretory apparatus induced by the increased serum prolactin level captures prolactin and TGF-b away from the regulated exocrine secretory pathway. Accordingly, the level of prolactin excreted in lacrimal gland fluid decreases, and TGF-b becomes scarcely detectable. Experiments that have not yet been published indicate that when ovariectomized rabbits are implanted with sustained-release pellets establishing pregnancy-like serum levels of estradiol and progesterone, the patterns of lymphocyte organization and of TGF-b and prolactin expression and localization change to resemble the patterns characteristic of pregnancy. Thus, estradiol, progesterone, or the two steroid hormones in concert act on ductal epithelial cells to increase their expression TGF-b. They may increase ductal epithelial cell prolactin expression either directly, or indirectly, that is, by increasing pituitary prolactin secretion (see Box 1). The increased level of prolactin then directs both mediators away from the regulated exocrine protein-secretion apparatus and directs prolactin into the novel paracrine apparatus. As discussed below, the changes that occur in the lacrimal glands during pregnancy are analogous to those which occur over roughly the same time in the mammary glands. While the lacrimal glands are accessory organ of the visual system, the mammary glands are accessory organs of the reproductive system. Both glands also are effector organs of the mucosal immune system. Other organs of the male and female reproductive systems also play parallel roles as adaptive mucosal immune system effector organs, and their immunophysiological functions are, likewise, influenced by the reproductive hormones.

Reproductive Hormone Influences on Other Mucosal Immune System Tissues Neither the testes nor the ovaries are normally populated by IgA+-plasmacytes. However, in males, IgA+ cells are abundant in the urethral glands and prostate; they are also present in the seminal vesicles in some species, but not others. Orchiectomizing male rats has little effect on the amount of IgA in the prostate and seminal

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vesicles; subsequent administration of DHT causes a slight increase of the IgA content of the prostate, but not the seminal vesicle. There is little pIgR expression in the testes, vas deferens, or epididymis, but a significant level of expression in the seminal vesicles and a 20-fold greater level in the prostate. Orchiectomy decreases pIgR expression, and subsequent administration of DHT increases it threefold in the seminal vesicles and fourfold in the prostate. Interestingly, estradiol has no effect on pIgR expression in the seminal vesicles but doubles it in the prostate. In females, IgA+ immune cells are abundant in the lamina propria of the fallopian tubes. They are sparse in the endometrium. Since endometrial gland epithelial cells contain IgM+ and IgA+, as well as J chain, it may be that the uterine lining secretes Igs derived primarily from the serum, rather than from local plasmacytes. In contrast, IgA+ cells are abundant within the epithelia and lamina propria of the endocervix, although somewhat less abundant in the ectocervix and vagina. Epithelial expression of pIgR roughly parallels the abundance of IgA+ cells. The level is significant in the fallopian tubes and endocervix. While there seems to be no clear evidence that pIgR is expressed in the ectocervix and vagina, the level of sIgA in cervical mucus increases just prior to ovulation and remains elevated throughout the luteal phase, and the uterine fluid contains a high level of sIgA throughout pregnancy. As the alveolar epithelium of the mammary glands develops during pregnancy, ductal epithelial cells are induced to express pIgR, and the glands’ stromal spaces become populated by dIgA+-plasmacytes. During lactation, the mammary epithelium secretes sIgA as well as lactoperoxidase and other innate mucosal immune effector molecules. Like the other changes occurring during lactogenesis, the induction of mucosal immune effector functions is controlled by interacting influences of estradiol, progesterone, and prolactin. Most studies of hormonal influences on the mammary glands have been motivated by interest in normal lactogenesis, lactation, and postweaning involution, and in mammary carcinoma – rather than focused on the mammary glands’ mucosal immune functions. This work has shown that the systemic hormones orchestrate lactogenesis and lactation, in part, by regulating the expression of autocrine/intracrine and paracrine mediators. The ductal network of the mammary gland develops during puberty, largely under the influence of estradiol. Prior to pregnancy, TGF-b – which is expressed both by ductal epithelial cells and periductal mesenchymal cells – exerts pro-apoptotic and antiproliferative influences that prevent development of the alveoli – and, during pregnancy, alveolar development depends on the concerted influences of prolactin and progesterone. Increasing progesterone levels increase the abundance of TGF-b, but

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they also increase expression of EGF, FGF-2, and TGFalpha (TGF-a) – which abrogate TGF-b’s anti proliferative and pro-apoptotic influences. Notably, the expression of prolactin by mammary epithelial cells also increases at this time. Despite its evident synergy with prolactin in promoting lactogenesis, the elevated level of progesterone inhibits lactation. Recent evidence suggests it does so by increasing expression of Wnt-5b, which is thought to maintain the undifferentiated state by promoting nuclear translocation of b-catenin, and by increasing expression of insulin-like growth factor-binding protein (IGFBP-5) – which suppresses insulin-like growth factor signaling. These inhibitory influences are removed and lactation becomes possible at parturition, when production of progesterone is abruptly suppressed. Certain of the estradiol-, progesterone-, and prolactininduced mediators that determine development of the mammary epithelium also determine expression of the mammary glands’ mucosal immune functions. As reviewed elsewhere in the encyclopedia, TGF-b typically acts as a differentiation factor for immature dIgA+-expressing plasmablasts, inducing them to mature into dIgA-secreting plasmacytes as they arrive at mucosal immune effector sites. Normal plasmacytes – like other bone-marrowderived cells – express an intrinsic apoptotic program, and their ongoing survival requires that this program be abrogated by survival signals from the local milieu. As noted above, prolactin plays a role in inducing ductal epithelial cells to express pIgR. Its known mitogenic influences on T cells and B cells suggest that it may also act as one of the factors which support the mature plasmacytes’ survival. Thus, prolactin may contribute to a counterpoise against plasmacyte’s apoptotic program as well as against the pro-apoptotic and antiproliferative influences TGF-b would otherwise exert on both the plasmacytes and the alveolar epithelium. Evidently, this counterpoise is maintained as the levels of TGF-b, prolactin, and other estrogen- and progesterone-dependent factors increase during pregnancy, and it supports expansion of the population of plasmacytes that will produce dIgA for secretion in the milk. Similar interactions between the sex steroids and prolactin may account for the reproductive hormones’ influences on the other mucosal effector organs of the female and male reproductive systems, and the data available so far indicate that they do so for the lacrimal glands, as well.

Counterpoises between Contradictory Signals Figure 2 summarizes the spatial and temporal actions of the differentiation and survival factors, as they are

organized in the lacrimal glands. The notions that plasmacytes express an intrinsic apoptotic program which must constantly be abrogated and that the steady-state pools of paracrine secretory products in lacrimal epithelial cells are small may help explain why they begin undergoing apoptosis so abruptly after ovariectomy. The pools of paracrine secretory products would deplete rapidly after the signals that support their ongoing expression are removed. Unpublished findings that prolactin immunoreactivity is present in the nuclei of plasmacytes seem to confirm that prolactin is one of the lacrimal epithelial paracrine mediators the influence plasmacytes. This may only be part of the explanation; however, and it is possible that testosterone, estradiol, and, perhaps, progesterone as well also might interact with prolactin and other survival factors to generate synergistic signals that maintain plasmacyte survival. While prolactin may be one of several factors that provide mitogenic signals abrogating TGF-b’s pro-apoptotic and antiproliferative influences, it appears that TGF-b may provide a counterpoise to prolactin’s lymphoproliferative and proinflammatory influences. As reviewed elsewhere in the encyclopedia, there is evidence that the transcytotic apparatus mucosal epithelial cells use to internalize dIgA and release sIgA into the fluid they produce inevitably secretes a significant burden of autoantigens to the underlying stromal spaces. Thus, newly matured dendritic cells that emigrate from the lacrimal glands to the draining lymph nodes carry with them lacrimal epithelial autoantigens. They process the autoantigens to generate epitopes that their surface MHC class-II molecules will present to CD4+ T cells, and they also release the autoantigens for sampling by IgM+-B-cell antigen receptors. There is now evidence that TGF-b induces immature dendritic cells to mature into immunosuppressive antigen-presenting cells that prevent proliferation of autoreactive lymphocytes within the lacrimal glands and draining lymph node. Moreover, it is possible that dendritic cells that have matured within the lacrimal glands might also function as tolerogenic antigen-presenting cells – inducing the generation of TH3 or TR1 regulatory cells. Microarray studies have clarified that the reproductive hormones influence the expression of other cytokines and growth factors apart from TGF-b and prolactin in the lacrimal glands. Further work will be needed to determine the extents to which the various mediators are expressed by infiltrating immune cells, acinar and ductal epithelial cells, and mesenchymal cells. Other factors in addition to the reproductive hormones are likely to influence epithelial expression of TGF-b and of prolactin and the plasmacyte survival factors. Nevertheless, the concept that the reproductive hormones orchestrate counterpoises between contradictory signals, summarized in Figure 3, may lead to detailed paradigms that explain why Sjo¨gren’s dacryoadenitis and the common histopathological syndrome


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Acinus

Plasmacyte

Interlobular duct

Mature dendritic cell

Plasmablast

Immature dendritic cell

Afferent lymph vessels Venule Dimeric IgA

Autoantigen

Survival factor

Secretory IgA

Maturation

Differentiation factor

Secretory component (SC)

Polymeric IgA receptor (pIgR)

Figure 2 Interlobular duct epithelial cells produce both differentiation factors, such as TGF-b and IL-10, which may also exert proapoptotic and immunoregulatory influences. They also produce survival factors, such as EGF, FGF, and prolactin, some of which also may exert mitogenic and proinflammatory influences. The counterpoises of contradictory influences maintain for the lacrimal glands’ exocrine functions as well as their mucosal immune functions. For example, TGF-b may support expression of the ductal epithelial phenotype, while FGF, EGF, and prolactin may support both survival of the ductal epithelium and development and survival of the acini. TGF-b induces plasmablasts to undergo terminal differentiation to dIgA-secreting plasmacytes, while prolactin may be one of several mediators that support the plasmacytes’ ongoing survival. TGF-b also induces immature dendritic cells that have taken up lacrimal autoantigens to differentiate as regulatory antigen presenting cells.

are both so much more prevalent among women, and why the onset of clinical dry eye disease is associated with events of the reproductive cycle and life cycle. There are several physiological states during which prolactin-mediated influences might become excessive with respect to the available counterpoises (see Box 1). Given the burden of epithelial autoantigens constitutively present in the stromal space of the lacrimal glands, one might predict that such states favor the accumulation of autoreactive T cells and B cells. In a direct experimental test of the hypothesis, an adenovirus vector was used to transiently increase prolactin expression in lacrimal glands of mature female rabbits. As has been reported in preliminary form, increased abundance of prolactin transcripts was accompanied by increased

abundances of mRNAs for IFN-g and TGF-a, as well as accumulation of large lymphocytic infiltrates and apparent formation of germinal centers. Moreover, the lymphocytic foci persisted for weeks after the prolactin mRNA levels returned to normal. Of interest also is a recent report that, after having a primary relative with an autoimmune disease, carrying a pregnancy to term is the second greatest risk factor for Sjo¨gren’s syndrome appears to accord with this prediction. Both findings suggest that autoimmune activation can be suppressed after systemic hormone levels have returned to normal, but that autoreactive memory cells may persist and become activated as the age-related loss of reproductive steroids changes the immunoregulatory signaling milieu within the lacrimal glands.

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Immunoregulation epithelial survival plasmacyte survival plasmablast differentiation

Atrophy

Inflammation

TGF-β, IL-10

Prolactin, EGF, FGF

Differentiation factors

Survival factors

Figure 3 The capacity of the lacrimal gland to perform its exocrine functions – that is, secretion of electrolytes, water, and proteins – and its mucosal immune functions – that is, maintenance of a population of mature, dIgA-secreting plasmablasts and transcytotic delivery of sIgA into the fluid being produced, while avoiding autoimmune inflammatory processes – depends on a system of counterpoises between contradictory influences. The reproductive hormones influence expression of many of the paracrine mediators that exert those influences.

See also: Adaptive Immune System and the Eye: Mucosal Immunity.

Further Reading Ariga, H., Edwards, J., and Sullivan, D. A. (1989). Androgen control of autoimmune expression in lacrimal glands of MRL/Mp-lpr/lpr mice. Clinical Immunology and Immunopathology 53: 499–508. Azzarolo, A. M., Eihausen, H., and Schechter, J. (2003). Estrogen prevention of lacrimal gland cell death and lymphocytic infiltration. Experimental Eye Research 77: 347–354.

Azzarolo, A. M., Mircheff, A. K., Kaswan, R. L., et al. (1997). Androgen support of lacrimal gland function. Endocrine 6: 39–45. Azzarolo, A. M., Wood, R. L., Mircheff, A. K., et al. (1999). Androgen influence on lacrimal gland apoptosis, necrosis and lymphocytic infiltration. Investigative Ophthalmology and Visual Science 40: 523–526. Bailey, J. P., Nieport, K. M., Herbst, M. P., et al. (2004). Prolactin and transforming growth factor-b signaling exert opposing effects on mammary gland morphogenesis, involution, and the Aky-forkhead pathway. Molecular Endocrinology 19: 1171–1184. Ding, C., Chang, N., Fong, Y. C., et al. (2006). Interacting influences of pregnancy and corneal injury on rabbit lacrimal gland immunoarchitecture and function. Investigative Ophthalmology and Visual Science 47: 1368–1375. Frey, W. H., Nelson, J. D., Frick, M. L., and Elde, R. P. (1986). Prolactin immunoreactivity in human tears and lacrimal gland: Possible implications for tear production. In: Holly, F. J. (ed.) The Preocular Tear Film in Health, Disease, and Contact Lens Wear, pp. 798–807. Lubbock, TX: Dry Eye Institute. Kolek, O., Gajowska, B., Godlewski, M. M., and Motyl, T. (2003). Antiproliferative and apoptotic effect of TGF-b1 in bovine mammary epithelial BME-UV1 cells. Comparative Biochemistry and Physiology C 134: 417–430. Mathers, W. D., Stovall, D., Lane, J. A., Zimmerman, M. B., and Johnson, S. (1998). Menopause and tear function: The influence of prolactin and sex hormones on human tear production. Cornea 17: 353–358. Mircheff, A. K., Wang, Y., de Saint Jean, M., et al. (2005). Mucosal immunity and self-tolerance in the ocular surface system. Ocular Surface 4: 182–193. Priori, R., Medda, E., Conti, F., et al. (2007). Risk factors for Sjo¨gren’s syndrome. Clinical and Experimental Rheumatology 25: 378–384. Richards, S. M., Liu, M., Jensen, R. V., et al. (2005). Androgen regulation of gene expression in the mouse lacrimal gland. Journal of Steroid Biochemistry and Molecular Biology 96: 401–413. Rosfjord, E. C. and Dickson, R. B. (1999). Growth factors, apoptosis, and survival of mammary epithelial cells. Journal of Mammary Gland Biology and Neoplasia 4: 229–237. Rudolph, M. C., McManaman, J. L., Hunter, L., Phang, T., and Neville, M. C. (2003). Functional development of the mammary gland: Use of expression profiling and trajectory clustering to reveal changes in gene expression during pregnancy, lactation, and involution. Journal of Mammary Gland Biology and Neoplasia 8: 287–307. Schechter, J., Carey, J., Wallace, M., and Wood, R. (2000). Distributions of growth factors and immune cells are altered in the lacrimal gland during pregnancy and lactation. Experimental Eye Research 71: 129–142. Sullivan, D. A., Kelleher, R. S., Vaerman, J. -P., and Hann, L. E. (1990). Androgen regulation of secretory component synthesis by lacrimal gland acinar cells in vitro. Journal of Immunology 145: 4238–4244. Suzuki, T., Schirra, F., Richards, S. M., et al. (2006). Estrogen’s and progesterone’s impact on gene expression in the mouse lacrimal gland. Investigative Ophthalmology and Visual Science 47: 158–168. Wang, Y., Chiu, C. T., Nakamura, T., et al. (2007). Traffic of endogenous, over-expressed, and endocytosed prolactin in rabbit lacrimal acinar cells. Experimental Eye Research 85: 749–761.

Lacrimal Gland Signaling: Neural D Zoukhri, Tufts University, Boston, MA, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Acinar cells – Highly polarized epithelial cells that form an acinus and whose primary function is to secrete proteins, electrolytes, and water. Exocytosis – The process in which molecules (such as secretory proteins) in a membrane-enclosed vesicle (secretory vesicle or granule) fuse with the plasma membrane and are then released outside the cell. Muscarinic receptors – A subtype of receptors for the neurotransmitter acetylcholine that is more responsive to muscarine than nicotine. Neurotransmitters – Chemicals released by neurons to modulate the function of a target cell. Preocular tear film – A complex and highly structured moist film which covers the bulbar and palpebral conjunctiva, and the cornea. It is composed of water, electrolytes, proteins, mucins, and lipids. Signal transduction – The biochemical events that conduct the signal of an external stimulus from the cell exterior, through the cell membrane, and into the cytoplasm.

Anatomy of the Lacrimal Gland The lacrimal gland is a compound tubuloalveolar serous gland composed primarily of acinar, ductal, and myoepithelial cells (Figure 1). Acinar cells account for over 80% of the cell type present in the lacrimal gland and form the site for synthesis, storage, and secretion of proteins. Several of these proteins have antibacterial or growth factor properties and are crucial to the health of the ocular surface. Acinar cells are highly polarized cells with tight junctions surrounding each acinar cell on the luminal side and thus separating the plasma membrane into apical (luminal) and basolateral (serosal) components. The basal portion of the cell contains a large nucleus, rough endoplasmic reticulum, mitochondria, and Golgi apparatus, while the apical portion is filled with secretory granules. Like the acinar cells, the duct cells are also polarized with the nuclei located basolaterally, whereas the rough endoplasmic reticulum and mitochondria are more apical. The primary function of the ductal cells is to modify the

primary fluid secreted by the acinar cells by absorbing or secreting water and electrolytes. The duct cells secrete a KCl-rich solution so that the final secreted lacrimal gland fluid is rich in K+. It has been estimated that as much as 30% of the volume of the final lacrimal gland fluid is secreted by the duct cells. The myoepithelial cells lie scattered between the acinar and ducts cells and the basement membrane and are interconnected by gap junctions and desmosomes. These cells are highly branched and contain multiple processes which surround the basal area of the acinar cells (Figure 1). The myoepithelial cells are thought to contract because they contain muscle contractile proteins (a-smooth muscle actin, myosin, and tropomyosin). The contraction of these cells would help expel the fluid out of the acini and the ducts onto the ocular surface. In support of a functional role of lacrimal gland myoepithelial cells, receptors and intracellular signaling molecules for parasympathetic neurotransmitters have been described. The lacrimal gland contains other cells: plasma cells, B and T cells, dendritic cells, macrophages, and mast cells. Immunoglobulin A (IgA)-positive plasma cells account for the majority of the mononuclear cells in the lacrimal gland. These cells synthesize and secrete IgA, which then is transported into acinar and ductal cells and secreted by these epithelial cells as secretory IgA.

Neural Control of Lacrimal Gland Secretion To ensure adequate production of the aqueous component of the preocular tear film, lacrimal gland secretion is under tight neural control. To this end, the lacrimal gland is densely innervated by the parasympathetic and sympathetic nervous system (Figure 2). Although scarce, sensory nerves are also present in the lacrimal gland (Figure 2). Nerves are located in close proximity to acinar, ductal, and myoepithelial cells, as well as blood vessels, and hence can control a wide variety of lacrimal gland functions. While each individual cell may not be innervated, gap junctions electrically and chemically connect cells within an acinus so that even noninnervated cells can respond to the neural stimulus. In the lacrimal gland, parasympathetic nerves contain the neurotransmitters acetylcholine and vasoactive intestinal peptide (VIP). Sympathetic nerves contain the neurotransmitters norepinephrine and neuropeptide Y (NPY).

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Sensory nerves contain the neurotransmitters substance P and calcitonin gene-related peptide (CGRP). Acetylcholine and VIP are potent stimuli of lacrimal gland protein and electrolyte/water secretion. Norepinephrine is also a potent stimulus of protein secretion, but a weak stimulus of electrolyte/water secretion. In contrast, NPY and CGRP are weak stimuli of protein secretion, while substance P does not appear to stimulate either protein or electrolyte/water secretion. The stimulation of lacrimal gland secretion occurs through a neural reflex arc originating from the ocular surface (Figure 3). Neural reflexes are initiated by stimulation of the afferent sensory nerves of the cornea and conjunctiva or by activation of the optic nerve in response to intense light. Efferent parasympathetic and sympathetic nerves of the lacrimal gland are then activated to release their neurotransmitters (Figure 3). The neurotransmitters interact with and activate specific receptors located on the basolateral membranes of acinar and duct cells, which then

initiates a cascade of intracellular events known as signal transduction. Activation of these signal transduction pathways induces fusion of the preformed secretory granules with the apical membrane to release secretory proteins into the lumen (Figure 3). To trigger electrolyte and water secretion, ion channels and pumps, located in the apical and basolateral membranes, are also activated.

Signal Transduction Pathways Activated in the Lacrimal Gland Signal transduction proceeds in three steps: (1) initiation of the signal by interaction of the ligand (neurotransmitter, neuropeptide, or hormone) with its receptor; (2) amplification of the signal through the interaction of the receptor/G protein/effector enzyme leading to the generation of second-messenger molecules; and (3) termination of the signal through the action of protein phosphatases and membrane pumps to bring the amount of phosphorylated proteins and ions, respectively, back to resting levels (Figure 4). Cholinergic Agonist-Activated Signal Transduction Pathways Acetylcholine, released from parasympathetic nerves, activates muscarinic receptors on the basolateral membrane of lacrimal gland cells. Of the five receptor subtypes (M1–5) identified, only the M3 or glandular subtype is present in the lacrimal gland. These receptors are coupled to the activation of phospholipases C and D (PLC and PLD, respectively) and the activation of the p42/p44 mitogen-activated protein kinase (p42/p44 MAPK, also known as extracellular signal-regulated kinase (ERK)) pathway (Figure 5).

Figure 1 Schematic of the lacrimal gland and photomicrographs showing the three major cell types that it is composed of. The acinar cells, which account for 80% of the cell type present in the lacrimal gland, and ductal cells were stained with hematoxylin and eosin. The myoepithelial cells were identified immunohistochemically (brown stain) using an antibody against a-smooth muscle actin.

PLC-coupled signaling pathway Lacrimal gland M3 receptors are coupled, via the Gprotein Gaq, to the effector enzyme PLC. Activated PLC hydrolyzes the plasma membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP2), to generate two

Figure 2 Photomicrographs depicting the innervation of murine lacrimal gland. Nerves were visualized using antibodies against the following neurotransmitters or enzymes: VIP for the parasympathetic nerves, dopamine b-hydroxylase for the sympathetic nerves, and CGRP for the sensory nerves.

Lacrimal Gland Signaling: Neural

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Parasympathetic Sympathetic

Parasympath

+ +

+

S ym

+

Acinus

Brain etic

pat het ic Lacrimal gland

Secretion Neurotransmitters Secretory granules

Optic nerve

Tears

Secreted proteins

Cornea Sensory nerves Figure 3 Schematic of the neural reflex arc that controls lacrimal gland secretion. Neural reflexes are initiated by the stimulation of the afferent sensory nerves of the cornea and conjunctiva or by activation of the optic nerve. Efferent parasympathetic and sympathetic nerves of the lacrimal gland are then activated to release their neurotransmitters. The neurotransmitters activate specific receptors located on the basolateral membranes of acinar cells to stimulate secretion.

Nerve ending

Neurotransmitter (a)

Signal initiation Ion channel

Receptor G Signal amplification

Effector enzyme

(c) Second messengers

(b)

Inactive substrate

Active substrate

3

Signal termination

Protein phosphatases Figure 4 Schematic depicting the three steps involved in signal transduction in response to a neural stimulus: (a) signal initiation by interaction of the neurotransmitter with its receptor; (b) signal amplification through the interaction of the receptor with the G protein and effector enzyme to generate second-messenger molecules; and (c) signal termination through activation of protein phosphatases and membrane pumps.

second-messenger molecules, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG; Figure 5). IP3, a water-soluble molecule, diffuses to the endoplasmic reticulum where Ca2+ is stored in an inactive, bound form. It also interacts with specific receptors located on the endoplasmic reticulum to release Ca2+ into the cytosol. Depletion of these Ca2+ stores leads to an increase in the influx of extracellular Ca2+ across the plasma membrane. The IP3 receptor is a homotetramer of 310 kDa each and constitutes one of the largest of all known ion channels. Binding sites for IP3 are located within the N-terminal

domain, whereas the C-terminal regions form the intrinsic Ca2+ channel. Multiple isoforms of IP3 receptor have been cloned. They share significant similarity to each other and are encoded by at least four genes. The activation of lacrimal gland cholinergic M3 receptors triggers a biphasic Ca2+ response: a rapid (usually referred to as peak) increase in [Ca2+]i due to IP3-induced release of Ca2+ from intracellular stores, followed by a slow and sustained (usually referred to as plateau) increase in [Ca2+]i due to influx of Ca2+ from the extracellular milieu. Both Ca2+ responses are necessary for cholinergic

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Parasympathetic nerves

Acetylcholine M3 Receptor

Ras Raf MEK

Gαq

Pyk2

DAG

PLC

PKC α, δ, ε

Src

Ca2+

IP3

PLD ER

ERK Ca2+ –

+

+ +

Exocytosis Figure 5 Schematic depicting the main signal transduction pathways activated by acetylcholine to stimulate protein secretion. The activation of lacrimal gland cholinergic receptors activates three main signaling pathways that either enhance (PKC, Ca2+) or attenuate (ERK) protein secretion. The net protein secretory output in response to acetylcholine stimulation will likely depend on the relative contribution of the stimulatory versus the inhibitory signal transduction pathways.

agonist-stimulated protein secretion since chelation of either the intracellular or the extracellular Ca2+ leads to complete inhibition of secretion. Ca2+ can stimulate secretion alone or do so by activating Ca2+ and calmodulindependent protein kinases to phosphorylate-specific substrates to cause secretion. The DAG formed from the hydrolysis of PIP2 activates protein kinase C (PKC, Figure 5). PKC is a family of closely related isozymes that has been divided into three categories based on structural and functional criteria. A first group, termed conventional PKCs (cPKCs), includes PKCa, -bI, -bII, and -g isoforms, which have a Ca2+- and DAG-dependent kinase activity. A second group, termed novel PKCs (nPKCs), includes PKCE, -d, -y, and -Z isoforms, which are Ca2+-independent and DAG-stimulated kinases. A third group, termed atypical PKCs (aPKCs), includes PKCz and -i/l isoforms, which are Ca2+- and DAG-independent kinases. Four isoforms of PKC are expressed in the rat lacrimal gland: one classical, PKCa; two novel, PKCd, and -E; and one atypical, PKCi/l. In an attempt to define the role that individual PKC isoforms might play in regulating lacrimal gland functions in response to cholinergic stimulation, isoform-specific peptide inhibitors of PKC were synthesized. These peptides were derived from the unique pseudosubstrate sequences of PKCa, -d, and -E, and were myristoylated at their N-terminus to make them cell permeant. Indeed, all PKC isoforms have a pseudosubstrate sequence in their N-terminal part, which interacts with the

catalytic domain to keep the enzyme inactive in resting cells. Using these peptides, it was shown that cholinergic agonists activate PKCa and -E to a larger extent and PKCd to a lesser extent, to induce protein secretion. It was also shown that PKCd and -E, but not -a, negatively modulate cholinergic-induced Ca2+ elevation in the lacrimal gland.

PLD-coupled signaling pathway PLD catalyzes the hydrolysis of membrane phospholipids (preferably phosphatidylcholine), producing phosphatidic acid and the free polar head group. Phosphatidic acid, by itself or after its conversion to DAG by a phosphohydrolase, is an important second-messenger molecule. Besides its hydrolytic activity, PLD possesses the unique ability to catalyze a transphosphatidylation reaction, in the presence of a primary alcohol, in which the phosphatidyl moiety of the phospholipid substrate is transferred to the primary alcohol producing the corresponding phosphatidylalcohol. Accumulation of such unique transphosphatidylation products has been used to detect PLD activity unambiguously in diverse cell types. Depending on the cell’s type, the receptor activation of PLD was shown to occur through mechanisms involving PKC activity, Ca2+, G proteins, or receptor-linked tyrosine kinases. Since PKC activation and Ca2+ mobilization are downstream to PLC stimulation, it has been suggested that PLD activation may be secondary to receptor activation of PLC.


Taking advantage of the transphosphatidyl reaction catalyzed by PLD, it was shown that the lacrimal gland contains a PLD activity. Cholinergic agonists, through the muscarinic receptor, stimulate both the hydrolytic activity of PLD to produce phopshatidic acid, as well as the transphosphatidyl reaction. However, if either Ca2+ is mobilized or PKC is activated, only the transphosphatidyl reaction is stimulated. This finding implied that cholinergic agonist activation of PLD in the lacrimal gland is not secondary to the activation of PLC by these agonists. MAPK-coupled signaling pathway

MAPK, also known as ERK, is a dual serine/threonine and tyrosine protein kinase. It is activated through phosphorylation by an MAPK kinase (known as MEK). MEK is also activated through phosphorylation by its upstream MAPK kinase kinase (known as Raf). Raf is activated when the small GTP-binding protein, Ras, is in its GTP-bound form. Depending on the cell’s type, Ras can be activated by several mechanisms, including the nonreceptor tyrosine kinases Pyk2 and Src, growth factors receptors, and PKC. In the lacrimal gland, the activation of MAPK attenuates protein secretion. The activation of MAPK by the M3 receptor was shown to involve the nonreceptor tyrosine kinases Pyk2 and Src, which in turn activate Ras and, ultimately, MAPK (Figure 5). Recent evidence showed that the activation of MAPK by cholinergic agonists is downstream of PLD activation. The mechanisms involved in PLD-mediated activation of MAPK in the lacrimal gland remain to be elucidated. The termination of the cholinergic signaling pathway involves receptor desensitization, activation of protein

phosphatases to dephosphorylate ERK and other phosphorylated substrates, and the activation of ion channels/ pumps to return the concentration of cytosolic Ca2+ to its resting levels. In summary, the activation of lacrimal gland cholinergic receptors in response to the parasympathetic neurotransmitter acetylcholine activates three main signaling pathways that either enhance (PKC, Ca2+) or attenuate (ERK) protein secretion (Figure 5). The net protein secretory output in response to acetylcholine stimulation will likely depend on the relative contribution of the stimulatory versus the inhibitory signal transduction pathways. VIP-Activated Signal Transduction Pathways VIP interacts with specific VIP receptors located on the basolateral membranes of lacrimal gland cells. Two types of VIP receptors have been identified, VIPRI and VIPRII, which are also known as VIPACR1 and VIPACR2, and both of them are expressed in the lacrimal gland, with VIPRI being the predominant type. Adenylate cyclase-coupled signaling pathway The VIP receptor uses the G protein Gas to activate the effector enzyme adenylyl cyclase (AC), which produces the second-messenger molecule, cyclic adenosine monophosphate (cAMP) (Figure 6). Molecular cloning has identified several isoforms of mammalian AC forming a family of at least 10 enzymes (ACI-X). There are at least three isoforms of AC (ACII, ACIII, and ACIV) present in the lacrimal gland, each having a unique localization. Although the regulation of AC enzymatic activity is complex and isoform specific, all AC isoforms are activated

Parasympathetic nerves

VIP

Ras Raf

87

VIP receptor I or II

Gαs

AC Inactive PKA

Ca2+ ATP

cAMP

R

C

R

C

MEK Active PKA

ERK –

+

C

R

C

R

Exocytosis Figure 6 Schematic depicting the main signal transduction pathways activated by the VIP to stimulate protein secretion. The activation of lacrimal gland VIP receptors activates two main signaling pathways that enhance protein secretion.

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by Gas. Increases in the intracellular levels of cAMP lead to activation of protein kinase A (PKA), a ubiquitous serine and threonine protein kinase. In its inactive state, PKA consists of a complex of two catalytic (C) subunits and two regulatory (R) subunits (Figure 6). Binding of cAMP to the R subunit alleviates an autoinhibitory contact that releases the active C subunit (Figure 6). The active kinase is then free to phosphorylate specific protein substrates to stimulate lacrimal gland protein and fluid secretion.

signal-terminating mechanisms include desensitization of the VIPR and AC, sequestration of the PKA C subunits by the naturally occurring protein kinase inhibitor (PKI), and activation of protein phosphatases. a1-Adrenergic Agonist-Activated Signal Transduction Pathways Norepinephrine, released from the sympathetic nerves, binds to a1- and b-adrenergic receptors on lacrimal gland cells. b-Adrenergic receptors are coupled to activation of AC to activate a cAMP-dependent signal transduction pathway, as discussed for VIP. Of the three a1-adrenergic receptor subtypes (a1A, a1C, and a1D) identified, only the a1D subtype is expressed in the lacrimal gland.

MAPK-coupled signaling pathway

Recently, it has been shown that addition of VIP, exogenous cAMP, or analogs that increase cAMP levels inhibited both basal as well as cholinergic induced activation of MAPK in the lacrimal gland. One implication of these findings is that it could help explain the well-documented synergistic effect that addition of a cAMP along with a Ca2+-/ PKC-dependent agonist have on lacrimal gland protein secretion. Indeed, cholinergic agonists activate MAPK, which attenuates protein secretion. When the cAMP pathway is activated, MAPK activity is inhibited; this should alleviate the inhibitory effect that MAPK has on secretion and as a result, protein secretion will be potentiated if both the Ca2+/PKC and the cAMP pathways are activated simultaneously. The termination of VIP-activated signaling pathways likely includes activation of the cAMP-phosphodiesterase, which converts cAMP to the inactive 5’-AMP. Other

Ca2+- and PKC-coupled signaling pathways

In most exocrine tissues, a1-adrenergic agonists activate the same pathway as cholinergic agonists (i.e., activation of PLC and PLD). Surprisingly, in the lacrimal gland, a1-adrenergic agonists do not activate PLC or PLD, although their activation leads to a slight increase in cytosolic Ca2+ and to activation of PKC isoforms (Figure 7). To date, the effector enzyme(s) activated by lacrimal gland a1D-adrenergic receptors to mobilize Ca2+ and activate PKC is still unknown. Although Ca2+ mobilization in the lacrimal gland in response to adrenergic agonist stimulation is well

Sympathetic nerves

EGF

Norepinephrine α1D-AR

EGFR Pro-EGF MMP

Ras

Sc

Raf

SOS G rb 2

Gαq

? Ca2+

PKC

h

eNOS

ε

α, δ

MEK NO ERK GC UTP

cGMP

+ –

+ – Exocytosis Figure 7 Schematic depicting the main signal transduction pathways activated by the norepinephrine to stimulate protein secretion. The activation of lacrimal gland a1D-adrenergic receptors activates stimulatory pathways, including PKCE and cGMP, and inhibitory pathways, including PKCa, PKCd, and ERK. It is likely that the net lacrimal gland protein secretion in response to sympathetic stimulation is a balance between these stimulatory and inhibitory signal transduction pathways.


documented, the mechanisms involved are poorly understood. A role for IP3 has been ruled out since adrenergic agonists do not increase its production as they fail to activate PLC. It has been proposed that cyclic ADP ribose, which activates ryanodine receptors to release Ca2+ into the cytosol, might be involved. Other investigators proposed that nitric oxide (NO)-induced generation of cyclic guanosine monophosphate (cGMP) is involved in a1adrenergic agonist-induced mobilization of Ca2+. The role of PKC in a1-adrenergic agonist-induced lacrimal gland protein secretion has been studied using the myristoylated pseudosubstrate-derived peptides. It was shown that a1-adrenergic agonists activate three PKC isoforms – PKCa, -d, and -E. Activation of PKCE enhances protein secretion, whereas activation of PKCa and -d attenuates protein secretion. This is in contrast to the stimulatory effect that PKCa and -d isoforms have on protein secretion when activated by cholinergic agonists. These findings imply that the effect (inhibitory or stimulatory) of a given isoform of PKC in the lacrimal gland is stimulus dependent and might be dictated by the cellular location of PKC isoforms. MAPK-coupled signaling pathway

Similar to cholinergic agonists, a1-adrenergic agonists activate MAPK to attenuate lacrimal gland protein secretion. However, in contrast to the cholinergic pathway, activation of MAPK by the a1D-adrenergic receptor does not involve the nonreceptor tyrosine kinases Pyk2 and Src, but involves activation of the epidermal growth factor receptor (EGFR; Figure 7). The EGFR, also known as Erb1, is the prototypical member of the ErbB/EGFR family of receptors which consists of three additional members (ErbB2–4). EGFR is a type 1 transmembrane tyrosine kinase receptor consisting of an extracellular domain (ligand-binding site), a transmembrane domain, and the carboxy-terminal, an intracellular domain containing the tyrosine kinase motif. In addition, the carboxy-terminal domain contains tyrosine residues that become phosphorylated following ligand binding and receptor dimerization. Following receptor activation, several exogenous substrates that contain either Src homology 2 (SH2) or protein tyrosine binding (PTB) motifs are recruited to specific phosphorylated tyrosine residue. In the lacrimal gland, the activation of the EGFR by a1-adrenergic agonists occurs through a process termed as transactivation and involves the activation of a metalloproteinase and shedding of EGF (Figure 7). Following activation, Shc (an SH2 motif-containing protein) is recruited to the EGFR, followed by recruitment of Grb2 and the guanine nucleotide exchange factor protein, SOS (Figure 7). SOS stimulates the exchange of GDP for GTP on Ras and hence leads to its activation. Activated Ras triggers the activation of the MAPK cascade, leading to activation of ERK (Figure 7). Similar to the cholinergic pathway, a1-adrenergic-activated ERK has been shown to attenuate lacrimal gland protein secretion.

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NO-coupled signaling pathway NO, along with L-citruline, is produced from L-arginine in the presence of O2- and NADPH-derived electrons. This reaction is catalyzed by the enzyme NO synthase (NOS). There are three well-characterized isoforms of NOS expressed by mammalian cells: neuronal NOS (nNOS also known as NOS1), inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3). Activation of nNOS and eNOS, but not iNOS, requires calmodulin and an increase in [Ca2+]. It has been shown recently that lacrimal gland a1-adrenergic receptors are coupled to the NO/cGMP pathway (Figure 7). Indeed, it was found that both nNOS and eNOS are expressed in the lacrimal gland. The addition of a1-adrenergic agonists led to generation of NO, presumably through activation of eNOS and not nNOS. NO, in turn, activates soluble guanylate cyclase to generate cGMP which enhances lacrimal gland protein secretion. The termination of the a1-adrenergic signaling pathway is likely to involve receptor desensitization, activation of protein phosphatases to dephosphorylate ERK and other phosphorylated substrates, and the activation cGMP-phosphodiesterase, which converts cGMP to the inactive 5’-GMP. In summary, the activation of lacrimal gland a1Dadrenergic receptors, in response to the sympathetic neurotransmitter norepinephrine, activates stimulatory pathways (including PKCE and cGMP) and inhibitory pathways (including PKCa, PKCd, and ERK; Figure 7). It is likely that the net lacrimal gland protein secretion in response to sympathetic stimulation is a balance between these stimulatory and inhibitory signal transduction pathways. See also: Adaptive Immune System and the Eye: Mucosal Immunity; Lacrimal Gland Hormone Regulation; Lacrimal Gland Overview; Tear Film.

Further Reading Botelho, S. Y., Hisada, M., and Fuenmayo, N. (1966). Functional innervation of the lacrimal gland in the cat. Archives of Ophthalmology 76: 581–588. Broad, L., Braun, F., Lievremont, J., et al. (2001). Role of the phospholipase C-inositol 1,4,5-trisphosphate pathway in calcium release-activated calcium current and capacitative calcium entry. Journal of Biological Chemistry 276: 15945–15952. Chen, L., Hodges, R. R., Funaki, C., et al. (2006). The effects of a1D-adrenergic receptors on shedding of biologically active EGF in freshly isolated lacrimal gland epithelial cells. American Journal of Physiology. Cell Physiology 291: C946–C956. Funaki, C., Hodges, R. R., and Dartt, D. A. (2007). Role of cAMP inhibition of p44/p42 mitogen-activated protein kinase in potentiation of protein secretion in rat lacrimal gland. American Journal of Physiology. Cell Physiology 293: C1551–C1560. Hodges, R. R. and Dartt, D. A. (2003). Regulatory pathways in lacrimal gland epithelium. International Review of Cytology 231: 129–196.

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Hodges, R., Rios, J., Vrouvlianis, J., et al. (2006). Role of protein kinase C, Ca2+, Pyk2 and c-Src in agonist activation of rat lacrimal gland p42/p44 MAPK. Investigative Ophthalmology and Visual Sciences 47: 3352–3359. Ota, I., Zoukhri, D., Hodges, R., et al. (2003). a1-Adrenergic and cholinergic agonists activate MAPK by separate mechanisms to

inhibit secretion in lacrimal gland. American Journal of Physiology. Cell Physiology 284: C168–C178. Wu, K., Jerdeva, G., da Costa, S., et al. (2006). Molecular mechanisms of lacrimal acinar secretory vesicle exocytosis. Experimental Eye Research 83: 84–96.

Lids: Anatomy, Pathophysiology, Mucocutaneous Junction T Wojno, The Emory Clinic, Atlanta, GA, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Actinic lesion – Dry, scaly, rough-textured patches that form after years of exposure to ultraviolet light, such as sunlight. Amblyopia – Disorder of the visual system that is characterized by poor or indistinct vision in an eye that is otherwise physically normal. Anisometropia – Condition in which the two eyes have unequal refractive power. Blepharitis – Chronic inflammation of the eyelids. Blepharoplasty – Surgical procedure intended to reshape the upper eyelid or lower eyelid by the removal or repositioning of excess tissue as well as by reinforcement of surrounding muscles and tendons. Dermatochalasis – Redundant, baggy eyelids. Ectopion – Outward malposition of the eyelid. Entropion – Inward malposition of the eyelid. Lagophthalmos – Inability to close the eye. Ptosis – Downward malposition of the upper eyelid. Strabismus – A condition in which the eyes are not properly aligned with each other. Trichiasis – Misdirected eyelashes.

Anatomy From a functional perspective, the upper lid can be divided into anterior, middle, and posterior lamellae (Figure 1). In the upper lid, the anterior lamella consists of the skin and orbicularis muscle while the posterior lamella consists of the conjunctiva, tarsus, levator, and Mu¨ller’s muscle. The middle lamella is the orbital septum and orbital fat. The thin eyelid skin covers the orbicularis muscle, which is functionally divided into the pretarsal, preseptal, and orbital parts. There is no discreet anatomic border to these components of the orbicularis. The levator muscle originates just superior to the annulus of Zinn at the orbital apex and changes from striated muscle to fibrous apponeurosis 15 mm above the superior border of tarsus. The levator inserts into the superior border and anterior surface of the tarsal plate. It is innervated by the third cranial nerve. Mu¨ller’s muscle is only 10–14 mm long and arises from the underbelly of the levator and inserts into the superior border of tarsus. It is composed of

smooth muscle fibers and is adrenergically innervated. The levator and Mu¨ller’s muscles function to open the upper lid while the orbicularis muscle closes it. The orbital septum is a thin, multilayered sheet of fibrous tissue separating the lid from the orbit. It arises from the superior orbital rim and inserts onto the levator aponeurosis 2–10 mm above the superior border of tarsus in Caucasians, 15 mm or more in blacks and at or below the superior border of tarsus in Asians. Small, fine, fibrous attachments extend from the levator to the subcutaneous tissue. These attachments and the insertion of the septum form the lid crease while the skin above the crease forms the lid fold. There are two fat pockets in the upper lid found nasally and centrally. The upper tarsus is a firm connective tissue usually 10–12 mm in height and 1 mm in thickness. The lower lid is likewise divided into three functional lamellae: an anterior layer of skin and orbicularis, a posterior layer of conjunctiva and lower lid retractors, and a middle layer of septum and orbital fat (Figure 2). The lower lid retractors are composed of the capsulopalpebral fascia (the equivalent of the levator in the upper lid) and the inferior tarsal muscle (the equivalent of the Mu¨ller’s muscle in the upper lid). The capsulopalpebral fascia is a fibrous band originating from the underbelly of the inferior rectus muscle that courses anteriorly, enveloping the inferior oblique, and inserts onto the inferior border of tarsus. It is functionally controlled by its origin from the inferior rectus muscle to retract the lower lid inferiorly when the eye looks downward, preserving unobstructed vision. The inferior tarsal muscle is often just scattered smooth muscle fibers intermixed with the capsulopalpebral fascia. As in the upper lid, the septum arises from the orbital rim and inserts on the inferior border of tarsus, often blending with the lower lid retractors. A lower lid crease occasionally exists but is usually less obvious. There are three fat pockets in the lower lid: nasal, central, and temporal (or lateral). The lower tarsus is usually 4–5 mm in height. The lid margin is the border between the anterior skin–muscle layer and the posterior tarsoconjunctival layer. There are two to three irregular rows of lashes, whose bulbs are embedded just below the skin surface within the orbicularis muscle. Posterior to the lash line are the meibomian gland orifices. These sebaceous glands are embedded within the tarsal plates and run the entire vertical length of the tarsus. There are about 25 of these glands in the upper lid and 20 in the lower lid. The mucocutaneous junction is just posterior to the meibomian

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Skin Subcutaneous tissue Preseptal orbicularis

Superior rectus muscle

Orbital septum Levator muscle

Preaponeurotic fat pad

Suspensory ligament of the upper fornix

Levator aponeurosis Fine attachment from levator aponeurosis to Müller’s muscle

Superior conjunctival fornix

Conjunctiva

Müller’s muscle Tarsus

Pretarsal orbicularis

* = Whitnall’s ligament Figure 1 Cross-section of the upper eyelid.

Conjunctiva Skin Inferior fornix

Pretarsal orbicularis Tarsus

Inferior rectus muscle

Suspensory ligament in the inferior fornix Inferior tarsal muscle Orbital septum Preseptal orbicularis Fat pad

Capsulo-palpebral fascia

Inferior oblique muscle Figure 2 Cross-section of the lower eyelid.

gland orifices. The gray line is a variably visible section of pretarsal muscle (muscle of Riolan) just anterior to the tarsus. Embedded within the lid margin are the apocrine glands of Moll and the sebaceous glands of Zeiss associated with the lash follicles. The upper and lower lids join laterally where the pretarsal heads of the orbicularis muscle form the lateral canthal tendon, which inserts into the orbital tubercle just posterior to the lateral orbital rim. Medially, the preseptal and pretarsal muscle form the medial canthal tendon,

whose anterior and posterior heads surround the lacrimal sac. The lacrimal puncta open on the lid margin 6 mm from the medial commissure of the lids.

Pathophysiology Dermatochalasis Dermatochalasis is the normal aging change in the upper and lower lids characterized by loose, redundant skin and


orbicularis muscle often with bulging of the orbital fat pockets (Figure 3). It may be associated with ptosis of the eyebrows and forehead relaxation. When severe in the upper lids, it can limit peripheral vision and obstruct the central visual axis. Lower lid dermatochalasis rarely affects the vision, but in rare cases, fat bulging can be so extreme so as to contact the patient’s glasses. Surgical treatment is aimed at removal of the excess skin, orbicularis, and fat.

Ptosis Lid ptosis is a lower-than-normal position of the upper lid margin (Figure 4). When the lid margin is 2 mm or less from the center of the pupil, the superior visual field is usually significantly obstructed. Ptosis is either congenital or acquired. Congenital ptosis is usually the result of a malformed levator muscle often with a family history. It is unilateral in 75% of cases and bilateral (although often

Figure 3 Dermatochalasis of all four eyelids.

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asymmetric) in 25% of cases. It is associated with anisometropia, amblyopia, or strabismus in 30% of cases. In general, the more severe the ptosis, the more dystrophic appears the muscle histologically. In cases of severe congenital ptosis, striated muscle fibers are usually completely absent, totally replaced by fibro-fatty connective tissue. Treatment for mild-to-moderate congenital ptosis is to perform levator resection surgery, wherein 12–18 mm of the distal levator and the underlying Mu¨ller’s muscle is resected and the cut end resewn to the superior border of tarsus. This effectively shortens the muscle, resulting in a higher resting level of the lid on the globe but does not improve the overall movement of the lid. The most common problems post-operatively are undercorrection, overcorrection, or asymmetry of the lids often necessitating reoperation. Surgery induces lagophthalmos that increases with the amount of levator resected. Surprisingly, if done during childhood, lagophthalmos is usually well tolerated throughout the patient’s lifetime. In severe congenital ptosis, the levator is usually so dystrophic that resection is ineffective. In such cases a sling must be performed. In this surgery, autogenous or banked fascia or some alloplastic material is sewn into the tarsus and then threaded under the skin to the frontalis muscle in the forehead. The patient then elevates the lid by contracting the frontalis muscle, which pulls up the lid margin. Most patients do so automatically, resulting in effective lid opening. Acquired ptosis, usually seen with aging, results from thinning or dehiscence of the levator aponeurosis from the tarsal plate. Any condition, however, that causes lid swelling or stretching (cataract surgery, trauma, contact lens wear, etc.) can result in acquired ptosis. Much less frequently, acquired ptosis is due to actual deterioration of the levator muscle or true muscular dystrophy. The usual treatment of acquired ptosis is to shorten the levator aponeurosis but to a much smaller degree than done with congenital ptosis (usually 4–10 mm). In adults, ptosis repair, when performed bilaterally, is often combined with blepharoplasty surgery for optimal cosmesis. Like in congenital ptosis, undercorrection, overcorrection, and asymmetry are common problems. In adults, however, adjustments can often be accomplished in the office under local anesthesia. Lagophthalmos is generally to be avoided in adult surgery, since the cornea is usually very intolerant of any chronic exposure and can rapidly result in discomfort and even ulceration. Retraction

Figure 4 Ptosis of the right upper lid.

The opposite of ptosis, retraction is abnormal elevation of the upper lid or downward positioning of the lower lid (Figure 5). Most cases of upper lid retraction are due to thyroid eye disease resulting from contracture of the upper and lower lid retractors secondary to inflammation.

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Figure 5 Retraction of all four lids secondary to thyroid eye disease.

Figure 6 Involutional entropion of the right lower lid.

The lower lids may also retract due to age, associated with poor support from the cheekbones. Retraction due to thyroid eye disease is often associated with exopthalmos or an abnormal anterior displacement of the globe, which further increases the stare – so characteristic of this disease. Patients with retraction often have lagophthalmos, resulting in corneal exposure and irritation. Lower lid retraction is often seen as a normal physiologic variant in people with shallow orbits, especially common in black patients. Retraction is occasionally seen with overexcessive skin excision in blepharoplasty surgery. Treatment of upper lid retraction in thyroid eye disease consists of graded recession of the levator–Mu¨ller’s muscle complex superiorly so as to drop the upper lid margin down. Some surgeons insert spacers (autologous or banked tissues) between the recessed, cut edge of the levator–Mueller’s muscle complex and the superior border of the tarsus when performing this surgery. In the lower lid, retraction is treated with recession of the lower lid retractors very frequently combined with spacer grafts for additional support of the lower lid. In the upper lid, gravity works in favor of the correction while it works against it in the lower lid. If the retraction is due to skin shortage after blepharoplasty, then skin grafting may be necessary.

distinctive rolled-in appearance and can be reduced by pulling the lid against the lateral orbital rim, effectively tightening the lid. Surgical correction is aimed at correcting the horizontal laxity by resection of the redundant lid margin with resuspension at the lateral canthus. The vertical lid laxity may be corrected by plication of the lower lid retractors to the inferior border of tarsus. An effective repair is one which combines both of these procedures often performed through a lower lid blepharoplasty incision. Involutional entropion usually does not occur in the upper lid. Such aging changes usually result in ptosis, as discussed above, or in lash ptosis – a downward angulation of the lashes due to relaxation of the anterior lamella of the eyelid in which the lash follicles are embedded. Lash ptosis is often corrected as part of an upper lid blepharoplasty procedure. Cicatricial entropion may occur in both the upper and lower lids. It is due to vertical shortening of the posterior lamella of the eyelid, the tarsoconjunctival layer. It may be due to autoimmune disorders of the mucous membranes, inflammation, infection, surgery, trauma, or long-term use of glaucoma drops. Often, however, the cause is unclear. All surgeries for cicatricial entropion can be conceptualized as falling into three categories. The first are those that act by outward rotation of the lid margin (prototypical Weis procedure) usually involving a full-thickness, horizontal blepharotomy 4 mm from the lid margin. The second category involves expanding the shortened posterior lamella of the eyelid with grafts (usually buccal mucosa, amniotic membrane, or banked sclera). The third category involves procedures that split the lid margin at the gray line often with insertion of a spacer material, such as mucous membrane, to thicken the lid margin to the point that the lashes no longer rub against the globe. In some cases, the lash-bearing segment of the lid margin may simply be resected after splitting the lid margin.

Entropion Entropion is an inward turning of the upper or lower lid margin (Figure 6). This results in the lashes rubbing on the globe, causing irritation and even corneal ulceration. Senile or involutional lower lid entropion is common with age and is due to excess horizontal (canthal tendons) and vertical laxity (the lower lid retractors). This may be intermittent at first but usually progresses to a chronic condition. The lid margin appears to have a


Trichiasis Often confused with entropion, trichiasis is inward misdirection of lashes against the globe in the presence of normal lid margin position. Trichiasis may, however, coexist with cicatricial entropion. Trichiasis is caused by the same factors that cause cicatricial entropion but is often idiopathic. Focal trichiasis is often treated by simple epilation of the offending lashes. If recurrent, electrolysis, cryotherapy, or laser may be used. Repeat treatment is often necessary since these modalities will kill the visible offending lashes but not the lashes that are about to bud. For large areas of the lid margin, surgery as outlined above for ciciatricial entropion may be needed. Alternatively, if focal, a segmental resection of the involved lid margin can be performed. Distichiasis Distichiasis is an additional row of lashes that grow from the meibomian orifices on the posterior lid margin. Such lashes will rub against the globe causing corneal irritation. Distichiasis may be congenital, often with a family history or acquired due to lid inflammation causing metaplasia of the cells in the posterior layer of the lid margin. It is treated with electrolysis or cryotherapy often after splitting the lid margin to prevent damage to the normal lashes. It may also be treated by direct surgical excision of the offending lashes. Ectropion Ectropion is outward rotation of the lower lid margin away from the globe (Figure 7). The exposure of the globe and the palpebral conjunctiva results in irritation, corneal damage, and keratinization of the conjunctiva. Epiphora, increased tear production, often results when the lower punctum stands off the globe and cannot adequately drain tears from the eye. Involutional ectropion occurs as an

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aging change secondary to horizontal lid laxity, mainly in the medial and lateral canthal tendons. Repair is accomplished by horizontal shortening of the redundant lid margin, usually at the lateral canthus, sometimes combined with vertical shortening of the lower lid retractors. When the problem is mainly medial, punctual ectropion, a spindle of conjunctiva and lower lid retractors is resected immediately below the lower punctum. Lower lid ectropion may coexist with retraction, as was discussed above. Involutional ectropion usually does not occur in the upper lid. Cicatricial ectropion is due to vertical shortening of the anterior, skin–muscle lamella of the upper or lower lid. It is usually secondary to trauma or skin disorders. In the upper lid, release of the scarred tissue is followed by skin grafting from the opposite upper lid, the retroauricular area, supraclavicular area, or forearm. In the lower lid, horizontal lid shortening is also often required since the chronically ectropic lid often has or develops a component of excess horizontal laxity. In the lower lid, a skin graft may be replaced by advancement of a skin–muscle flap from the surrounding area. Paralytic ectropion is due to paralysis of the seventh nerve. There is also lagophthalmos due to inability of the upper and lower lids to close. Exposure can be severe and can lead to corneal ulceration. Conservative treatment consists of ocular lubrication, moist chamber devices and lid taping. Tarsorrhaphy may be needed if the cornea dries out in spite of conservative measures. For those cases in which seventh nerve function will not return a gold weight or spring may be placed in the upper lid to counter the lagophthalmos. The lower lid usually needs to be tightened horizontally and often lifted vertically with a posterior lamellar graft or suspension sling of fascia or silicone. Although such treatments are helpful, patients never have a normal blink with any of these procedures. Floppy Lid Syndrome This uncommon syndrome is most frequently seen in obese, middle-aged males (Figure 8). The upper lid is extremely lax and spontaneously everts often while the patient sleeps and rubs against the pillow. Those affected have a severe papillary conjunctivitis, ropey mucoid discharge, and irritation. Floppy lid syndrome is accompanied by sleep apnea and hypertension. Symptoms can sometimes be controlled by having the patient wear a Fox shield over the eye while sleeping to reduce nocturnal eversion of the lid. Most often, horizontal upper lid shortening is needed along with tightening of the lower lid for control of symptoms. Common Malignant Eyelid Tumors

Figure 7 Involutional ectropion of the lower lids, worse on the right.

Basal cell carcinoma (BCC) is the most common eyelid malignancy accounting for 85–90% of such lesions.

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Figure 8 Demonstration of the floppy eyelid syndrome on the left.

BCC is an actinic lesion and is more common in fairskinned individuals on sun-exposed parts of the body. It is most common on the lower lid, followed by the medial canthus, upper lid, and lateral canthus. The tumor is classically a firm, nodular lesion with a central ulcer and pearly, vascular border (Figure 9). It may also present as an irritating, erythematous patch or rarely be entirely subcutaneous. Lesions of the medial canthus have a propensity for deep invasion into the eye socket. BCC only rarely metastasizes and generally grows very slowly. The preferred treatment is complete excision with frozen section control or by Mohs micrographic surgery usually performed by a specially trained dermatologist. It provides the best cure rate (98–99%) while saving as much normal tissue as possible. Excision is followed by reconstruction of the eyelid defect or occasionally by spontaneous healing if the defect is small or does not involve the lid margin. Radiation therapy is an alternative to surgery but not as effective due to the relatively high recurrence rate (5–20%). Recurrence is also difficult to detect after radiation due to tissue alteration from the treatment. Cryotherapy is sometimes used for small lesions but has similar problems with a relatively high recurrence rate (20–30%) and depigmentation and atrophy of treated tissue. Squamous cell carcinoma (SCC) accounts for about 5% of periocular malignancies. It too is an actinic lesion, being more common on sun-exposed areas of fair-skinned individuals. Again, the lower lid is the most common location. It most frequently presents as an erythematous, thickened patch with erosion of the involved tissue (Figure 10). A nodular subtype is occasionally observed. It may arise from preexisting actinic keratoses, which undergoes malignant transformation 20% of the time. SCC can metastasize through the regional lymphatics or spread along involved sensory nerves. Mohs removal and reconstruction are the preferred therapy. Radiation

Figure 9 Nodular basal cell carcinoma of the left lower lid.

Figure 10 Squamous cell carcinoma of the left medial canthus.

therapy is much less effective in SCC and thus is used only for palliation of surgically unresectable tumors. Topical therapy with 5-fluorouracil or imiquimod is a frequent treatment of the premalignant actinic keratosis. Sebaceous cell carcinoma accounts for about 5% of periocular malignancies but is increasing in frequency, which may be due in part to better pathological diagnosis. It arises from the meibomian glands, the glands of Zeiss, and sebaceous glands of the caruncle. It presents as a nodular lesion, which is often mistaken for a chalazion or a diffuse intraepithelial pattern that looks like a chronic conjunctivitis (pagetoid spread) (Figure 11). Because of this, the diagnosis is often delayed until biopsied. It is capable of both regional lymphatic and vascular spread. It is considered deadlier than SCC. Mohs microsurgery may be effective in tumor removal if the tumor is nodular, but pagetoid spread and skip lesions often necessitate wide sampling of the bulbar and palpebral conjunctiva (map biopsy). If diffusely spread, orbital exenteration is


Figure 11 Pagetoid spread of sebaceous cell carcinoma of the conjunctiva of the left lower lid.

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Figure 12 Malignant melanoma of the right lower lid.

usually required. Sebaceous cell carcinoma is relatively radio resistant and responds poorly to chemotherapy. Malignant melanoma accounts for less than 5% of periocular malignancies, but it too is increasing in frequency (Figure 12). It too is an actinic lesion but other less well-defined causes play a role. It arises spontaneously usually as a nodule or in an existing nevus or area of lentigo maligna (intraepithelial tumor). It too is capable of spread through the regional lymphatics and the blood stream. Surgical excision is the treatment of choice, but requires permanent section histology to adequately assess tumor margins. Because of this, the resection of the tumor is often spread over several days (Slow Mohs) followed by reconstruction when margins are clear. Benign Eyelid Tumors A variety of benign lesions are found on the lids and periocular skin. The vast majority are of minimal functional significance, but patients frequently request removal for cosmetic reasons. The most common are nevi, inclusion and glandular cysts, seborrheic keratoses, verruca, skin tags, and benign glandular tumors. Simple excision can usually be carried out in the office under local anesthesia. Inflammatory and Infectious Disorders of the Lids Chalazion is a lipogranuloma of one of the meibomian glands of the tarsal plate. They arise relatively rapidly over a period of a few days, often with inflammation and discomfort. They can progress to form a chronic peasized, firm nodule in the lid (Figure 13). The initial treatment is to hot compress the involved lid frequently in the first few days in hopes of opening the obstructed gland. If not effective many will resolve spontaneously over the next several weeks to few months. Patients often

Figure 13 Chalazion of the right upper lid.

request removal for cosmetic reasons. This can usually be done in the office with local anesthesia. An alternative is to inject the chalazion with steroid, which is effective about 50% of the time. Hordeolum is a staphylococcal infection of one of the sebaceous glands of the lid. It presents as an acutely swollen, erythematous, painful nodule on the lid margin (external hordeolum) or on the palpebral conjunctiva (internal hordeolum). Hot compress, topical antibiotics, and surgical drainage are effective. Oral antibiotics may be needed if cellulitis occurs. Blepharitis is the most common inflammatory disorder of the eyelids characterized by redness, swelling, and irritation with visible crusting. It is a chronic condition characterized by intermittent exacerbations. There are three subtypes, but many patients show combinations of all three. In staphylococcal blepharitis, chronic colonization of the lid margin leads to inflammation from the bacterial toxins and antigens. Slit-lamp examination

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reveals characteristic white collarettes around the base of the lashes. In seborrheic blepharitis, there are greasy scales (scurf) found on the lashes and often associated seborrheic dermatitis of the face and scalp. The third form, posterior blepharits (posterior lid margin disease, meibomian gland dysfunction, and meibomianits) is characterized by a change in the normal clear meibomian gland secretion to a thick, cloudy to yellow, oily discharge. Posterior blepharitis is often associated with acne rosacea and chalazia. All forms of blepharitis lead to chronic redness of the conjunctiva, sometimes with papillary hypertrophy. The tear film is often unstable as manifested by a rapid tear break-up time. Dry eye is a frequent association. In addition to the above findings, slit-lamp examination may reveal punctate epithelial erosions (PEEs) of the cornea and marginal corneal erosions due to staphylococcal hypersensitivity. There may be small, rounded domes on the meibomian orifices, manifestations of the thick, inspissated secretions. Digital pressure on the lid may cause the meibomian glands to express material that can, in severe cases, have a cheesy consistency. Treatment consists of warm compresses to thin the meibomian secretions and eyelid scrubs with baby

shampoo or commercially available products to clean the lid margin and express the meibomian glands. Topical antibiotic drops or ointment reduces the bacterial colonization of the lid margin and topical steroids help to control the inflammation. Oral, low-dose tetracycline (50 mg doxycycline per day) is helpful in reducing the meibomian gland discharge and normalizing the pH of the tear film. Oral erythromycin can be used if the patient is intolerant to tetracyclines, pregnant, nursing, or under the age of 12. See also: Eyelid Anatomy and the Pathophysiology of Blinking; Lacrimal Gland Overview; Tear Drainage.

Further Reading Jordan, D. R. and Anderson, R. A. (2000). Surgical Anatomy of the Ocular Adnexa: A Clinical Approach. San Francisco, CA: American Academy of Ophthalmology. Stewart, W. B. (2000). Surgery of the Eyelid, Orbit, and Lacrimal System. San Francisco, CA: American Academy of Ophthalmology.

Overview of Electrolyte and Fluid Transport Across the Conjunctiva O A Candia and L J Alvarez, Mount Sinai School of Medicine, New York, NY, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Short-circuit current – The short-circuit current (Isc) across tissues isolated within an Ussing chamber is defined as the charge flow per time, per the crosssectional area of the epithelium that is exposed to the bathing solutions, when the tissue is short-circuited by clamping the transepithelial voltage (PDt) to zero with an external circuit. The Isc is a current that circulates through the tissue and the external circuit thereby completing a closed loop. Because of this, the Isc enters the tissue across one surface and leaves across the other. Monitoring the Isc provides a continuous measure of the net charged flow of current across the transcellular pathways of the tissue. There is no net flow across the paracellular pathways in the short-circuited condition if the tissue is bathed bilaterally with solutions containing identical ionic concentrations. Adding drugs that affect the ionic channels or electrogenic elements within the membranes of the epithelium will affect the magnitude of the Isc. Transepithelial resistance – An epithelium can be considered to be comprised of an arrangement of resistance elements or resistors. This arrangement is most simply modeled in a multilayered epithelium as two resistors in series, namely Ra (the resistance of the apical membrane) and Rb (the resistance of the basolateral membrane). These resistances are shunted by a parallel resistor, Rshunt, which is the cumulative resistance of the paracellular pathways. As such, transepithelial resistance (Rt) is defined as follows: Rt ¼

ðRa þ Rb Þ Rshunt Ra þ Rb þ Rshunt

In the conjunctiva, Rshunt tends to be lower than the transcellular pathway. This leads to small measured changes in Rt when drugs that selectively affect either Ra or Rb are added. For example, potassium channel blockers should selectively increase Rb, but the effect on the measured Rt parameter is relatively smaller in the conjunctiva than in the corneal epithelium, which is a tight epithelium, that is, a tissue with a high Rshunt. Transepithelial voltage – Epithelial cells transport ions and thereby generate a transepithelial voltage (PDt). The generation of PDt requires (i) an asymmetric distribution of ion channels and

electrogenic transporters on the apical and basolateral sides of the tissue, and (ii) the presence of tight junction proteins between adjacent cells in the superficial epithelium to impede the flow of ions along the paracellular pathways. Unidirectional fluxes – Across epithelia isolated in Ussing-type chambers, a unidirectional flux of a substance (e.g., a radiolabeled electrolyte or water molecule) is its rate of translocation across the tissue from the bathing solution within one hemichamber to the contralateral bath, disregarding any counterbalancing flux in the opposite direction. In practice, unidirectional fluxes are measured in the apical-to-basolateral direction, and again in the basolateral-to-apical direction. A difference in the magnitude of these two unidirectional fluxes provides a measure of the net flux, for example, there is a net chloride flux across the conjunctiva in the basolateral-to-apical direction. Ussing chamber – A device designed by Hans Ussing in 1951 to originally study vectorial ion transport across the frog skin. It has since been modified (i.e., an Ussing-type arrangement) by many investigators to characterize electrolyte transport across various epithelial tissues including epithelia of the eye. This approach has significantly contributed to our understanding of how electrolytes are transported. The Ussing-type methodology entails two aspects. One is the chamber itself, which is constructed to hold the dimensions of a particular tissue, and enable it to be bathed bilaterally. The second aspect is the external electrical circuitry, which can be designed to measure transepithelial voltage, resistance, and current. The effects of pharmacological agents on the electrical parameters generated by the epithelium can be studied by applying test compounds unilaterally to either the apical-side or basolateral-side baths.

Introduction The conjunctiva and the corneal epithelium together form the ocular surface. The conjunctiva (from late Latin, feminine of conjunctivus, or conjoining) is in essence a connection (conjunction) between the eyelids, the sclera of the eyeball, and the cornea. It lines the posterior surface of the

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eyelids (the palpebral conjunctiva) and the exposed, anterior aspect of the globe (the bulbar conjunctiva). The latter is loosely attached to the sclera of the eyeball, and translucent, thereby exposing the so-called ‘white of the eye’; it merges with the corneal epithelium at the limbus, which constitutes the edge of the cornea. The palpebral conjunctiva is tightly adherent to the eyelid. The space, lined by the conjunctiva, between the lids and the globe is known as the conjunctival sac. The bottom of the sac, which is unattached to the eyelids or to the eyeball, is known as the fornix, forniceal region, or conjunctival fold. The untethered nature of the fornix allows the eyeball to move freely. The conjunctival sac varies in size depending upon the degree to which the lids are open. The depths of the unextended sac in humans are about 14–16 mm superiorly and 9–11 mm inferiorly. The total surface area of the conjunctiva is about 9 and 17 times larger than that of the cornea in rabbits and humans, respectively. The lacrimal glands, which secrete tears, open into the superior fornix. The palpebral conjunctiva contains the openings of the lacrimal canaliculi, which allow tears within the conjunctival sac to drain into the nasal cavity. The vasculature of the palpebral conjunctiva is clearly visible within the tissue upon examining the posterior surface of the eyelid. In contrast, the bulbar conjunctiva is normally colorless, unless its vessels are dilated as a result of inflammation (conjunctivitis). The conjunctival vessels arise from a peripheral palpebral arcade and from the anterior ciliary arteries. Blood comes mostly from the orbit, but anastomoses with the facial system. Conjunctival innervation is mediated by the ophthalmic division of the trigeminal nerve. The conjunctiva is extensively innervated with adrenergic, cholinergic, and peptidergic fibers identified in various species. In general, the largest numbers of nerves present are sympathetic, with fewer parasympathetic and sensory nerves. The parasympathetic nerves contain the neurotransmitters acetylcholine and vasoactive intestinal peptide; the sympathetic nerves, norepinephrine and neuropeptide Y; and the sensory nerves, substance P and calcitonin-gene-related peptide. The major roles of the conjunctiva are: (1) to contribute to tear production by secreting electrolytes and fluid; (2) to modify the composition of the tear film by secreting mucins and lipids, and absorbing various organic compounds found in tears; and (3) to contribute to the resistance of the eye to infection by providing protection against microorganisms. The conjunctiva is comprised of an epithelium and an underlying stroma. The epithelium is embryologically related to, and anatomically continuous with, the epithelium of the upper airway. Within the conjunctival epithelium are goblet cells, which are specialized epithelial cells that function as unicellular mucus glands. The goblet cells secrete the

mucin component of the tear film, which consists of three layers, each of which is secreted by different cells. Secreted mucins constitute the inner layer of the tear film and serve as wetting agents that keep the apical, hydrophobic aspects of the corneal and conjunctival epithelia hydrated. The middle, aqueous layer of the tear film contains water, electrolytes, immunoglobulin A (IgA), glucose, and proteins (including antibacterial enzymes). This layer is primarily secreted by the main and accessory lacrimal glands; the latter glands of Krause and Wolfring flank the main lacrimal duct near the superior fornix. It is possible that the conjunctiva also contributes to this layer under basal conditions when the lacrimal glands are not stimulated. The outer, lipid layer of the tear film contains a fat mixture that is secreted by the meibomian glands that line the eyelids. This layer functions to reduce evaporation of the aqueous layer. Underlying the conjunctival epithelium, the connective tissue contains blood vessels, nerves, conjunctival glands, mast cells, and leukocytes including macrophages. The latter, defensive cells can be recruited in large numbers to an injury site on the ocular surface due to disruption of the barrier properties of the epithelium. They may also release paracrine-signaling agents that affect the transport properties of the epithelium, and certain leukocyte populations can also serve as antigen-presenting cells. Recent work has characterized the active transport properties of the conjunctival epithelium. The epithelium is capable of transporting fluid as a consequence of a sufficiently high water permeability bestowed by endogenous water channels (aquaporins) and transepithelial solute movement due to active transport mechanisms. This article includes a synopsis of the current understanding of the electrolyte and fluid transport across the conjunctiva.

Conjunctival Epithelium A primary role of all epithelial tissues, including those in the conjunctiva, is the absorption and/or secretion of fluid. In brief, two main elements are necessary for fluid movement across a membrane or a set of membranes: (1) the driving force represented by an osmotic gradient (or hydrostatic pressure), and (2) a water pathway represented by water channels (aquaporins) and the lipid bilayer. Thus, all fluid secretion or reabsorption is a consequence of the osmotic gradient created by active electrolyte transport, with the direction of fluid movement identical to that of the net transepithelial solute transport. To date, extensive, functional characterizations of the electrolyte transport properties of the conjunctival epithelium have been done only on the isolated rabbit conjunctiva. The epithelium of this species exhibits mechanisms that simultaneously mediate Na+ absorption and Cl secretion. The relative proportions of these oppositely directed functions varies considerably from one individual rabbit conjunctival

Overview of Electrolyte and Fluid Transport Across the Conjunctiva

specimen to another, for reasons that are unknown, but in general Cl transport is predominant. On average, the ratio of Cl secretion to Na+ absorption is about 1.5 to 1, suggesting that the rabbit epithelium can function primarily as a chloride-secreting epithelium potentially capable of moving fluid into the conjunctival sac. However, it must also be noted that a small proportion of conjunctival specimens exhibited a Na+ absorptive rate larger than the rate of Cl secretion. Morphologically, the epithelia of the rabbit bulbar, forniceal, and palpebral regions have distinct appearances (Figure 1). The bulbar epithelium appears columnar and thinner than the other sections with goblet cells present. It is as thick as two to three cell layers and packed irregularly. In the forniceal area, the number of cell layers increases to three or four with a greater abundance of goblet cells. From this region to the lid margin, a transition within the palpebral epithelium is evident with the number of goblet cells diminishing, and the epithelial

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cells becoming more stratified. Species differences in morphology among mammalian conjunctivae have been described. Excluding variations in the number of goblet cells, the epithelial cells within each conjunctival region appear homogeneous, which suggests that both absorptive and secretory activities coexist within an individual epithelial cell. If so, the conjunctival epithelium exhibits a rare property among epithelia in that the transport functions for Na+ absorption and Cl secretion are not segregated in distinct cell types, and the transport rates for these oppositely directed functions are nearly equivalent in isolated conjunctivae under in vitro conditions. Regardless of the normal, physiologic direction of fluid movement across the human conjunctiva, it is clear that inhibiting reabsorption and/or stimulating secretion may have a beneficial effect by increasing the aqueous layer of the tear film in individuals with a tear-fluid deficit due to various lacrimal gland deficiencies.

G

(d)

(a)

G G

(e)

(b)

G

50 µm (c)

(f)

Figure 1 Histological sections of the rabbit limbal and conjunctival regions. (a) The limbal epithelium upon a highly vascularized stroma. (b) The bulbar conjunctival epithelium. There are goblet cells (G) present among the epithelial cells. (c) The forniceal epithelium nearest the bulbar region. (d) Another section of the forniceal epithelium, within which the goblet cells become more numerous relative to the bulbar region. The forniceal epithelium is thicker than the bulbar epithelium. (e) The palpebral epithelium near the forniceal region that is characterized by a decrease in the number of goblet cells and abundant lymphoid tissue. (f ) The palpebral epithelium closer to the eyelid margin. It is more stratified than the other conjunctival regions and devoid of goblet cells. Adapted from Wei, Z. G., Sun, T. T., and Lavker, R. M. (1996). Rabbit conjunctival and corneal epithelial cells belong to two separate lineages. Investigative Ophthalmology and Visual Science 37: 523–533.

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Bioelectric Studies on the Isolated Rabbit Conjunctiva The ionic transport systems potentially mediating the absorptive and/or secretory functions of an epithelium can be efficiently characterized by isolating the epithelium under an Ussing-type arrangement. With this method, a flat piece of an epithelium is dissected with some of its underlying connective tissue or stroma maintained in place to provide structural support. The thickness of the entire dissected preparation is usually about 1 mm, of which about 0.05 mm represents the epithelial cellular compartment. The isolated tissue is then positioned between two hemichambers, which when closed together result in the tissue serving as a partition separating the cavities of the two hemichambers. In this situation, the apical surface of the epithelium interfaces with the cavity of one hemichamber, while the basal surface attached to the underlying stroma interfaces with the contralateral chamber. Each hemichamber is then filled with a physiological solution to simultaneously bathe the two surfaces of the in vitro preparation. The rabbit conjunctiva serves as a fairly good specimen for this approach given its relatively large surface area, as well as the fact that the conjunctival sac can be removed nearly intact as a cylinder and then cut longitudinally to convert it to a flat epithelium that is easily mounted between Ussing-type hemichambers. Typically, about 0.5 cm2 of cross-sectional area of tissue is bilaterally exposed to the bathing solutions within the chambers. A negative aspect of this approach with the conjunctiva is that the epithelium seems to be relatively frail (when compared to the corneal epithelium) as its integrity deteriorates following several hours of isolation within the chambers. Nevertheless, useful electrophysiological experiments can be designed and informative data are obtainable. Upon isolation of an epithelial preparation, such as the conjunctiva, within the divided chambers, a potential

Apical aspect

Na Glucose

difference (PD) develops across the tissue. The PD is a consequence of the active transport of electrolytes by the epithelium, which spends metabolic energy to maintain ionic gradients between the cellular compartment and the extracellular bath. An epithelium will exhibit a negative intracellular voltage with respect to both the apical-side bathing solution and the stromal-side (basolateral) solution. Typically, the positive voltage of the stromal-side bath (PDs) with respect to the cellular compartment occurs because of the electrogenic Na+–K+ ATPase, which extrudes three Na+ ions for two imported K+ ions, and the fact that the cellular K+ ion concentration is above equilibrium, so that K+ will constantly efflux through K+ channels toward the stromal (basolateral) bath (Figure 2 shows an overview of the major transport elements found in the conjunctival epithelium). The Na+–K+ ATPase functions incessantly to maintain cellular K+ above equilibrium. The positive voltage of the apical-side bath (PDa) with respect to the cellular compartment is less positive than PDs. As such, a transepithelial PD (PDt) exists, which equals the difference between the PDs across the respective contralateral surfaces of the epithelium (PDt = PDa – PDs; and has a negative sign with PDs taken as reference). PDa is less positive with respect to the cellular compartment than is PDs because of the efflux of Cl via channels in the apical domain toward the apical-side bath, and in the case of the conjunctiva, an influx of Na+ also occurs via electrogenic transporters that are in the apical membrane (Figure 2). PDt can only exist if tight junctions are present in the epithelium, which is the case in the conjunctiva. These elements are located between the lateral membranes of the most superficial epithelial cells and form a resistance barrier that impedes the diffusion of ions from the contralateral bathing solutions through the paracellular pathways between the epithelial cells. Without the presence of tight junctions, ionic flows in the paracellular pathway would

Basolateral aspect

H

H2O H2O Na

Na

K

Cl H2O Na CI channels NSCC? include CFTR and CLCA AQP5 on apical surface

Na

K

Cl

Cl Cl

Na K H2O

HCO3

cAMP- and Ca-activated K channels are present

AQP3 on basolateral surface

Figure 2 Summary cartoon of the major transport elements found in the rabbit conjunctiva with water fluxes indicated with double arrows. NSCC, nonselective cation channels; CFTR, cystic fibrosis transmembrane conductance regulator (which has chloride channel activity); CLCA, calcium-activated chloride channel; AQP5, aquaporins homolog type 5; AQP3, aquaporins homolog type 3.


short-circuit PDt, because PDa and PDs would still exist and result in a net movement of anions from the apical-side bath to the stromal-side bath through the paracellular pathways, and a net movement of cations in the opposite direction through the paracellular pathways. The Ussing-type chambers used to isolate the conjunctiva have ports for inserting electrodes into the bathing solutions to directly measure PDt. In addition, there are ports for current-sending electrodes. These are used to short-circuit PDt with an external circuit connected to an automatic voltage clamp that constantly maintains PDt ¼ 0 mV. The amount of current needed for this is continuously recorded and known as the short-circuit current (Isc). The Isc represents a real-time measure of the net transepithelial movement of electrolytes across the transcellular pathways of the tissue due to metabolically dependent active transport. As PDt is maintained at 0 mV, there is no net movement of electrolytes through the paracellular pathways, even if tight junctions were, in principle, not present. Transepithelial electrical resistance (Rt) can be determined by applying Ohm’s law to the measured PDt (under open-circuit conditions) and the measured Isc (under short-circuited conditions), or by measuring the amount of current necessary to offset the short-circuited condition by a few mV for a few seconds. Either approach gives identical measures of Rt with the conjunctiva. As alluded to above, the integrity of the conjunctival epithelium appears to degenerate following a prolonged period in the chamber. This is observed as a spontaneous, gradual decline in Rt. It appears that there is a loss in paracellular resistance (i.e., tight-junction structure may change with time in vitro) since the Rt decline occurs in the presence of a steady Isc. As noted, under the shortcircuited conditions, increases in paracellular ion movement do not result in a net flow across this pathway given the absence of a PD across the epithelium and identical electrolyte concentrations on each side of the preparation. As such, the Isc continues to measure net transcellular flow of electrolytes in experiments with symmetrical solutions. However, changes in conjunctival Rt in response to the additions of various drugs frequently underestimate changes in membrane resistance. This is because transcellular resistance is proportionally larger than paracellular resistance, which means that large changes in transcellular resistance are recorded as smaller changes when measuring conjunctival Rt. Nonetheless, the initial measurements of conjunctival Rt upon the isolation of fresh preparations within the divided chambers provide a good indication of the barrier properties of the epithelium. This is because the electrical resistances of both the cell membranes as well as the paracellular pathways contribute to the Rt measurement. The paracellular pathways of the in situ conjunctiva at the ocular surface allow for the passive diffusion of

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hydrophilic solutes across the conjunctiva. Passive paracellular transport of electrolytes across the conjunctiva in vivo, which is analogous to the open-circuit situation in vitro, occurs because of gradients created by the transcellular mechanisms. In addition, cell-impermeable, hydrophilic solutes applied to the conjunctival sac may diffuse across the epithelium through the paracellular route. The transepithelial permeability of such solutes decreases with increased solute size. Tight junctions located at the apical-most aspect of an epithelium create the major barrier for the movement of solutes across all epithelia including the conjunctiva. However, the paracellular route varies considerably among epithelia in terms of permeability to solutes and electrical resistance. Rt measurements can range at least 1000-fold between highly resistant and so-called leaky epithelia. In some tissues, electron microscopy studies have correlated the ultrastructure of the tight junctions with the measured Rt values obtained in vitro. In general, the number of tight-junction strands along the apical-basal axis is proportional to the junctional resistance. Rt values from freshly isolated rabbit conjunctival epithelia are in the range of 1–2 kO cm2, with many studies reporting an average value of 1 kO cm2. This suggests that the conjunctival epithelium is a moderately tight epithelium. For comparison, Rt measurements of the rabbit corneal epithelium and rabbit corneal endothelium are 7–9 kO cm2 and 0.01–0.07 kO cm2, respectively. As such, the electrical resistances of the corneal epithelium and endothelium vary over a range of about 100-fold, and the conjunctiva exhibits an intermediary value. The fact that the measured Rt of the freshly isolated conjunctiva is so high also indicates that tight junctions must exist between the surface goblet cells and the most superficial stratified epithelial cells. An explanation as to why Rt declines with prolonged time in vitro remains to be evinced, but barrier resistance is physiologically regulated in other systems. The stable Isc recorded during the spontaneous Rt decline indicates that the epithelial cells have remained metabolically viable.

Electrolyte Transport Systems of the Rabbit Conjunctiva The bioelectrical approach discussed above has been used to determine the major electrolyte transport systems present in the rabbit conjunctival epithelium. In work done to date, the short-circuiting methodology was used to characterize transcellular transport. Such transport is energy dependent, and controlled by the tissue-specific profile of transporters and channels along the apical and basolateral membranes of the epithelium. The conjunctival apical membranes interface with the tears and the basolateral membranes interface with the paracellular pathways from the tight junctions to the basement membranes at the

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stroma. Transport mechanisms in the tissue are generally uncovered by electrolyte substitution experiments, and the application of relatively specific drugs (i.e., channel blockers, channel openers, transporter inhibitors, etc.) to the bathing solutions of conjunctiva isolated in the divided chambers. In some cases, the identity of channels and transporters that were identified in electrophysiological experiments were corroborated with immunoblotting and immunohistochemcial observations. Identical to other Cl-secreting epithelia, the rabbit conjunctival epithelium has a basolateral bumetanidesensitive Cl uptake process (mediated by the Na+–K+–2Clcotransporter, NKCC1) positioned in series with apical Cl channels, including cystic fibrosis transmembrane conductance regulator (CFTR). In addition, Na+/H+ and Cl/HCO 3 exchangers exist in parallel in the basolateral membrane and can also mediate Cl uptake (Figure 2). Oppositely directed, electrogenic Na+ reabsorption is amiloride-insensitive, indicating the absence of the major epithelial Na+ channel (ENaC) at the apical surface, and occurs through a Na+-dependent cotransporter for glucose. Nonselective cation channels (NSCCs) were identified in whole-cell patch clamping of freshly isolated conjunctival epithelial cells and such channels may also reside at the apical surface. A transcellular Na+ movement occurs because the apical uptake mechanisms for Na+ exist in series with the basolaterally located Na+–K+ pump (Figure 2). Other electrogenic Na+-dependent uptake mechanisms at the apical surface were demonstrated by adding the transported substrates to the apical bath. Transport systems for amino acids, nucleosides, L-lactate, and di- and tri-peptides were evidenced in this manner because such compounds are normally not included in the physiological solutions used to bathe the in vitro preparations. The addition of such compounds to the apical bath results in a shortcircuit current stimulation. In the cases of the amino acids and nucleosides, the largest Isc stimulations occurred with L-arginine and uridine, respectively. The roles of these carriers are not firmly established. It is thought that these transport systems may clear the tear fluid of these compounds in either nonphysiologic or pathophysiological states of the ocular surface when excess amounts of such solutes might have leaked into tears. Results from protocols for immunoblotting and the immunofluorescent labeling of frozen sections from separately isolated bulbar and palpebral regions of the conjunctival epithelium indicated that the proteins for the Na+–glucose cotransporter, Na+–K+ ATPase, and Na+–K+–2Cl cotransporter are uniformly distributed throughout the conjunctiva. These observations suggest that despite stark differences in the regional morphology of the bulbar and palpebral regions, the entire conjunctival epithelium exhibits the elements for transepithelial Na+ and Cl transport.

Regulation of Epithelial Ion Transport in Rabbit Conjunctiva Currently, information on the regulation of electrolyte transport by the conjunctival epithelium is somewhat limited. This is because the characterization of the macroscopic electrolyte transport properties of this tissue, as measured in bicameral Ussing-type chambers, was begun relatively recently. Hence, many fundamental aspects of the tissue have not been elucidated. One underlying rationale for studying conjunctival transport is to define the secretory functions of the epithelium. This effort could prove to have utility in ameliorating complications from dry-eye diseases, and some progress has been made in this regard. Because of the large surface area of the conjunctival epithelium, active transport by conjunctiva with accompanying fluid secretion could, hypothetically, contribute a significant fraction of tear production, which is normally provided in healthy individuals by the lacrimal gland. Upon stimulation, the transepithelial conjunctival contribution could be greater. As commonly found in Cl-secreting epithelia, the exposure of the conjunctiva to secretogogues that increase either cell calcium or cyclic adenosine monophosphate (cAMP) stimulates transepithelial Cl fluxes and the Isc. The latter intracellular messenger can be increased in the conjunctival epithelium with forskolin (a direct stimulator of adenylyl cyclase), dibutyryl-cAMP (a cell permeable form of cAMP), 3-isobutyl-1-methyl-xanthine (IBMX, a nonselective phosphodiesterase inhibitor), rolipram (an inhibitor specific for cAMP-phosphodiesterase type IV), or epinephrine (a nonselective adrenergic agonist). In addition, these agents also increase the transconjunctival Isc under Cl-free conditions indicating that the increased cAMP levels also stimulate the Na+ absorptive activity of the epithelium. The increase in Na+ absorption has been attributed to a protein kinase A (PKA)-regulated, bariuminhibitable, basolateral K+ conductance in the rabbit conjunctival epithelial cells. One or more different types of K+ channels that have not yet been identified may mediate this K+ conductance. The stimulation of the PKA-gated K+ channels hyperpolarizes the negative cell potential relative to the bathing solutions and favors both the uptake of Na+ across the apical face and the efflux of Cl into the tears. There is evidence that apical Cl channels are also gated by PKA, particularly in the case of CFTR. In the shortcircuited conjunctiva, cAMP has a central role in coordinating simultaneous changes in apical Cl and basolateral K+ conductances to enable stimulations in the transcellular transport of Cl in the stromal-to-apical direction and of Na+ in the opposite direction. Should both absorptive and secretory mechanisms coexist within the same cell, such cAMP-evoked stimulations of the in vivo conjunctiva would, in principle, deplete the epithelium of KCl and reduce cellular volume, while Na+ and Cl move in opposite directions both trans- and


paracellularly. Experimental measurements of net water fluxes across the isolated conjunctiva (under open-circuit conditions) indicate an increased fluid movement in the stromal (basolateral)-to-apical direction in response to cAMP, likely due to the higher rate of Cl secretion relative to Na+ absorption. Fluid flow occurs under open-circuit conditions, which is the situation in vivo. The dominant transport system, apparently Cl, will be transported mainly transcellularly, while Na+ will reverse from its net tear-to-stroma direction found under shortcircuit conditions to move paracellularly as a companion ion to neutralize the Cl charge and create a possible isotonic fluid at the apical surface. In open circuit, there would still be a transcellular movement of Na+ toward the stroma, and a paracellular movement of Cl toward the stroma, but the magnitude of these flows will be less than the net of Na+ and Cl secreted into tears (Figure 3). cAMP stimulates all flows and increases the net. Other effective secretogogues in the rabbit conjunctiva are: (1) 1-ethyl-2-benzimidazolinone (EBIO), a Cl and K+ channel opener that elicits electrophysiological effects similar to those of cAMP, although different subtypes of channels are likely involved; and (2) the nucleotide uridine 50 -triphosphate (UTP), which stimulates Cl secretion through P2Y2 receptors upon exogenous application to the apical-side bath. Of these, only the latter has been tested on net fluid movement across isolated conjunctivae and found to be a useful stimulant. Recently, synthetic P2Y2 agonists (e.g., diquafosol tetrasodium, which is also known as INS365) have been studied in clinical trials. Such agents are administered 4–5 times daily, and there is a time-dependent loss of efficacy that is observable in the data produced by such trials. This may be because P2Y2-receptor activation is often transitory due to the nature of the Ca2+ signal itself (through the phospholipase C-sensitive calcium signaling pathways) and the fact that purinergic agonists produce receptor desensitization from which recovery is slow. Yet currently, the use of purinergic agonists appears a suitable approach to palliate dry eye because of not only the stimulatory effects on conjunctival Cl secretion and fluid transport, but also the fact that purinergics serve as mucin secretogogues from conjunctival goblet cells. As such, purinergics appear to have utility in conserving the composition of the tear film. The established receptors that stimulate electrolyte and fluid secretion in the stromal-to-tear direction under open-circuit conditions are schematically presented in Figure 4. The specific channel subtypes activated by calcium and cAMP remain to be conclusively identified.

Fluid Transport Studies across Isolated Rabbit Conjunctiva Two commonly used methods to measure water fluxes across various epithelia have been applied to the excised,

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Figure 3 A simplified model of the sodium and chloride flows across the rabbit conjunctiva under open-circuit conditions, which are analogous to the in vivo situation. Some transporters present in the epithelium have been omitted for clarity. Cl1 is the transcellular efflux of chloride via chloride channels in the apical domain. Cl2 is the paracellular movement of chloride in the stromal-to-tear direction. Cl3 is the paracellular movement of chloride in the tear-to-stromal direction. Na1 is the sodium efflux mediated by the sodium-potassium ATPase pump. Na2 is the paracellular movement of sodium in the tear-to-stromal direction, while Na3 is the paracellular movement of sodium in the opposite direction. In open-circuit, the tear-side (apical) bath will have a negative potential relative to that of the basolateral-side bath. Cations will thus flow in the paracellular pathways toward the tear side, while anions will flow in the opposite direction. There is also the possibility that some potassium will move along the paracellular pathways toward the tears. The sodium–potassium–chloride cotransporter in the basolateral membranes drives the transcellular movement of chloride. The net flux of sodium and chloride into the tears results in a net fluid transport across the conjunctiva. Flux relationships are indicated at the bottom of the figure by equations.

isolated rabbit conjunctiva: (1) unidirectional/diffusional flow with tritiated water (3H2O); and (2) net water flow by volumetric procedures. With method 1, the diffusion permeability coefficient, Pdw , is expressed in cm s1 and given by: Pdw ¼ Jdw =A Vw Cw

where, A is the area of the membrane (cm2), Vw is the partial volume of water (cm3 mol1), Cw is the concentration of water (mol cm3), and Jdw is the measured unidirectional H2O flux in cm3 s1. In this case, a two-compartment chamber is used. The tissue is mounted between compartments; 3H2O is added to one side and samples are taken periodically from both

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Figure 4 Cartoon of the established receptors that stimulate electrolyte and fluid secretion in the stromal-to-tear direction under open-circuit conditions, which are analogous to the in vivo situation. Some transporters present in the epithelium have been omitted for clarity. The specific channel subtypes activated by calcium and cAMP remain unverified. Abbreviations: UTP (uridine 50 -triphosphate); INS365 (a synthetic activator of the P2Y2 receptor); P2Y2 (a receptor subtype for the pyrimidine, UTP, that triggers calcium signaling via the phosphoinositide pathway); CLCA2 (a calcium-activated chloride channel subtype thought to be present in the conjunctiva); CFTR (cystic fibrosis transmembrane conductance regulator, which mediates a cAMP-gated chloride conductance via PKA); SK4 (a potassium channel subtype thought to be present in the conjunctiva that is activated by calcium, possibly via calmodulin, a calciumregulatory protein); KvLQT (a potassium channel protein subunit linked to cAMP-activated potassium conductances). The presence of the above channel subtypes has been suggested from the expression levels of message in gene microarray assays of human conjunctiva, but the operational existence of the putative channel proteins has not yet been confirmed in functional experiments.

sides to determine the diffusion of 3H2O, which is proportional to Jdw. With method 2, the osmotic permeability coefficient, Pf , is expressed in cm s1 and can be calculated from the following expression: Pf ¼ Jv =A Vw DCs

where Jv (the measured net H2O flux) is expressed in cm3 s1 and DCs is the difference in solute concentration (mol cm3). For this approach, a two-compartment arrangement could also be used, along with a graduated capillary tube, or appropriate detection system, in order to directly measure Jv as a function of time. Unidirectional fluxes of water determined with 3 H2O (method 1) are usually large and similar in both directions. Thus, a small difference (the net volumetric flow, or Jv, which is detected directly with method 2) is difficult to detect by method 1 and is usually not calculated as a difference between two unidirectional fluxes. For example, in the case of the conjunctival epithelium, unidirectional water fluxes (Jdw) across the tissue are statistically identical in either direction, and have a magnitude 60-fold larger than the reported values for the net flux

(Jv) of fluid secreted to the tear side by the isolated conjunctiva (4–6 ml h1 cm2, using method 2 (a volumetric approach). Because of this discrepancy in magnitude, it is unfeasible (if not impossible) to calculate Jv as the difference between the two, relatively large, unidirectional fluxes in the opposite directions. However, method 1 (a diffusional approach) is useful for determining the effects of agents or various experimental conditions on water permeability (Pdw); because although labeled water will cross cell membranes via all available pathways – lipid bilayer, aquaporins, and other channels, the measurements of Jdw, which reflect Pdw, change equally in both directions when an experimental maneuver changes the water permeability of the epithelium. From diffusional water fluxes (Jdw) and mannitol fluxes it was determined that the conjunctival apical surface is highly permeable to water, and that the transepithelial water permeability (104 cm s1) exceeded the paracellular permeability (106 cm s1). A recently described element contributing to the water permeability of the apical surface is the water channel homolog known as aquaporin type 5 (AQP5). Generally, epithelia exhibit distinct AQPs in the apical and basolateral domains, and in the case of the conjunctiva, AQP3 is expressed in the lateral membranes. Together, AQP5 and AQP3 may be necessary in the conjunctiva for transepithelial fluid transport. AQP5 could serve as a potential target for pharmacological upregulation to enhance fluid secretion given that cAMP via PKA activity has been reported to increase the expression levels of this water channel at both transcriptional and posttranscriptional levels in other cell systems. From measurements of Jv, a spontaneous fluid transport across the isolated conjunctival epithelium in the basolateral-to-apical direction has been described, a property consistent with the more dominant Cl secretory activity of the tissue. As noted, the reported fluid secretion rates were 4–6 ml h1 cm2. This flow was dependent upon transepithelial electrolyte transport given its abolition by ouabain, sensitivity to K+ channel blockade, and Cl dependency. In addition, in experiments that increased the Na+ absorptive activity by raising the glucose concentration (to 25 mM) of the apical bath, fluid transport was inhibited by 77%; an inhibition that did not occur with a similar concentration of mannitol. Studies found that the fluid transport rate was increased (50–100 %) by Cl secretogogues that included purinergic agonists acting via P2Y2 receptors. As purinergic agonists stimulate mucin secretion by conjunctival goblet cells, it seems plausible that the roles of epithelial Cl transport include the hydration of mucins upon release. Overall, the conjunctival epithelium has sufficient water permeability and the transporters necessary to contribute significant fluid to the tear film (50 ml h1 based upon its total surface area). This level of fluid flow is sufficiently large that it may represent a baseline tear


secretion beyond that contributed by the lacrimal gland, which mediates reflex tearing under neuronal control. It is not yet clear if the innervation of the conjunctiva directly regulates the rate of fluid transported across the conjunctiva in vivo. However, the transport systems of the conjunctiva can potentially be manipulated pharmacologically. See also: Antigen-Presenting Cells in the Eye and Ocular Surface; Cornea Overview; Corneal Angiogenesis; Imaging of the Cornea; Stem Cells of the Ocular Surface.

Further Reading Anderson, J. M. (2001). Molecular structure of tight junctions and their role in epithelial transport. News in Physiological Sciences 16: 126–130. Bron, A., Tripathi, R., and Tripathi, B. (eds.) (1997). Wolff’s Anatomy of the Eye and Orbit, 8th edn. London: Chapman and Hall. Candia, O. A. (2004). Electrolyte and fluid transport across corneal, conjunctival and lens epithelia. Experimental Eye Research 78: 527–535.

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Dartt, D. A. (2004). Control of mucin production by ocular surface epithelial cells. Experimental Eye Research 78: 173–185. Gipson, I. K. and Arguëso, P. (2003). Role of mucins in the function of the corneal and conjunctival epithelia. International Review of Cytology 231: 1–49. Hosoya, K., Lee, V. H., and Kim, K. J. (2005). Roles of the conjunctiva in ocular drug delivery: A review of conjunctival transport mechanisms and their regulation. European Journal of Pharmaceutics and Biopharmaceutics 60: 227–240. Kaufman, P. L. and Alm, A. (eds.) (2003). Adler’s Physiology of the Eye, Clinical Application, 10th edn. St. Louis, MS: Mosby. Li, H., Sheppard, D. N., and Hug, M. J. (2004). Transepithelial electrical measurements with the Ussing chamber. Journal of Cystic Fibrosis 3(supplement 2): 123–126. Nichols, K. K., Yerxa, B., and Kellerman, D. J. (2004). Diquafosol tetrasodium: A novel dry eye therapy. Expert Opinion on Investigational Drugs 13: 47–54. Oen, H., Cheng, P., Turner, H. C., Alvarez, L. J., and Candia, O. A. (2006). Identification and localization of aquaporin 5 in the mammalian conjunctival epithelium. Experimental Eye Research 83: 995–998. Tiffany, J. M. (2008). The normal tear film. Developments in Ophthalmology 41: 1–20. Ussing, H. H. (1949). Transport of ions across cellular membranes. Physiological Reviews 29: 127–155. Wei, Z. G., Sun, T. T., and Lavker, R. M. (1996). Rabbit conjunctival and corneal epithelial cells belong to two separate lineages. Investigative Ophthalmology and Visual Science 37: 523–533.

Conjunctival Goblet Cells R R Hodges and D A Dartt, Schepens Eye Research Institute, Boston, MA, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Dry eye – A multifactorial disorder of the tear film characterized by either decreased tear production or increased tear evaporation. Glycosylation – The process by which a carbohydrate is added to protein. Goblet cells – Specialized epithelial cells that secrete mucins. Mucins – Large, highly glycosylated proteins. Signal transduction – The process by which a cell converts a signal or stimulus from outside the cell into a functional change.

Goblet cells are columnar epithelial cells that synthesize and secrete mucins, for example, the gel-forming mucin MUC5AC. These cells were originally termed goblet cells because of their distinctive goblet-like shape (Figure 1). The basal portion is narrow and shaped like the stem of a goblet containing the nucleus and organelles, while the apical portion of the cell is shaped like a cup due to the presence of numerous secretory granules. Goblet cells are found in all wet-surfaced epithelia such as the respiratory, gastrointestinal, and reproductive tracts, and the conjunctiva and are surrounded by stratified squamous epithelial cells. Goblet cells of the respiratory, gastrointestinal, and reproductive tracts have been extensively studied with regard to secretion and proliferation in healthy and diseased states. Conjunctival goblet cells have not been studied as extensively, but much information is available. The purpose of this article is to examine the current knowledge of conjunctival goblet cells.

Goblet Cell Development In humans, conjunctival goblet cells begin to appear in the eighth to ninth week of gestation in the region of the lid margin. By the 11th to 12th week, mature goblet cells can be seen containing secretory granules in the palpebral conjunctiva. Goblet cells appear in the bulbar conjunctiva around the 20th week. In the developing chick, goblet cells appear 2 days after hatching in the fornix and 3 days after hatching in the palpebral and bulbar conjunctivae. In rats, messenger ribonucleic acid (mRNA) expression for the conjunctival goblet cell-specific mucin, MUC5AC, first appears 1 day after birth, before eyelid

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opening. This expression was detected in cells in the fornix region. By day 13 (before eyelid opening), a few round-shaped cells in the fornix expressed cytokeratin 7 (a cytokeratin expressed in goblet cells of adult rats) and were positive for alcian blue/periodic acid Schiff ’s (AB/PAS) reagent, a stain that that binds to sialomucins synthesized and secreted by the goblet cells. These cells also bind Ulex europaeus agglutinin I (UEA-I), which is a lectin that binds specific glycoprotein moieties present in adult goblet cells of the rat. In humans, the lectin Helix pomatia agglutinin (HPA) specifically binds to the goblet cell secretory products. On day 17, single and small clusters of these cells were seen distributed in the fornix and palpebral regions. Goblet cell clusters were seen by day 60. In addition to the appearance of the secretory product, by day 17, immunofluorescence microscopy showed nerves surrounding the basolateral portion of goblet cell clusters and the muscarinic receptor subtypes M2 and M3 and b1- and b2-adrenergic receptors were located on the goblet cells. The presence of nerves and neurotransmitter receptors on goblet cells around the time of eyelid opening (12–15 days after birth) implies that mucin secretion is regulated as the eyes open (Figure 2). There is considerable evidence that stem cells of the conjunctival epithelium, including goblet cells, are distinct from the stem cells of the cornea. This is despite the fact that conjunctival cells can, in the case of a corneal wound involving the limbus, rapidly migrate over the wound and eventually form an epithelium similar to the normal cornea that does not contain goblet cells. However, when bulbar, fornical, and palphebral conjunctivae were grown separately, each with 3T3 feeder cells, they did not express the cornea-specific cytokeratin pair of K3/K12. In addition, when cultured conjunctival cells were injected into athymic mice, the epithelial cyst formed contained epithelial and goblet cells. Given these data, it is thought that conjunctival stem cells are different from stem cells of the cornea. It is well established that corneal stem cells reside in the limbus, the area between the cornea and bulbar conjunctiva. The location of conjunctival stem cells is not as clear. In the mouse and rabbit, it is believed that the stem cells reside in the fornix based on the fact that slowcycling cells are clustered in the fornix, and these cells do not incorporate tritiated thymidine or bromodeoxyuradine that label dividing cells. In addition, cells in the fornix have the highest rate of proliferation in vitro. Cells grown from the bulbar conjunctiva and fornix had the same proliferative capacity as cells grown from

Conjunctival Goblet Cells

Figure 1 An electron micrograph of rat conjunctival goblet cells. Numerous secretory vesicles can be seen in the apical portion of the cells, while nuclei can be seen in the apical portion. Magnification 6000. Reprinted from Dartt, D. A. Regulation of mucin and fluid secretion by conjunctival epithelial cells. Progress in Retinal and Eye Research 21: 555–576. With kind permission of Elsevier.

Figure 2 Co-localization of the M3 muscarinic receptor with goblet cells from the developing rat conjunctiva. Sections from a 17-day-old rat conjunctiva were incubated with an antibody against M3 muscarinic receptor (shown in green) and the lectin UEA-I conjugated to rhodamine (shown in red) which is specific for rat goblet cells. Goblet cells were visualized with differential interference contrast microscopy. Arrows indicate the presence of M3 muscarinic receptor subjacent to the secretory vesicles (shown in red). Magnification 200.

the corneal limbus. Cells from all areas of the fornix and bulbar conjunctiva were capable of undergoing 80–100 cell divisions before reaching senescence, similar to limbal cells. Interestingly, goblet cells were present during the entire life span of the cultures of fornix and bulbar conjunctivae and the number of goblet cells increased during cultivation of the cell cultures. As goblet cells were present during the entire culture time, it seems likely that conjunctival stem cells are bipotent, that is, capable of differentiating into either goblet or stratified squamous cells. It is not known what causes the stem cell to differentiate

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into either cell type. Possible explanations include genetic programming as it was demonstrated that conjunctival cells with high proliferative capacity differentiate into goblet cells at specific times in their cell cycle of duplication. The first goblet cells appear after a cell has achieved 45–50 doublings and, subsequently, another 10–20 doublings before senescence occurs. It is also possible that environmental effectors such as cytokines or growth factors play a role in the differentiation of a goblet cell. The number of goblet cells in the adult conjunctiva varies depending upon the location in the conjunctiva and species. For example, in the rabbit: (1) the tarsal conjunctiva contains the highest number of goblet cells while the bulbar conjunctiva contains the least, (2) the goblet cells of the tarsal conjunctiva were larger than those present in the bulbar conjunctiva, and (3) conjunctival goblet cells appeared as single cells interspersed throughout the stratified squamous epithelial cells. In contrast, in the rat: (1) the fornix contained the most goblet cells while very few were seen in the bulbar and limbal conjunctivae and (2) goblet cells appear as clusters with as many as 10 goblet cells present in some clusters in the fornix (Figure 3). Goblet cells in the human conjunctiva tend to appear as single cells (Figure 4), similar to the rabbit, though in certain areas, the density of goblet cells is high enough such that they appear to be clustered. The highest goblet cell density in human conjunctiva is in the inferior palpebral conjunctiva. It has been proposed that clusters of goblet cells, such as those that occur in rats, arise from the fact that goblet cells divide several times before senescence giving rise to the clusters. In contrast, in species such as rabbit and human, where goblet cells occur as single cells in the conjunctiva, differentiation into goblet cells occurs after the cell has undergone terminal differentiation.

Function of Conjunctival Goblet Cells The tear film is a thin layer of fluid that covers the ocular surface. Tears are secreted in response to decreased humidity, bright light, mechanical stimulation, bacterial and viral pathogens, and other environmental factors. The tear film is a stratified fluid layer consisting of three layers: (1) the outermost, which is a lipid layer secreted by meibomian glands of the upper and lower eyelids and is thought to be a barrier to evaporation; (2) the middle, which is an aqueous layer secreted essentially by the main and accessory lacrimal glands and contains water, electrolytes, and proteins such as growth factors and antibacterial proteins necessary for the health of the ocular surface; and (3) the innermost, which is a mucin layer, containing mucins that are not only secreted mainly by the conjunctival goblet cells, but also the stratified squamous cells of the cornea and conjunctiva. The mucin layer moves freely over the ocular surface toward the nasolacrimal

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Figure 3 Goblet cell clusters in rat conjunctiva. Rat conjunctival sections were incubated with the EGF receptor (shown in red) the lectin UEA-I conjugated to fluorescein isothiocyanate (FITC; shown in green) which is specific for rat goblet cells (left). Flat mounts of rat conjunctiva were prepared and stained with AB/PAS (right). Goblet cells appear as pink-to-purple cells. These micrographs indicate the clusters of goblet cells in rat conjunctiva. Magnification 200.

Figure 4 Goblet cell in human conjunctiva. Human conjunctival sections were incubated with an antibody specific to cytokeratin 7 (shown in red) which is specific for goblet cells (left). Impression cytology samples of human conjunctiva were prepared and stained with AB/PAS (right). Arrows indicate goblet cells. These micrographs indicate that goblet cells occur as single cells. Magnification 200.

duct with the blink providing the mechanism of movement. As it moves, it traps debris and pathogens. The function of conjunctival goblet cells is to synthesize and secrete mucins onto the ocular surface. Mucins are large, highly glycosylated proteins containing tandem repeats of amino acids that are rich in serine and threonine. Owing to the large amount of glycosylation, mucins are highly negatively charged molecules that are believed to be a barrier to pathogens. The glycosylation moieties are quickly hydrated upon exiting the cell, causing them to swell. Thus, mucins provide lubrication, water retention, and a barrier to infectious agents. Members of the mucin family can be subdivided based on whether they are secreted from the cell (secretory) or remain associated with the plasma membrane (membrane associated). While other members of the mucin family, namely MUC1, MUC2, MUC4, and MUC16, are present in the conjunctiva, only the gel-forming, secretory mucin MUC5AC has been identified in conjunctival goblet cells.

MUC5AC is a large gel-forming mucin that is closely related to the other gel-forming mucins MUC2, MUC5B, and MUC6. It contains four cysteine-rich domains (D domains), a tandom repeat region that can be duplicated 17–124 times, as well as a cysteine knot along with an additional cysteine region. The D domains, based on their large number of cysteines, form disulfide bridges with other MUC5AC molecules to form a large gel-like association of mucin molecules (Figure 5).

Control of Goblet Cell Proliferation and Mucin Secretion It is vital for the mucin in the tear film be of sufficient quantity and quality. The quantity of mucin depends on the: (1) number of goblet cells present (proliferation or differentation), (2) amount of mucin synthesized and


stored in secretory granules, (3) rate of mucin secretion, and (4) rate of mucin degradation. There are no studies on the rate of mucin synthesis or mucin degradation.

and HB-EGF increased goblet cell proliferation through activation of the EGF receptor. There are four types of EGF receptors, namely ErbB1 (the EGF receptor), ErbB2 (HER), ErbB3, and ErbB4. Each receptor binds to specific members of the EGF family. Activated EGF receptors form homo- or heterodimers and then recruit adaptor molecules including Shc/Grb2, phosphoinositide-3 kinase (PI-3K), phospholipase C (PLC)g, p38 mitogen-activated protein kinase (MAPK), and c-jun NH (2)-terminal kinase ( JNK). Each of these kinases initiates a cascade of kinases leading to a cellular response. The downstream kinase activated by Shc/Grb2 is the extracellular-related kinase 1/2 (ERK1/2); the downstream kinase activated by PI-3K is AKT; the downstream kinase activated by PLCg is protein kinase C (PKC); and the downstream kinase activated by JNK is c-jun (Figure 6). Further investigation of the role of ERK in EGFstimulated conjunctival goblet cell proliferation demonstrated that EGF increased the number of cells expressing ERK in their nucleus in a biphasic manner. Under basal conditions, ERK is present in the cytosol. Upon stimulation, ERK translocates to the nucleus where it phosphorylates proteins necessary for proliferation. The first, major peak occurs 1 min after the addition of EGF to cultured rat goblet cells. The number of goblet cells with ERK present in the nucleus returned to basal levels before increasing again approximately 18 h after the addition of EGF. The second peak corresponded with the appearance

Goblet Cell Proliferation With the development of a method to culture conjunctival goblet cells, studies of goblet cell proliferation are now underway. One important stimulus of goblet cell proliferation is the epidermal growth factor (EGF) family as measured by both a cell proliferation assay kit and by immunoflouresence microscopy using the an antibody against Ki-67, a protein known to be present in cells that have entered the cell cycle. EGF, transforming growth factor a (TGFa), heparin-binding EGF (HB-EGF), and heregulin are present in rat conjunctiva as well as cultured rat goblet cells as determined by reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analysis, and immunofluorescence microscopy. EGF, TGFa,

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Figure 6 A schematic representation of the EGF pathway leading to goblet cell proliferation. EGF receptor dimerizes upon EGF binding, recruiting the adaptor molecules leading to cell proliferation. PI-3K, phosphoinositide-3 kinase; PLCg, phospholipase Cg; JNK, c-Jun NH (2)-terminal kinase; ERK1/2, extracellular-related kinase 1/2; PKC, protein kinase C; PIP2, phosphatidylinositol bisphosphate; IP3, inositol trisphosphate; DAG, diacylglycerol; PKC, protein kinase C; MEK, mitogen-activated kinase kinase; MEKK-1, mitogenactivated kinase kinase-1; MKK-7, mitogen activated kinase kinase-7.

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of Ki-67, indicating that goblet cells were starting to proliferate. The ERK inhibitor U0126, added 20 min prior to EGF, inhibited both peaks of the translocation of ERK as well as goblet cell proliferation. Interestingly, inhibition of the second peak of ERK translocation with U0126 also prevented EGF-stimulated proliferation. In addition to the activation of ERK, EGF also activates PKC isoforms -a and -e to, in turn, stimulate proliferation. Most proliferation studies to date have been performed on cultured goblet cells from rat. Several studies have examined the proliferation of human goblet cells in comparison to rat cells, and demonstrated that human goblet cells respond similarly to EGF. For example, EGF stimulates human goblet cell proliferation in a similar time- and concentration dependency. In both cell types, EGF activates ERK and PKC to stimulate proliferation. Due to the difficulty of obtaining a human conjunctiva and culturing human goblet cells, rat goblet cells are an excellent model for studying human goblet cell proliferation. Goblet Cell Mucin Secretion Goblet cell secretion occurs through an apocrine mechanism. In this mechanism, most or all the secretory vesicles fuse with one another upon stimulation and subsequently with the apical membrane releasing the mucin into the extracellular space. Therefore, the amount of mucin released is dependent upon the number of cells responding to a given stimulus. A strong stimulus would involve responses from more goblet cells than a weak stimulus. Taking advantage of this property, conjunctival samples can be treated with histochemical stains that recognize mucins in goblet cells, and the number of goblet cells can be counted. A decrease in the number of goblet cells would indicate an increase in secretion. Another method to measure conjunctival goblet cell secretion is based on the fact that the lectins UEA-I and HPA bind to specific carbohydrate residues found in MUC5AC, depending on the species. The amount of secreted mucin can be determined by an enzyme-linked lectin assay (ELLA) and Western blot or dot-blot analyses. Using the histochemical method of staining for mucins in a conjunctival button, it was demonstrated, for the first time, that goblet cell mucin secretion is neurally mediated, as a wound to the central cornea induced goblet cell mucin secretion. Sensory nerves in the cornea were activated by the wound causing a neural reflex arc in which the parasympathetic nerves that surround the goblet cells released their neurotransmitters to stimulate mucin secretion. These results were confirmed when parasympathetic nerves containing the neuropeptide vasoactive intestinal peptide (VIP) were demonstrated to be present in the conjunctiva surrounding the goblet cells subjacent to the secretory granules. Addition of exogenous VIP or

the cholinergic agonist carbachol (an analog of the parasympathetic neurotransmitter acetylcholine) stimulated goblet cell secretion. Other compounds which have been shown to stimulate conjunctival mucin secretion include activators of the purninergic receptor subtype P2Y2, such as ATP, UTP, and INS365, the neurotrophins nerve growth factor, and bone-derived neurotrophic factor, and the drug OPC-12759, an antigastric ulcer drug. Interestingly, no sympathetic neurotransmitter has been shown to stimulate goblet cell secretion despite the presence of the receptors for these neurotransmitters on goblet cells. The signal transduction pathways utilized by cholinergic agonists have been well studied. Cholinergic agonists bind to M2 and M3 muscarinic receptors which are present on conjunctival goblet cells. Classically, these receptors activate PLC, which hydrolyzes phosphatidylinositolbisphosphate into 1,4,5-inositol trisphosphate (IP3) and diacylglycerol (DAG) (Figure 7). IP3 induces the release of Ca2þ from the endoplasmic reticulum into the cytosol. It is not known if this occurs in goblet cells; however, what is known is that an increase in intracellular Ca2þ alone is sufficient to cause mucin secretion from conjunctival goblet cells. Calcium can also activate Ca2þ-/calmodulindependent protein kinase. However, inhibitors of Ca2þ/ calmodulin protein kinase do not have any effect on cholinergic agonist-stimulated mucin secretion. DAG is a phospholipid activator that along with Ca2þ activates PKC. Though a direct role for PKC involvement in conjunctival goblet cell mucin secretion has not been demonstrated, activators of PKC (phorbol esters) do stimulate secretion. In addition to the Ca2þ pathway, cholinergic agonists in goblet cells activate a second pathway leading to protein secretion. This pathway involves the transactivation of the EGF receptor through the stimulation of the focal adhesion kinase Pyk2. Pyk2, in turn, activates the nonreceptor tyrosine kinase p60Src. Pyk2 and p60Src are activated in goblet cells by Ca2þ and PKC as the addition of either a calcium ionophore or a phorbol ester increases phosphorylation (activation) of Pyk2 and p60Src and the use of PKC inhibitors inhibits cholinergic agonist-stimulated Pyk2 and p60Src phosphorylation (Figure 7). This again implies that PKC plays a vital role in conjunctival goblet cell secretion. The Pyk2/p60Src complex then transactivates the EGF receptor, recruiting the adaptor molecules Shc, Grb2, and the Ras guanine nucleotide exchange factor Sos. Sos binds to and activates the low molecular weight guanosine triphosphate (GTP)ase, Ras, causing the exchange of guanosine diphosphate (GDP) for GTP to activate Ras. Ras initiates another kinase cascade of Raf (also known as MAPK kinase kinase), mitogen-activated kinase kinase (MEK) (MAPK kinase), and ERK 1/2 (p44/p42 MAPK). Upon stimulation by cholinergic agonists, ERK activates proteins in the cytosol, leading to secretion. Inhibition of ERK inhibits cholinergic agonist-stimulated mucin secretion (Figure 7).


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Figure 7 A schematic representation of the cholinergic pathway leading to goblet cell mucin secretion. Muscarinic receptors activate phospholipase C (PLC) to generate the production of inositol trisphosphate (IP3), which releases intracellular Ca2þ and diacylglycerol (DAG), which activates protein kinase C (PKC). The EGF receptor (EGFR) is transactivated leading to the activation of Ras, Raf, mitogen-activated kinase kinase (MEK), and ERK 1/2.

In contrast, when ERK is activated by exogenous EGF, ERK 1/2 translocates to the nucleus where it stimulates proliferation. The signaling pathways of the other compounds that stimulate mucin secretion are not well studied. Receptors for VIP (VPAC1 and VPAC2) are present on goblet cells both in vivo and in vitro. It is not known if exogenous VIP (which causes mucin secretion) acts through these receptors. As it is well established in other tissues that VIP increases the intracellular cyclic adenosine monophosphate (cAMP) concentration as well as the intracellular [Ca2þ], it is likely that a similar mechanism occurs in goblet cells. It is not know if the EGF family stimulates mucin secretion. As these growth factors activate ERK, which plays an important role in both secretion and proliferation, it is likely that they do. It is interesting that many of the same proteins and kinases are used by EGF to stimulate proliferation and by cholinergic agonists to stimulate mucin secretion. ERK1/2 is known to phosphorylate over 100 proteins. In order to confer specificity, ERK1/2, as is the case for many signaling molecules, is organized through the use of scaffold proteins. These proteins bind to two or more components of a signaling pathway in close proximity to assist with their interactions. Scaffolding proteins also target signaling molecules to particular areas of the cell in order to phosphorylate specific substrates. They can prevent cross talk between pathways. As the scaffolding proteins are also regulated, their stability can affect the duration of the signal. Several different scaffolding proteins have been identified in mammalian cells including kinase suppressor of Ras, MEK partner-1,

Morg1, IQGAP1, and b-arrestins 1 and 2. Not surprisingly, scaffolding proteins are required for the translocation of ERK1/2 to the nucleus and other proteins are responsible for maintaining ERK1/2 in the cytosol. In fact, ERK1/2 translocation to the nucleus requires phosphorylation by MEK, which causes conformational changes in ERK1/2, allowing for the formation of ERK dimers. This dimerization facilitates the translocation of phosphorylated ERK1/2 into the nucleus. In contrast, PEA-15, a small phosphoprotein, has been shown to anchor ERK1/2 in the cytosol.

Clinical Implications of Mucin Deficiency on the Ocular Surface The presence of goblet cells in the conjunctiva is of great importance to the health of the ocular surface. Dry eye, which is characterized by a deficient tear film or excessive evaporation, is a multifactorial disorder that causes damage to the ocular surface. One characteristic of dry eye is a decrease in the number of mucin-containing goblet cells. All in vivo studies to date identify goblet cells by their secretory product, either by staining with AB/PAS or by the presence of MUC5AC within the cell, and should be termed filled goblet cells. Thus, a decrease in the number of filled goblet cells in the conjunctival tissue indicates that goblet cells have secreted. Under chronic conditions, the decrease in filled goblet cells could indicate repeated stimulation such that mucin synthesis is unable to keep pace with secretion or indicate a loss of goblet cells through either a decrease in goblet cell proliferation,

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dedifferentiation, or an increase in goblet cell death. It is difficult to know whether the loss of goblet cells is a cause of the disease or a result of it. Indeed, the mechanism by which goblet cells are lost from the conjunctiva has not been studied. In animal models of dry eye, proteins involved in apoptosis have been shown to be upregulated in conjunctival epithelial cells, though the effects on the goblet cells themselves were not clear. The loss of goblet cells in dry eye patients warrants further examination. The presence or absence of conjunctival goblet cells has been examined in different types of dry eye. These have been described below.

Sjo¨gren Syndrome Sjo¨gren syndrome is an autoimmune disease characterized by profound lymphocytic infiltration of the lacrimal and salivary glands, resulting in dry eye and dry mouth. It is not known whether the changes seen in the conjunctiva are a result of the autoimmune disease itself or as a result of the change in tears due to lacrimal gland destruction. Though the changes in the conjunctiva are relatively mild compared to the destruction of the lacrimal glands, there is a decrease in the number of mucin-containing goblet cells in patients with Sjo¨gren syndrome, as determined by histochemical staining methods. In addition, the amount of MUC5AC mRNA is decreased in patients with Sjo¨gren syndrome and the amount of MUC5AC detected in tears of these patients is also decreased.

Vitamin A Deficiency Vitamin A is vital for the health of the ocular surface as it is essential for the development of goblet cells in the conjunctiva. Vitamin A deficiency has been shown to cause keratinization and squamous metaplasia of the conjunctiva. The lack of goblet cells in the conjunctiva in vitamin-A-deficient patients has been documented through the use of impression cytology and histochemical staining methods. It has also been demonstrated that in vitamin-A-deficient rats, not only were there no mucincontaining goblet cells, but the mRNA for MUC5AC and MUC5AC protein also disappeared 20 weeks after the initiation of a vitamin-A-deficient diet. Reintroduction of vitamin A into the diet has been shown to increase the number of conjunctival mucin-containing goblet cells.

Topical Preservatives Preservatives, such as benzalkonium chloride, are often found in artificial tears and medications such as those used to treat glaucoma and anti-inflammatory medications. These patients often report the signs and symptoms of dry

eye. Benzalkonium chloride has been shown to decrease the number of mucin-containing goblet cells present in the conjunctiva. Ocular Cicatrical Pemphigoid Mucous membrane pemphigoid is an autoimmune disease that is characterized by blisters in the mucous membranes of the body including the mouth, nose, trachea, and conjunctiva. When the conjunctiva is involved, it is known as ocular cicatrical pemphigoid (OCP). This condition causes chronic conjunctivitis and, eventually, complete keratinization of the conjunctiva, resulting in severe dry eye. It has been observed that the mucin-containing goblet cell number is reduced in the later stages of the disease, likely as a result of the keratinization. Interestingly, it has been shown that the expression of a specific glycosyltransferase present exclusively in goblet cells in normal human conjunctiva is altered in late stages of OCP. This enzyme, which is responsible for the glycosylation of mucins, is expressed in stratified squamous cells in areas of the conjunctiva that were nonkeritinized in OCP patients, and, as the disease progressed, the expression disappeared. Laser-assisted In Situ Keratomileusis Patients undergoing laser-assisted in situ keratomileusis (LASIK) often experience dry eye symptoms. These symptoms are usually temporary, but can develop into chronic dry eye. It is known that the number of mucincontaining goblet cells decreases significantly within 1 week postsurgery, but returns to preoperative levels by 3 months after surgery. Diseases and disorders of goblet cells are a result not only of mucous underproduction, but also of mucous overproduction. Ocular Allergies The symptoms of allergic conjunctivitis include mucus production, ocular itching, foreign-body sensation, tearing, hyperemia, chemosis, and lid edema. Traditionally, allergic eye disease has been classified into: seasonal allergic conjunctivitis (SAC), perennial allergic conjunctivitis (PAC), vernal keratoconjunctivitis (VKC), atopic keratoconjunctivitis (AKC), and contact-lens-induced papillary conjunctivitis (CLPC). Each of the five categories of allergic conjunctivitis has a distinct pathology. In SAC and PAC, conjunctival inflammation is mild and of short duration. In VKC and AKC, conjunctival inflammation has an unclear history of exposure to allergen, is more severe, and lasts longer than SAC and PAC. A common symptom of allergic conjunctivitis in the human is alteration in mucus production. One hypothesis as to how mucous production is altered in allergic conjunctivitis is that activation of sensory nerves in the cornea


and conjunctiva, manifested by itchiness, foreign-body sensation, and increased tearing, could occur. This, in turn, could activate the efferent parasympathetic and sympathetic nerves that surround conjunctival goblet cells to release the neurotransmitters acetylcholine and VIP, which are known to stimulate conjunctival goblet cell secretion. When goblet cells secrete, all secretory granules are released at once. Under these chronic conditions, the decrease in filled goblet cells could indicate repeated stimulation with mucin synthesis unable to keep pace with secretion. In support of this, a decrease in filled conjunctival goblet cells in VKC and AKC patients, respectively, compared to control subjects, has been noted. In addition, the amount of MUC5AC RNA in the conjunctiva was decreased in AKC and VKC patients versus control. The excess mucus seen on the conjunctiva in individuals with VKC is a stringy mucus and does not necessarily represent goblet cell MUC5AC, but could be stratified squamous cell membrane-spanning mucins, poorly hydrated mucins, or mutated mucin variations. Additional study of the role of goblet cell mucin secretion in different types of ocular allergy is warranted. Results from animal models of allergic conjunctivitis, which most closely mimics SAC and PAC, found that the number of filled goblet cells decreased for 6 h after the final allergen challenge. Over 48 h, the number of filled goblet cells returned toward control values, indicating that goblet cell mucin secretion increased during delayed hypersensitivity, but that goblet cells refilled after allergen removal. MUC5AC RNA was also depleted by the final challenge and recovered at 6 h, indicating that the delayed hypersensitivity depletes MUC5AC RNA; however, it recovers quickly to begin synthesizing MUC5AC to refill the goblet cells.

Summary Much information has been gathered regarding the role of conjunctival goblet cells in the health of the ocular surface. Goblet cell proliferation, mucin synthesis, and secretion control the amount of mucin present on the ocular surface. These processes are regulated as growth factors increase proliferation and neural stimulation causes mucin secretion. However, many questions remain. Are the three processes that regulate mucin amount coordinated so that

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one stimulus activates all three? While some of the intracellular pathways leading to proliferation or secretion are known, it is not known if these pathways are altered in diseases. The life span of a conjunctival goblet cell as well as the mechanisms by which goblet cells are lost in disorders of the tear film are unknown. It is also not known if goblet cells are able to secrete mucins multiple times, perhaps refilling secretory granules, in response to either a sustained signal or multiple stimuli. Further research into these questions may lead to the development of treatments for many of the ocular surface diseases. See also: Defense Mechanisms of Tears and Ocular Surface; Ocular Mucins; Overview of Electrolyte and Fluid Transport Across the Conjunctiva.

Further Reading Dartt, D. A. (2004). Control of mucin production by ocular surface epithelial cells. Experimental Eye Research 78: 173–185. Gipson, I. K. and Argueso, P. (2003). Role of mucins in the function of the corneal and conjunctival Epithelia. International Review of Cytology 231: 2–49. Gipson, I. K., Hori, Y., and Argueso, P. (2004). Character of ocular surface mucins and their alteration in dry eye disease. Ocular Surgery 2: 131–148. International Dry Eye Workshop (2007). The definition and classification of dry eye disease. Report of the definition and classification subcommittee of the International Dry Eye Workshop (2007). Ocular Surgery 5: 75–92. Lavker, R. and Sun, T-T. (2003). Epithelial stem cells: The eye provides a vision. Eye 17: 937–942. Pellegrini, G., Golisano, O., Paterna, P., et al. (1999). Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. Journal of Cell Biology 145: 769–782. Ramos, J. W. (2008) The regulation of extracellular signal-regulated kinase (ERK) in mammalian cells. International Journal of Biochemistry and Cell Biology, 40: 2707–2719. Rios, J. D., Forde, K., Diebold, Y., et al. (2000). Development of conjunctival goblet cells and their neuroreceptor subtype expression. Investigative Ophthalmology and Visual Science 41: 2127–2137. Sellheyer, K. and Spitznas, M. (1988). Ultrastructural observation on the development of the human conjunctival epithelium. Graefe’s Archive of Clinical and Experimental Ophthalmology 226: 489–499. Shapiro, M. S., Friend, J., and Thoft, R. A. (1981). Corneal reepithelialization from the conjunctiva. Investigative Ophthalmology and Visual Science 21: 135–142. Shatos, M. A., Rios, J. D., Tepavcevic, V., Kanno, H., Hodges, R. R., and Dartt, D. A. (2001). Isolation, characterization, and propagation of rat conjunctival goblet cells in vitro. Investigative Ophthalmology and Visual Science 42: 1455–1464.

Ocular Mucins M Berry, Bristol Eye Hospital, Bristol, UK ã 2010 Elsevier Ltd. All rights reserved.

Glossary Alternative splicing – A variation mechanism in which linear combinations of exons are translated, resulting in a variety of mature products encoded by a single gene. Atomic force microscopy (AFM) – A technique of high-resolution imaging through the measurement of forces between atoms in the sample and those on the instrument tip. Glycan – The oligosaccharide portion of a glycoconjugate. Glycocalyx – An outer, carbohydrate-rich coating on the surface of cells. Glycoforms – Variations in the amount or compositions of oligosaccharides decorating the same peptide core. Meibomian glands – The special sebaceous glands at the rim of the eyelids that supply the lipid layer of the tear film. Mucins – A family of large, heavily glycosylated molecules, with most glycan chains O-linked to the peptide core. Persistence length – A measure of polymer stiffness; it is the length over which correlations in the direction of the tangent are lost. Reptation – Movement of a long polymer parallel to itself, similar to the movement of a snake. Tandem repeats – The adjacent repetition of a pattern of two or more nucleotides. Worm-like model – A model for the behavior of semiflexible polymers, considered continuously flexible. Young’s modulus – A measure of elasticity, defined as the ratio of stress to strain.

Introduction In addition to the well-defined anatomical blind sac formed by the cornea and conjunctiva, the meibomian and lacrimal glands, the ocular surface comprises a mucosal immune system, rich neural and endocrine loops, as well as the blink reflex. As with other mucosal systems the ocular surface is further integrated into the adaptive immunity of the organism, and into the microbial richness

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of the outer environment. An additional and specific requirement of the ocular surface is the maintenance of transparency that applies to the cornea as well as to the preocular fluid. Bathing the exposed part of the outer eye is a complex fluid whose elements are secreted by the wet epithelia, and the lacrimal and meibomian glands. Mucins are the main component of a mucus gel and responsible for its viscoelastic properties. They form a dynamic matrix wetted by a plasma dialysate enriched by secretions from the lacrimal glands and topped by a layer of waxes and lipids originating in the meibomian glands. This fluid is periodically sheared and mixed by the movement of the lids during blinking. Underneath the mucous gel, the epithelial glycocalyx anchors the tear film to the ocular surface: mucins and glycoproteins are the major components of this layer.

Mucin Architecture A very rich glycosylation, with most sugar chains O-linked through N-acetylgalactosamine (GalNAc) to serine or threonine in the peptide core, is diagnostic of mucins. Sugar chains tend to be clustered in discrete regions resulting in concentrations of negative charges. In these high-charge regions the peptide core is rich in serine, threonine, and proline (PTS domains), and repeated sequences of aminoacids (variable number of tandem repeats, hence VNTR domains) are present, specific to the encoding gene. Other regions are less richly glycosylated and contain relatively more N-linked glycan chains, the latter necessary for mucin transit through intracellular microtubules during synthesis. In humans, ocular mucin regions of tens of nanometers seem to be almost naked: the molecular diameter measured in liquid with atomic force microscopy (AFM) is not significantly higher than that of aminoacids in a helix (Figure 1(a)), giving mucin polymers the appearance of strings of small beads, small beads of dense bottlebrushes. Toward the N- and C-termini of mucins, the arrangement of moduli is similar to those found in proteins involved in coagulation (von Willebrand factor, VWF domains), cysteine knots, or SEA (sea urchin sperm protein, enterokinase, and agrin) domains. These are useful in tracing the evolution of mucin genes. Genes encoding for PTS domains and multiple VWF domains (D1–D2–D3 PTS, as in secreted mucins) can be found early in the evolution of metazoa, preceding hemostasis or coagulation

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Figure 1 Architecture of ocular mucins deposited on mica (a) Portions of a hydrated human conjunctival mucin molecule imaged with AFM (A, B). The respective height profiles (C, D), highlight the short oligosaccharides of ocular mucins. All axes are in nanometres. Reproduced from McMaster, T. J. (1999). Atomic force microscopy of the submolecular architecture of hydrated ocular mucins. Biophysical Journal 77: 533–541. With permission from Biophysical Society. (b) Schematic of a mucin monomer, containing von Willebrand factor domains toward the C- and N-termini (D1, D2, D0 , D3, and D4, B, C, and CK, indicated by green labels) and central mucin domains (yellow). A more detailed schematic of PTS domains reveals unique sequences (brown-filled octagons) and tandem repeat regions (yellow octagons) interspersed with cysteins (blue). Most secreted mucins polymerize by disulfide-bonded linear concatenation of such monomers.(c) Schematic of a cell-surface-associated mucin, containing an SEA domain (sea urchin sperm protein, enterokinase, agrin) within which there is a proteolytic cleavage site, a transmembrane domain (TD), and a cytoplasmic tail (CT).

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(Figure 1(b)). SEA domain mucins appear in vertebrates, while the auto-catalytically cleaved SEA domain is restricted to mammals (Figure 1(c)). Regions where the peptide core is extended by the insertion of the first sugar (GalNAc) and repulsion between negatively charged sugar chains alternate with more flexible polypeptide chain stretches, suggesting that mucins behave in solution like stiff random coils. Mucins occupy a large volume which indicates interpenetration of molecular domains at relatively low polymer concentrations. Using the worm-like model, calculations indicate that human ocular mucins are more flexible than DNA molecules of similar length; mucin persistence length is 35 nm and that of DNA is 50 nm. Polymer conformation and stiffness, for example, of human ocular MUC5AC glycoforms, are greatly influenced by the degree and nature of post-translational glycosylation. Mucin architecture is determinant of mucin role and function at the mucosal surface and in the gel. The organ, developmental state, and physiological status, in turn, affect mucin expression and details of glycosylation. Ocular mucins have short oligosaccharide chains: in humans they are mostly less than six sugars long, negatively charged and terminated in sialic acid, with fucosylation representing less than one-fifth of the sugars (Figures 2 and 3). In dogs and rabbits, glycans are mainly neutral and terminated in fucose and/or GalNAc. The short oligosaccharides might be related to transparency and (relatively fast) turnover of mucins, and terminal sugars to environmental microbiota. Mucin Families Mucin genes appeared through a combination of moduli existing in other proteins. The close connection between

mucin structure and function gives rise to a classification necessarily reflecting both. Surface-associated mucins Some mucins spend part of their life anchored into the apical cell membrane before they are shed into the luminal, that is, tear, fluid. Formerly known as membranebound mucins, they are now called cell-surface-associated mucins. These are heterodimers, with a large mucin subunit outside the cell, a (mostly hydrophobic) membranespanning region and an intracellular tail. A number of subfamilies are represented at the ocular surface: the mammalian-specific MUC1 with its SEA domain; the MUC16 that contains multiple SEA domains, not all of which are cleaved; and the MUC4 that has VWD but neither cysteine-rich domains nor SEA. Shedding of these mucins is thought to cause changes in the neighboring membrane domains, potentially transferring information to the cell interior. A further possibility is that information is conveyed through the cytoplasmic tail to the cytoskeleton. An important result of surface mucin release in the tear fluid is the renewal of the glycocalyx and the tear fluid itself. This group of mucins is heterogeneous and most genes also encode splice variants that are secreted: MUC4, a cell-surface mucin in normal cornea and conjunctiva, is a goblet cell mucin in some pterygia (Figure 4). MUC1/SEC, a splice variant of MUC1 that lacks the transmembrane domain and, therefore, results in a soluble, secreted form of MUC1, is present in human cornea and conjunctiva. Secreted mucins Milliseconds after the secretion of mucins stored in granules, often in specialized epithelial cells, their volume

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Increasing negative subunit charge Figure 2 Monosaccharide composition of human ocular mucins. The increase in sialic acids, mono- or di-acetylated (NeuAc, or Neu-Ac2) did not parallel the increase in negative subunit charge (A> type III) and sulfated PGs. As judged by collagen fibril diameter and density, ECM usually first reconstitutes to the most mature and most fibrotic stage in the portion of the wound just under the epithelium and then it advances progressively to deeper levels. Additionally, during the evolution of maturation, the hypercellularity and hypervascularity decreases due to apoptosis and tensile strength increases due to increased maturity acquired collagen fibril cross-linking. The mechanism of late disappearance of myofibroblasts is uncertain, but probably is due to a combination of apoptosis and dedifferentiation of cells back to quiescent fibrocytes. In humans, fibrotic scarring commonly causes major medical problems in a variety of tissues. In the eye, fibrotic scarring causes visual impairment or even blindness. In the peripheral and central nervous systems, glial scarring prevents neuronal reconnections and hence blocks

reparative attempts at restoring normal neuronal function. In gastrointestinal and reproductive organs, strictures and adhesions can give rise to serious or life-threatening conditions, such as infertility, bowel obstruction, or chronic pain. In ligaments and tendons, fibrotic scarring restricts motility and decreases strength. Additionally, if the fibrotic repair response is incomplete, becomes excessive, or fails to appropriately terminate, pathologic wound healing occurs resulting in unhealed wounds, hypertrophic scars, or keloid scars, respectively. Overexpression and release of pro-fibrotic TGF-b1 and b2 cytokines, upregulation of TGF-b receptors RI and RII, and/or the formation of a positive, autoinducible feedback loop have been suggested as the primary reasons for excessive fibrotic scarring in hypertrophic or keloid scars. The potential for TGF-b autoinduction is usually self-limited in nonpathologic fibrotic repair responses through a negative feedback loop that terminates further TGF-b expression and release from that cell. In adult humans, the degree of fibrotic scarring versus tissue regeneration or tissue fibrosis depends on the following five factors: tissue site, sex, race, age, and magnitude and contamination of the wound. For tissue site, the gums, liver, and skin can either regenerate or scar, depending on the context of the injury. Most other tissues or organs in the body just repair themselves through tissue fibrosis or

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fibrotic scarring, which is body site dependent. The deltoid, sternum, and conjunctiva scar more than the face, abdomen, legs, and cornea. Concerning sex, fertile females scar more than postmenopausal females and all males as estrogen has a major stimulatory effect on the fibrotic repair response. For race, darker pigmented individuals scar more than less pigmented individuals. Concerning age, young people in their teens and 20s scar more than infants and children, who have an immature immune response, and both heal better than older individuals, whose inflammatory response becomes sluggish and less effective with age and whose mesenchymal cells become senescent. Finally, the magnitude of injury and wound contamination alter the wound-healing response as larger, more inflamed, more contaminated wounds scar more than smaller, uncontaminated wounds.

Ocular Wound Healing The ocular fibrotic wound-repair response is quite similar to that seen in other tissues in the human body. In the eye, fibrotic scarring most commonly occurs in the conjunctiva, cornea, iris, lens, retina, and choroid (Figure 2). In vascularized ocular tissues, like the conjunctiva, iris, choroid, or even the cornea or retina after having chronic ocular disease, fibrovascular scars are basically granulation tissue. Scars normally develop in response to vascular stromal injury whereby serum plasma or blood leaks into

the wound resulting in a clot. This initiates a fibrovascular repair response predominantly through a platelet-derived growth factor (PDGF)-mediated platelet degranulation signaling pathway resulting in massive release of PDGF into the wound along with some TGF-b release. However, the eye also has several avascular transparent tissues (e.g., cornea, lens, vitreous, and portions of the retina) that also can repair themselves through fibrosis, albeit through four less intense TGF-b-mediated signaling pathways. TGF-b-mediated wound healing occurs because TGF-b and its associated receptors are constitutively expressed in the normal state by almost all cells residing in or on ocular tissues. The external ocular surface has other factors to additionally consider in that it is bathed in a tear film composed of lacrimal gland secretions that are rich in latent TGF-b1. Similarly, internal intraocular structures are bathed in aqueous humor composed of ciliary and lens epithelial secretions that are rich in latent TGF-b2. Latent TGF-b2 is thought to be one of the principal factors promoting the normal naive immune status of intraocular tissues, including the cornea, due to its ability to maintain resident tissue immune cells in an immature state. Secondarily, latent TGF-b2 has a direct immunosuppressive effect whereby it inhibits immune cell proliferation and inflammatory cell cytokine and GF release. At the concentration levels found in the normal aqueous humor, latent TGF-b2 is also thought to participate in the inhibition of angiogenesis into transparent parts

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Figure 2 After injury, tissue fibrosis or fibrotic scarring occurs in the cornea, conjunctiva, iris, lens, retina, or choroid. The origin of the mesenchymal repair cell types (activated fibroblasts, myofibroblasts) in each case is either from local fibrocytes (keratocytes, subconjunctival fibrocytes), local epithelial cells (lens epithelium, retinal pigment epithelium), local endothelial cells (corneal endothelium), or local smooth muscle cells that are associated with blood vessels (iris, choroid). The lens undergoes only avascular fibrotic scarring, while conjunctiva, iris, and choroid only undergo fibrovascular granulation tissue scarring; the cornea and retina can undergo both, depending on whether preexisting corneal or retinal disease is present or on the context and severity of the initial injury.

Corneal Scars

of the eye, although a primary role for latent TGF-b2 in preventing vascularization in the cornea remains controversial. In various ocular diseases or after injury, TGF-b release and subsequent extracellular activation usually initiates a fibrotic repair response. The TGF-b-mediated fibrotic repair response can lead to significant visual impairment. TGF-b can cause overcorrection, undercorrection, or regression of refractive effect leading to refractive instability. TGF-b can also cause loss of refractive function by inducing irregular astigmatism or it can cause loss of transparency by inducing corneal haze, cataract, capsular fibrosis, or vitreous opacification. Tractional distortion of ocular structures, such as proliferative vitreoretinopathy, or other ocular complications, such as wound dehiscence, epithelial ingrowth, infection, hypotony (low intraocular pressure), epiretinal membranes, or retinal detachment, can also be caused by TGF-b . Thus, this molecule is a subject of intense research. Avascular transparent tissue fibrosis or fibrotic scarring in the cornea, lens, and retina is perhaps even more intriguing to researchers since it provides a unique opportunity to directly examine the cell biology during wound healing caused by the four TGF-b-mediated signaling pathways in isolation from the PDGF-mediated platelet degranulation signaling pathway.

Corneal Wound Healing Although corneal neovascularization resulting from chronic ocular disease, such as corneal infection or chemical injury, may lead to a fibrovascular repair response (Figure 3, far-right), this type of repair response will not be focused on in this article. Instead, this article will strictly focus on the avascular corneal fibrotic repair response (Figure 3, middle). In the latter repair response, all cases have in common early inflammation that is minimally present up to 2 weeks after injury and maximally

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present up to 1 month after injury, at least some transient myofibroblast differentiation that is minimally present up to 4–6 months after injury and maximally present longterm, and the long-term presence of disorganized collagenous ECM (Figure 4).

Cell–Cell Communication Cytokines and GFs Corneal wound healing is an exceedingly complex process mediated by autocrine (affecting the same cell), juxtacrine (affecting adjacent similar cell types), or, most commonly, paracrine (affecting other adjacent cell types) cell–cell interactions involving cytokines and GFs produced by epithelial, stromal keratocyte, endothelial, immune, lacrimal gland, and corneal nerve cells (Figure 4). The paracrine interactions usually affect cells at most 50–75 mm from one another, while the other modes of interactions operate over shorter distances. The earliest event involves cell-mediated release of several cytokines and GFs by directly injured resident tissue cells or secretion by resident tissue cells just adjacent to the injury site and their subsequent extracellular activation. In the normal, uninjured state, the participating cytokines and GFs are expressed constitutively and stored intracellularly by the corneal epithelium, corneal nerves, keratoctyes, and corneal endothelium. These cytokines play a vital role in the maintenance of normal corneal health, structure, and function as some are secreted in a limited controlled fashion, mostly in the latent or inactive form. The adjacent tear film produced by the lacrimal gland and other ocular surface epithelia and the aqueous humor produced by ciliary and lens epithelial cells’ secretions are another rich source of cytokines and GFs in the normal, uninjured state since these fluids supply various nutrients to the avascular cornea that otherwise would not be available. After wounding activates this direct resident tissue injury-induced

Neutrophil Macrophage

Myofibroblast

Myofibroblast Activated keratocyte Macrophage

Activated keratocyte Keratocyte

Neovascularization Keratocyte

Platelets Keratocyte

Figure 3 Schematic diagrams demonstrating the histology of the cornea during the active wound-healing phase, approximately 1 week after injury when re-epithelization has just been completed and epithelial thickness restored, in normal uninjured cornea (far-left), avascular fibrotic scarring (middle), and fibrovascular granulation tissue scarring (far-right). If these wounds eventually reach the conclusion of the remodeling phase (not shown), the middle illustration would end up with corneal tissue fibrosis or fibrotic scarring, whereas the far-right illustration would end up with fibrovascular corneal scarring.

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Corneal tissue injury Epithelial

Stromal

Endothelial

Direct injury-induced cytokine and GF release or secretion: TGF-β>>PDGF

Epithelial cell migration until re-epithelization

Endothelial cell migration Keratocyte apoptosis Persistent corneal edema or stromal in-growth

Persistent epithelial defects or epithelial cell ingrowth

Proliferation resumes and epithelial thickness returns to normal

Compensatory basal epithelial cell size changes and/or changes in the number of cell layers in the epithelium

Endothelial cell junctions re-established and pump function resumes Inflammation

Attachment complexes Keratocyte proliferation (i.e., hemidesmosomes, and migration anchoring fibrils, and anchoring plaques) re-synthesized

Recurrent corneal erosions

Transient differentiation into activated keratocytes Inadequate cell-cell TGF-β signaling ± ECM suppressive factors

Hypercellularity of wound with transient myofibroblastic differentiation Provisional matrix synthesis and deposition Prolonged or excessive cell-cell TGF-β signaling Adequate cell-cell TGF-β signaling ± ECM amplifying factors

Early provisional matrix synthesis and deposition and later replacement by prolonged or excessive collagen and sulfated PG synthesis and deposition as well as chronoic myofibroblastic differentiation

Chronic mechanical irritation to keratocytes

Persistent keratoyte activation and extracellular lipid collection = stromal crystalline-like deposits

± ECM suppressive/amplifying factors

Early provisional matrix synthesis and deposition and later replacement by collagen and sulfated PG synthesis and deposition

Hypocellular primitive matrix

Wound remodeling Wound remodeling

Hypercellular fibrotic scar

Persistent corneal edema

Injury-induced inflammatory cell-stromal interactions: macrophages (TGF-β>PDGF)

Epithelial- or endothelial−stromal interactions Tear film- or aqueous humor−stromal interactions

Normocellular or hypercellular tissue fibrosis

Steroids MMC

Figure 4 Diagram of normal corneal wound-healing pathways in black and abnormal or pathologic healing pathways in blue. Depending on the context of the injury and the molecular signaling pathways invoked, three types of long-term corneal stromal scars are possible: hypocellular primitive matrix, normocellular-to-hypercellular tissue fibrosis, and hypercellular fibrotic scars. The most functionally optimal type is that of normocellular-to-hypercellular tissue fibrosis as it is macroscopically transparent, yet still somewhat strong in tensile strength (30%). Imbalances in the fibrotic repair response result in the other two stromal scar types. An incomplete fibrotic repair response results in a hypocellular primitive matrix scar, which is transparent, but very weak in tensile strength (2–3%). An excessive, nonpathologic fibrotic repair response results in a hypercellular fibrotic scar, which is hazy, yet the strongest in tensile strength (40%). Note that complete tissue regeneration is not possible in the in vivo adult human cornea, but if it could be manipulated to occur it would be the best option since it would be both transparent and back to normal tensile strength (100%). Topical corticosteroid drops suppress (i.e., decreases inflammation and blunts the intensity of some remaining wound-healing steps) the wound-healing pathways in both the epithelium and stroma (stroma > epithelium), while single intraoperatively applied mitomycin C (MMC) appears to block only the stromal wound-healing pathway by preventing keratocyte proliferation. Adapted from Dawson, D. G., Edelhauser, H. F., and Grossniklaus, H. E. (2005). Long-term histopathologic findings in human corneal wounds after refractive surgical procedures. American Journal of Ophthalmology 139: 168–178.

cytokine and GF cascade, supplemental exposure to additional cytokines and GFs may occur by four dynamic signaling pathways (Figure 4): epithelial– or endothelial–stromal interactions, inflammatory cell–stromal interactions, tear film– or aqueous humor–stromal interactions, and keratocyte mechanotransduction. The major pertinent cytokines and GFs studied to date in regard to corneal wound healing include connective tissue growth factor (CTGF), epithelial growth factor (EGF), fibroblast growth factor (FGF), interleukin-1

(IL-1), nerve growth factor (NGF), PDGF, TGF-a, TGF-b, tumor necrosis factor-alpha (TNF-a), and vascular endothelial growth factor (VEGF). Currently, TGF-b is thought to be the most important one of this group in regard to stimulating a fibrotic repair response. The major resident cellular source of TGF-bs in the normal, uninjured cornea is the epithelium, which primarily expresses TGF-b2, while keratocytes and endothelial cells express only a little TGF-b1. No TGF-b3 expression has been found in the adult human cornea.

Corneal Scars

Resident cell surface TGF-b receptor sites are most numerous on keratocytes, while epithelial and endothelial cells have very little. After injury, increased expression of both TGF-b1 and -b2 and their associated receptors occurs, with TGF-b2 predominating in epithelial–stromal interactions (stroma within 75 mm from the epithelium) and TGF-b1 predominating in endothelial–stromal interactions (stroma within 75 mm from the endothelium). In contrast, stromal injury more than 75 mm from either surface invokes only slightly increased TGF-b1 expression and, most importantly, receives no paracrine supplementation from epithelial– or endothelial–stromal interactions. Although the normal tear film contains high concentrations of latent TGF-b1, immediately following corneal epithelial injury, stimulation of the corneal nerve-lacrimal gland reflex arc causes hypersecretion of tears and increased secretion of several pro-fibrotic cytokines and GFs, such as PDGF-BB, NGF, and additional TGF-b1, all of which are normally stored intracellularly in lacrimal gland cells. The higher amounts of latent TGF-b1 as well as the newly secreted PDGF-BB and NGF are then activated by plasmin or MMPs, which are both released or secreted by injured or healing corneal epithelial cells into the wound. This corneal nerve-lacrimal gland reflex arc secretion and protease release usually returns to baseline levels within 7 days of epithelial defect closure; hence it is a transient event. Other cytokines or GFs from the group listed above have intriguing complementary or antagonizing effects to TGF-b’s pro-fibrotic effects. As discussed in the ocular fibrovascular repair response section, PDGF’s pro-fibrotic effects are almost identical to TGF-b’s pro-fibrotic effects. In the normal, uninjured cornea, small amounts of PDGFBB are produced by the corneal epithelium that reacts with its receptors, which are most highly concentrated in the keratocytes of the stroma and the corneal endothelium. Also, a limited amount of PDGF-BB is secreted by macrophages and lacrimal gland cells. After epithelial injury, the expression, release, and activation of PDGF-BB increases in parallel to that of TGF-b. NGF, a trophic neuropeptide important in maintaining corneal epithelial health, is another GF that is complementary to TGF-b’s profibrotic effects as NGF is known to directly stimulate myofibroblastic transformation, but without the proliferative or ECM deposition effects of TGF-b. CTGF is also complementary to TGF-b as it too stimulates tissue fibrosis. However, CTGF is only potently expressed when stimulated by TGF-b, suggesting CTCF mediates several downstream actions of TGF-b. Thus, CTGF may be more important in chronic stages of fibrotic scarring as opposed to initiation or early regulation of the fibrotic repair response. Alternatively, CTGF may just synergize with TGF-b’s pro-fibrotic effects since CTGF binds to TGF-b and potentiates TGF-b binding to TGF-b type II receptors. In contrast, TNF-a directly antagonizes TGF-b’s profibrotic effects by inhibiting the TGF-b/Smad pathway.

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This direct antifibrotic effect may seem counterintuitive since TNF-a is one of the major pro-inflammatory cytokines of the cornea. As can be appreciated, a correct combination and temporal sequencing of the various cytokines and GFs is required for proper healing to occur. Imbalances can result in incomplete wound repair, such as (1) persistent epithelial defects and corneal ulceration or melting, or (2) excessive pathological tissue fibrosis, such as stromal outgrowth, fibrodegenerative pannus formation, stromal ingrowth, or retrocorneal membrane formation. TGF-b signaling pathways Both historical clinical anecdotal observations and recent experimental investigations have suggested that epithelial–stromal interactions are the primary mediator of the avascular fibrotic repair response in the cornea. Thus, epithelial cell-derived substances substitute for those produced by platelets in the skin and other vascular tissues. The cornea consists of three cellular layers consisting of epithelium, interstitial stroma with interspersed keratocytes, and endothelium and two acellular layers consisting of an epithelial BM/Bowman’s layer complex and an endothelial Descemet’s membrane. Although Bowman’s layer is not re-formed if damaged, both BM layers are re-formed if damaged because they are produced by epithelial or endothelial cells. Both acellular layers seem to function clinically as physiological barriers to separate the three cellular layers from direct contact or communication with one another to maintain normal corneal homeostasis. Disruption of the epithelial BM/Bowman layer complex induces maximal epithelial–stromal interactions that are intense enough even in a minimal injury state to cause a fibrotic repair response. This is seen clinically with bullous keratopathy – a pathological condition in which small blisters (vesicles or bullae) are formed in the corneal epithelium due to endothelial dysfunction, at the host–graft interface after penetrating keratoplasty (aka full-thickness corneal transplantation; a surgical procedure where a damaged or diseased cornea is replaced by full-thickness donated corneal tissue), or with Bowman’s layer breaks (a degenerative disorder of the cornea in which structural changes cause it to thin and change in shape resulting an outward bulging of the tissue) as a subepithelial fibrotic scar, stromal outgrowth, or fibrodegenerative pannus formation (fibrocollagen connective tissue that proliferates in the anterior layers of the cornea in degenerative corneal disease). In some cases of bullous keratopathy, there may even be an no obvious microscopic full-thickness Bowman’s layer break, but rather just separation of the of epithelium and its associated BM from Bowman’s layer due to severe epithelial edema causing subepithelial bullae or blisters. In this extreme example of epithelial–stromal interactions, the keratocytes apparently migrate through trigeminal nerve fiber perforation sites found in Bowman’s layer to the

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subepithelial space created by the edema, where they undergo a fibrotic repair response. Separation or breaks in Descemet’s membrane provide a similar trigger for causing a fibrotic repair response in the posterior cornea through endothelial–stromal interactions. For example, with penetrating corneal injury or breaks in Descemet’s membrane, whether caused by trauma, disease (keratoconus), or surgery, endothelial–stromal interactions stimulate fibrotic scarring resulting in a supraendothelial fibrotic scar, stromal ingrowth, or retrocorneal membrane formation, depending on the degree of apposition of the broken Descemet’s membrane wound margins. Therefore, current evidence suggests that the main TGF-b pro-fibrotic signaling pathway to block or suppress to prevent or reduce the chance for fibrotic scarring is that of direct epithelial–stromal or endothelial–stromal interactions. Three other TGF-b pro-fibrotic signaling pathways are also present in the cornea and can initiate an avascular fibrotic repair response, but more often they just sustain the response already initiated by epithelial- or endothelial–stromal interactions. As already discussed earlier, a robust inflammatory cell response is part of a normal or nonpathologic fibrotic repair response. However, along with the advantages of stimulating an immediate innate immune response and thus preventing or at the very least lowering the risk for infection, leukocytes also secrete many pro-fibrotic cytokines and GFs, such as TGF-b and, to a lesser extent, PDGF. These pro-fibrotic factors are disadvantageous because they cause keratocytes in the corneal wound to undergo a higher degree of tissue fibrosis, which can lead to chronic nonpathologic scarring or even pathologic scarring. Usually after sterile clean injury, the inflammatory response in the avascular cornea is quite mild compared to that of the skin or other vascular tissues. However, dirty injuries or wounds complicated by infection or with preexisting immune-related disease may make the inflammatory response so massive that it becomes the primary pro-fibrotic signaling pathway resulting in varying degrees of severe corneal scarring. Another TGF-b pro-fibrotic signaling pathway was already discussed in the the section entitled ‘Cytokines and GFs’ when discussing the epithelial cell injury-induced corneal nerve-lacrimal gland reflex arc. These tear film–stromal interactions are usually transient (Balb/c>C57BL/6. With most strains of HSV-1, corneal infection requires surface abrasion to facilitate entry of the virus into corneal epithelial cells. An epithelial lesion then forms as a result of virus replication in and destruction of epithelial cells. The lesions are transient, resolving by 4 days post infection (dpi), with eradication of replicating HSV-1 by 7–8 dpi. Healing of the lesions is aided by the rapid turnover of corneal epithelial cells.

Eradication of Replicating HSV-1 from the Corneal Epithelium Viral clearance appears to be largely a function of the innate immune system, consistent with the rapid kinetics of virus elimination. Evidence suggests a role for neutrophils, natural killer (NK) cells, and gd T cells in the eradication of replicating virus from the cornea. These cells are attracted to and activated within the cornea as a result of the release of cytokines, including cyclooxygenase 2, prostaglandin E2, type 1 interferon, interleukin (IL)-1, IL-6, IL-17, granulocyte macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor (TNF); and chemokines including CXCL10 (IP-10), CXCL9 (MIG), CXCL2 (MIP-2), and CCL3 (MIP-1a). The production of these cytokines and chemokines appears to be initiated by ligation of tolllike receptors (TLRs), especially TLR2 and TLR9, and in response to cytokines including IL-17, IL-6, and interferongamma (IFN-g). The initial neutrophilic infiltrate peaks in HSV-1 infected mouse corneas around 4 dpi, and declines between 6 and 8 dpi, coincident with the peak and decline of viral titers in the mouse cornea. The adaptive immune system appears to play a less significant role in the initial clearance of HSV-1 from the infected cornea following primary infection. However, it is noteworthy that severe combined immune-deficient (SCID) mice that lack an adaptive immune system do not fully eradicate replicating virus from the cornea. Accordingly, activated CD4 T cells have been detected within the infected cornea by 3 dpi, although their role in inhibiting HSV-1 replication within the cornea has not been established. It is possible, and indeed likely that the failure of SCID mice to eradicate replicating HSV-1 from the cornea during primary infection results from their inability to fully establish and maintain viral latency in the trigeminal


ganglion. Replicating virus in the trigeminal ganglion could then be transported back to and shed into the cornea, perpetuating the corneal infection. The role of T cells in clearing virus from the corneal epithelium following reactivation has not been studied. Antibodies also appear to play an important role in the clearance of HSV-1 from the cornea during both primary and recurrent disease.

HSV-1 Colonization of Sensory Ganglia During the course of virus replication in epithelia of the cornea, oral, or nasal passages of mice, the virus gains access to the termini of sensory neurons and is transported to neuronal nuclei in trigeminal ganglia. There it replicates briefly and then establishes a latent infection that is characterized by retention of a functional viral genome without production of infectious virions. Evidence suggests that control of acute HSV-1 replication and establishment of latency in trigeminal ganglia is mediated largely by an innate response of macrophages and NK cells mediated by TNFa, IFN-g, and nitric oxide. However, complete eradication of replicating virus requires reinforcement by CD8+ T cells and gd T cells. Latently infected neurons within the ophthalmic branch of trigeminal ganglia then serve as a source of virus for recurrent corneal disease. HSV-specific CD8+ T cells then remain in the trigeminal ganglion in close apposition to infected neurons for the life of the mouse. These CD8+ T cells express a persistent activation phenotype and have been shown to inhibit HSV-1 reactivation from latency both in vivo and in ex vivo ganglionic cultures. A similar association of activated CD8+ T cells with latently infected neurons has also been established in human ganglia. Moreover, psychological stress, which is associated with recurrent herpetic disease in humans, causes a transient compromise of the function of ganglionic CD8+ T cells and HSV-1 reactivation from latency in trigeminal ganglia of mice. These findings suggest that maintenance of HSV-1 latency and prevention of recurrent shedding of virus at mucosal surfaces such as the cornea requires constant immunosurveillance by CD8+ T cells.

Herpes Stromal Keratitis Following resolution of the epithelial lesion, corneal clarity is reestablished in mice. However, by 7–10 dpi corneal opacity develops accompanied by ingrowth of blood vessels into the normally avascular cornea, and massive leukocytic infiltration. These are manifestations of HSK, which in mice progresses from a non-necrotizing form with little epithelial involvement to severe necrotizing keratitis in which stromal inflammation gives rise to epithelial necrosis. The necrotizing form represents about 7% of human HSK, with 88% exhibiting the non-necrotizing form, and

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5% exhibiting a mixture of both forms. It is not clear if the reduced incidence of necrotizing HSK in humans relative to mice is due to medical management of inflammation in human corneas or a reflection of fundamentally different pathogenic mechanisms in most human cases of HSK. T Cells in HSK Early studies revealed that HSK failed to develop in athymic nude mice that lacked T cells, but could be reconstituted in these mice by T-cell adoptive transfer. The CD4+ T cell population appears to be the predominant mediator of HSK in most mouse models, with CD8+ T cells tending to reduce disease severity. However, CD8+ T cells can also mediate a milder and more transient form of HSK when mice are deficient in CD4+ T cells or infected with certain strains of HSV-1. Human corneas with HSK contain both CD4+ and CD8+ T cells that are specific for HSV antigens, though the relative contribution of these cells in the progression or resolution of HSK cannot be evaluated. Recently, a population of CD4+ T cells referred to as natural T regulatory cells (nTregs) that co express CD25 and the forkhead/winged-helix transcriptional regulator, FoxP3 were shown to moderate the severity of HSK. The fact that these nTregs maintain the capacity to regulate HSK when expanded in vitro and adoptively transferred into infected mice offers a potentially useful therapeutic modality. Cytokines and Their Role in HSK Cytokines provide a means of communication among leukocytes and between leukocytes and parenchymal cells at sites of infection and/or antigen deposition. Under optimal conditions cytokines orchestrate an immunoinflammatory response that eliminates a pathogen with minimal damage to host tissue. In HSK resolution of the immunoinflammatory lesions in the cornea is accompanied by scar tissue formation. This trade-off of strength for clarity is unacceptable in a tissue within the visual axis. Moreover, the fact that in both mice and humans HSK can develop in the absence of replicating virus in the cornea renders HSK a purely immunopathological process. Thus, regulating the production and/or function of the cytokines that orchestrate HSK would reduce tissue damage without compromising protection of the host. Most studies in the murine models have implicated the Th1 cytokines as playing a primary role in the pathogenesis of HSK. The prototypic Th1 cytokine, IFN-g, aids neutrophil extravasation into the cornea at least in part through upregulation of platelet/endothelial cell adhesion molecule 1 (PECAM-1, CD31) on local vasculature. Th1 cells also produce IL-2 that has been shown to directly or indirectly regulate the migration of neutrophils into the central cornea during HSK. The Th1-associated cytokine

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Immune Regulation of the Cornea and Conjunctiva and Its Dysregulation in Disease

IL-12 also contributes to HSK development, presumably through its capacity to regulate IFN-g production. The cytokine IL-6 also plays a central role in orchestrating HSK in part through its ability to induce the neutrophil chemokine MIP-2 (CXCL2). Recently, the functional repertoire of CD4+ T cells was expanded to include Th17 cells. The IL-17 cytokine family represents the signature cytokines of Th17 cells, and is now implicated in a variety of inflammatory processes and autoimmune diseases. IL-17 is produced in both human and mouse corneas during HSK. Both human and mouse keratocytes express receptors for IL-17, and engagement of this receptor induces their production of neutrophil chemotactic factors as well as GM-CSF. The chemotactic factors attract neutrophils into the cornea while GM-CSF activates them and maintains their viability. Recent studies support a role for IL-17 in neutrophilic infiltration during the early stages of HSK. The requirement for IL-17 is transient and presumably superseded by Th1 cytokines as HSK progresses. Regulating the production or blocking the function of the Th1 and Th17 cytokines offer potential therapeutic benefit in preventing the progression of HSK. In addition, the Th1/Th2/Treg cytokine IL-10 has a proven capacity to ameliorate HSK. The general anti-inflammatory properties include inhibition of the production and function of IFN-g. Antigen-Presenting Cells in HSK Antigen-presenting cell (APC) refers to the ability of certain cells, including macrophages, dendritic cells, and B lymphocytes to take up, process, and present antigenic peptides to naive CD4+ T cells in the context of major histocompatibility complex class II (MHC class II) and co-stimulatory molecules. The once prevalent view that the normal cornea is devoid of these specialized cells has now been dispelled by several recent histological studies of mouse and human corneas. Dendritic cells (DCs) are present in the basal layer of the corneal epithelium and macrophages are present throughout the corneal stroma, with the density of both cell types gradually diminishing from the peripheral to the central cornea. During HSV-1 infection of the corneal epithelium, the resident DCs are reinforced by immigrants from the limbus. Studies employing ultraviolet irradiation to diminish DCs or cautery to increase their presence in the cornea prior to HSV-1 infection suggested a role for these cells in directly or indirectly inducing a delayed-type hypersensitivity response in lymphoid organs. Since DCs exhibit transient survival following HSV-1 infection, it is likely that either noninfected corneal DCs acquire viral antigens from corneal epithelial cells for direct presentation to T cells in the lymph nodes, or infected corneal DCs travel to the lymph nodes where they are phagocytosed by

resident DCs and viral antigens are cross-presented to T cells. The DC depletion studies also suggested an important role for corneal DC in presenting HSV-1 antigens to CD4+ T cells upon infiltration of the infected cornea. This is consistent with the observation that CD4+ T cells induce a second and more profound infiltration of DC into the cornea at the time of onset of HSK. It should be noted, however, that the method of DC depletion employed in these early studies was not specific, and the role of DC and DC subpopulations in the efferent and afferent limbs of the CD4+ T cell response in HSK awaits confirmation in studies employing specific depletion strategies now available. Although many cell types can be induced to express MHC class II antigens, professional APCs are uniquely able to co-express the co-stimulatory molecules that are required for the activation of naive and, in many cases, effector CD4+ T cells. In a mouse model of HSK, blocking the interaction of the ligands B7-1 (CD86) and B7-2 (CD80) on APCs with the co-stimulatory molecule CD28 on CD4+ T cells locally within the cornea inhibited the HSK progression. In contrast, ligation of the inhibitory receptor program death 1 (PD-1) by its ligand B7-H1 (CD274) appears to inhibit HSK progression, though it is not clear if this effect is exerted in the cornea or lymphoid organs. HSK progression was not influenced by blocking the interaction of the APC activating receptor OX40 by its T-cell ligand OX40L (CD154). These studies suggest that local inhibition of co-stimulatory molecule or augmentation of inhibitory molecule signaling in CD4+ T cells might hold therapeutic potential in the management of HSK. Chemokine Involvement in HSK Chemokines are chemotactic cytokines that activate and direct the migration of leukocytes into sites of inflammation. Chemokines not only orchestrate the innate immune response that eradicates replicating HSV-1 from the cornea (see above), but also have a central role in regulating HSK. Defining the role of chemokines in inflammatory processes is complicated by the fact that they are pleiotropic, have overlapping functions, share receptors, and can bind to multiple receptors. Not surprisingly, studies of their involvement in HSK, have in some cases, produced conflicting results. While IP-10 (CXCL10) and MCP-1 (CCL2) appear to be important for directing CD4+ T cells into the cornea, IP-10 might also inhibit neovascularization (see below). The chemokine CCL20, expressed by HSV-1-infected corneal epithelial cells and corneal keratocytes when stimulated by IL-1b and TNFa, appears to be involved in DC infiltration during both epithelial lesions and the secondary infiltration during HSK development. The chemoattractants MIP-2 (CXCL2), MIP-1a (CCL3), and RANTES (CCL5) have all been implicated in guiding neutrophils into the cornea


during HSK. A variety of neutralizing antibodies, and peptide and nonpeptide inhibitors of these chemokines have been developed and might have therapeutic potential in managing HSK, though their successful use will likely be complicated by the redundant nature of the chemokines. Angiogenesis in HSV-1 Stromal Keratitis Neovascularization of the normally avascular cornea appears to be a requisite step in the development of severe HSK in mice. Both vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 have an important role in the neovascularization of corneas with HSK. Production of these factors is induced by IL-1, IL-6, and MIP-2. Corneal cell production of soluble VEGF receptors prevents vascularization of the cornea during steady state. Clearly, the capacity of the soluble VEGF receptor to neutralize VEGF and prevent its binding to VEGF receptors on the limbal vasculariture is overwhelmed by the amount of VEGF produced in HSV-1 infected corneas. Neovascularization provides enhanced corneal access of blood-borne leukocytes that mediate HSK, and blocking neovascularization effectively prevents HSK progression. Based on the established roles for vascularization and VEGF in mouse models of HSK, and the prominent presence of corneal vessels in necrotizing keratitis in humans, therapeutic approaches geared toward blocking vascularization hold great promise in the management of HSK.

Models of HSK Pathogenesis Despite extensive advances in our understanding of the pathogenesis of HSK in mice, one of the most basic questions (viz., what stimulates the CD4+ T cells that mediate HSK) remains unanswered. Three models of CD4+ T-cell activation in HSV-1 infected corneas have been advanced and supported by experimental data. These include: (1) bystander activation by the cytokine milieu present in the cornea, (2) autoimmunity to corneal tissue resulting from molecular mimicry by viral proteins, and (3) virus-specific activation. The concept that bystander activation of CD4+ T cells mediated by the cytokine milieu within the infected cornea is sufficient to induce HSK was proposed based on findings in mice whose T cells all express transgenic Tcell receptors (TCRs) specific for ovalbumin (ova). When the corneas of these mice were infected with HSV-1, they developed HSK that was mediated by ova-specific CD4+ T cells. However, these mice failed to control HSV-1 replication in the cornea, and succumbed to infection early during HSK development. Indeed, when HSV-1 replication was controlled in the corneas of these with kinetics similar to that of immunologically normal mice,

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the mice failed to develop HSK. Thus, in immunologically normal mice (and presumably people) bystander activation of CD4+ T cells might contribute to, but is not itself sufficient for, the development of HSK. Other studies incorporating BALB/c mice that are congenic for the IgH locus concluded that HSK develops as a result of an autoaggressive attack of CD4+ T cells on corneal tissue. This autoimmune response was induced by a peptide contained in the viral UL6 protein that mimicked a sequence in a corneal protein and in the Ig heavy chain of one of the congenic strains. The BALB/c strain that expressed the mimicked peptide in its Ig heavy chains did not respond to the peptide due to clonal deletion, and were highly resistant to HSK. In contrast, the congenic strain that failed to express the UL6-mimicked peptide in its Ig heavy chain generated a response to UL6 that crossreacted with a corneal protein. The authors concluded from these findings that HSK is an autoimmune disease arising from antigenic mimicry by the HSV-1 UL6 protein. Unfortunately, these intriguing findings were not reproduced in subsequent studies by another group, and no UL-6-specific or cornea-specific CD4+ T cells have been isolated from mouse or human corneas with HSK. Thus, the involvement of autoimmunity in HSK remains uncertain. A third possibility is that reactivity of HSV-specific CD4+ T cells to HSV antigens within the cornea triggers HSK. This hypothesis is consistent with the fact that HSV-1-specific CD4+ T cells have been isolated from both human and murine corneas at varying stages of keratitis. Further support for this concept came from a study in which partial tolerization of CD4+ T cells to HSV-1 antigens was associated with reduced severity of HSK. However, no studies to date have established a clear role for HSV-1-specific CD4+ T cells in directly mediating HSK. Based on the available data, it is likely that the initiation of HSK results from stimulation of HSVspecific CD4+ T cells in the cornea, while bystander activation might contribute to the progression and chronicity of the inflammation.

Epidemiology and Pathogenesis of Human HSK Clinically, ocular HSV-1 infection remains a significant cause of visual impairment worldwide. In developed nations, there are approximately 8.4–13.2 new ocular HSV-1 infections per 100 000 person-years with an overall incidence, including recurrences, of 20.7–31.5 episodes per 100 000 person-years. Perhaps the most serious manifestation of ocular HSV-1 infection is the potentially blinding HSK, which accounts for only 2% of initial HSV-1 ocular presentations but represents 20–61% of recurrent disease.

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Multiple forms of HSK exist and no classification system is universally accepted (here we use a classification system described by Liesegang). HSK commonly presents as a non-necrotizing or immune form, but can also present as the rarer but more serious necrotizing form. Based on limited data, it appears that both the necrotizing and immune presentations of human HSK share the features of neovascularization and leukocytic infiltration seen in the mouse model. Since HSK in the mouse progresses from disease resembling immune HSK to disease resembling necrotizing HSK, it is reasonable to propose that the two forms represent a continuum rather than distinct pathologies. However, there are important differences between the human and mouse HSK, including the fact that HSK rarely leads to corneal perforation in humans, while perforation is a frequent occurrence in the mouse model. Moreover, human HSK is often associated with replicating HSV-1 in the cornea and overlying epithelial lesions, whereas replicating HSV-1 is typically eradicated from the mouse cornea prior to the onset of HSK.

of CD4+ T-cell pathways necessary for production of inflammatory cytokines, such as IL-2 and IFN-g. Furthermore, CsA inhibits corneal neovascularization in murine models of HSK. Successful medical treatment of human HSK has important implications concerning the theories of HSK pathogenesis. CsA functions to inhibit TCR-induced calcineurin signaling that leads to production of inflammatory cytokines, such as IL-2. Therefore, effective treatment of HSK with CsA implicates TCR signaling, favoring CD4+ T-cell activation by viral or autoantigens, but not by cytokines. Studies in mice emphasize an important role for neovascularization in HSK progression. Mouse studies also implicate VEGF as a critical regulator of vascularization associated with HSK. The current availability of therapeutic agents such as Avastin and Leucentis that inhibit vascularization by blocking VEGF offers the potential for exciting new treatment alternatives.

Management of Human HSV Stromal Keratitis and Implications on Pathogenesis

Acknowledgments

Management of HSK is complicated by the fact that the cells that are responsible for eliminating the virus also contribute to immunopathology in the cornea. Treating the inflammation alone can significantly exacerbate viral replication. In contrast, treating with antivirals alone can lead to uncontrolled inflammatory damage to the cornea. The current standard of care for both non-necrotizing and necrotizing HSV stromal keratitis includes topical corticosteroids and topical antivirals. Corticosteroids are used to diminish the immunopathological component of stromal disease, while the antivirals prevent further viral replication. However, corticosteroids applied to the ocular surface can lead to adverse side effects including development of cataracts or glaucoma. A less well-studied treatment option for patients suffering from HSV stromal keratitis is topical cyclosporin A (CsA) in addition to topical antivirals. Several small noncontrolled trials in humans with HSK suggested that treatment with CsA may be beneficial in cases resistant to corticosteroids. Additionally, studies using murine models of HSK revealed that CsA effectively reduced stromal inflammation and haze in a dose-dependent manner. Similar to corticosteroids, CsA also functions to dampen the immune system. CsA blocks transcriptional activation

The authors would like to thank Kira Lathrop, MAMS, for technical assistance within this article. See also: Adaptive Immune System and the Eye: T CellMediated Immunity; Antigen-Presenting Cells in the Eye and Ocular Surface; Avascularity of the Cornea; Corneal Epithelium: Response to Infection; Immunopathogenesis of Pseudomonas Keratitis.

Further Reading Biswas, P. S. and Rouse, B. T. (2005). Early events in HSV keratitis – setting the stage for a blinding disease. Microbes and Infection 7: 799–810. Lepisto, A. J., Frank, G. M., and Hendricks, R. L. (2007). How herpes simplex virus type 1 rescinds corneal privilege. In: Niederkorn, J. Y. and Kaplan, H. J. (eds.) Immune Response and the Eye, vol. 92, pp. 203–212. Karger: Basel. Liesegang, T. J. (1999). Classification of herpes simplex virus keratitis and anterior uveitis. Cornea 18: 127–146. Metcalf, J. F., Hamilton, D. S., and Reichert, R. W. (1979). Herpetic keratitis in athymic (nude) mice. Infection and Immunity 26: 1164–1171. Sheridan, B. S., Knickelbein, J. E., and Hendricks, R. L. (2007). CD8 T cells and latent herpes simplex virus type 1: Keeping the peace in sensory ganglia. Expert Opinion on Biological Therapy 7: 1323–1331.

Immunopathogenesis of Onchocerciasis (River Blindness) E Pearlman, Case Western Reserve University, Cleveland, OH, USA K Gentil, University of Bonn, Bonn, Germany ã 2010 Elsevier Ltd. All rights reserved.

Glossary Microfilariae – First stage (L1) larvae of filarial nematodes including Onchocera volvulus, the causative agent of river blindness. TLR (toll-like receptor) – A family of surface and endosomal receptors that are expressed on mammalian cells, including the cornea. These receptors recognize microbial products and transmit cell signals that culminate in the elaboration of chemokines and cytokines. Wolbachia – Obligate intracellular bacteria that exist as endosymbionts in filarial nematodes including Onchocerca volvulus.

Onchocerciasis (river blindness) remains endemic in a number of sub-Saharan African countries and has foci in Yemen and in Latin America. Most recent (2006) estimates indicate that there are 37 million individuals infected with Onchocerca volvulus. The life cycle of all filarial nematodes includes transmission through insect vectors, with Simulium blackflies transmitting O. volvulus. First stage larvae (microfilariae, L1) are ingested during a blood meal and migrate through the insect gut, thorax and into the salivary gland having undergone two molts to the third-stage larvae (L3). Infective L3 enter the mammalian host during a second insect blood meal, where they develop to L4 stage and then adult males and females. Adult male and female worms live for over 10 years in subcutaneous tissues, producing millions of microfilariae over their lifespan. Microfilariae can survive for over 1 year in the skin, and can enter the anterior and porterior segments of the eye. While alive, microfilariae appear to cause minimal damage, and individuals can be very heavily infected; however, when the larvae die either by natural attrition or following chemotherapy, the host immune response causes acute and chronic tissue damage, with severe onchodermatitis in the skin, visual impairment, and blindness. Figure 1 shows examples of sclerosing keratitis and of cellular infiltration and vascularization in the corneal stroma of an infected individual in west Africa.

The Role of Endosymbiotic Wolbachia Bacteria in Onchocerciasis Intracytoplasmic Rickettsia-like bacteria were first described in filarial nematodes in 1977, and later identified

as Wolbachia pipientis. Although 75% of insect species and a number of crustaceans harbor Wolbachia as endosymbionts, filarial nematodes are the only group of parasitic worms that are infected, most likely because they are the only nematode group with an obligate insect host. In filarial nematodes, Wolbachia are detected with in cells in the hypodermis and uterus, in immature microfilariae in the uterus, and in mature microfilariae in the skin and cornea. The bacteria are most numerous in the mammalian host compared with the insect stage, and appear to have an essential, though poorly understood, role in nematode embryogenesis. Antibiotic (Doxycycline) treatment of filaria-infected individuals effectively sterilizes the adult females, reducing overall microfilaria numbers in the skin and blocking disease transmission.

Role of Wolbachia in Pathogenesis – Evidence from Infected Individuals The role of Wolbachia in the pathogenesis of filarial disease has been implicated from observations made after antifilarial therapy. Elevated Wolbachia DNA and even intact Wolbachia are detected in the blood, and are associated with the proinflammatory cytokines seen in patients with post-treatment side effects such as fever, edema, and headache. Using quantitative PCR for Wolbachia surface protein (WSP) gene, which is present as a single copy per organism, the number of bacteria per worm was found to be similar in all insect stages of B. malayi. However, within 7 days in the mammalian host, bacteria numbers increased 600-fold and showed a high ratio of Wolbachia/nematode DNA in L4 larvae, indicating rapid bacterial replication within the worms. This Wolbachia load is maintained in adult males, but increases in females during embryogenesis. Wolbachia are therefore a target for antibiotic treatment, and patients given a course of doxycyline in addition to annual ivermectin treatment have reduced systemic microfilariae, and significantly fewer adult worm number in subcutaneous nodules. Wolbachia appear to mediate recruitment of neutrophils, as the number of neutrophils in nodules from doxycycline-treated individuals is greatly reduced compared with untreated individuals. An additional line of evidence for a role for Wolbachia in the pathogenesis of onchocerciasis relates to earlier studies showing that two strains of O. volvulus that differ in virulence exist in West Africa based on DNA probes using a noncoding repeat sequence, and the strain

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Figure 1 Ocular onchocerciasis. (a) Blind individual in endemic region of Cote D’Ivoire, West Africa, 1999, with both corneas opaque as a result of infection with Onchocerca volvulus (photograph by Eric Pearlman); (b) sclerosing keratitis, showing central region of corneal opacification and neovascularization (with permission from Dr Hugh Taylor); (c) corneal section showing intact microfilariae (arrow) in the corneal stroma. Blood vessels (V) and cellular infiltrate are also visible (from Armed Forces Institute of Pathology).

shown to cause more severe ocular disease has significantly higher Wolbachia loads compared with the second, less virulent strain, indicating a correlation between virulence and Wolbachia in ocular onchocerciasis. Taken together, these findings strongly support the notion of an important role for Wolbachia in the proinflammatory response and pathogenesis of onchocerciasis.

The Role of Wolbachia in the Pathogenesis of ocular onchocerciasis – Lessons from the Murine Model of O. volvulus/Wolbachia Keratitis Microfilariae invade both the anterior and the posterior segments of the eye. In the latter case, they cause uveitis and chorioretinitis, resulting in loss of vision; therefore, although Onchocerca keratitis is more frequent and more readily detectable, corneal transplants are not conducted as the patients generally also have posterior segment disease. Because eyes from human cases of onchocerciasis are not available, the host response to Onchocerca has been examined in the skin of infected individuals. As such, Onchocerca infected individuals show microfilariae in the dermis surrounded by neutrophils, eosinophils, or macrophages. The likely explanation is that neutrophils surround recently dead and degenerating worms in the skin, whereas macrophages and eosinophils migrate to the site at later

time points. Our findings in a murine model of Onchocerca keratitis shows that neutrophils surround microfilariae in the cornea within 24 h and immunogold labeling of the major Wolbachia surface protein shows neutrophils in close proximity to Wolbachia (Figure 2). Using this mouse model of O. volvulus keratitis in which filaria/Wolbachia extracts are injected into the corneal stroma, St. Andre and colleagues demonstrated that endosymbiotic Wolbachia bacteria are essential for the pathogenesis of O. volvulus keratitis as: (1) O. volvulus from individuals depleted of Wolbachia by antibiotic treatment do not induce corneal inflammation; (2) related filarial species containing Wolbachia induce keratitis in contrast to filarial species lacking Wolbachia; and (3) isolated Wolbachia induce neutrophil recruitment to the corneal stroma.

Wolbachia and TLRs in the Cornea TLRs are surface and endosomal receptors that are expressed in the cornea and respond to microbial products such as lipopolysaccharide (TLR4) and lipoproteins (TLR2). TLR2 forms heterodimers with TLR1 or TLR6 to initiate signaling through adaptor molecules and induce nuclear factor kappa B (NFkB) translocation to the nucleus, and results in production of pro-inflammatory and chemotactic cytokines. Our findings using gene knock-out mice clearly demonstrate that either O. volvulus extracts


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(e) Figure 2 Presence of endosymbiotic Wolbachia bacteria in microfilaria and adult female worms. C57BL /6 mice were injected into the corneal stroma with microfilariae, corneas were removed after 4 h or 18 h and thin sections were immunostained with anti-Wolbachia Surface Protein (WSP) and visualized with IgG conjugated to 15 nm gold particles. Sections were counterstained with uranyl acetate and lead citrate, and examined by electron microscopy. (a, b) 4 h after injection. WSP was clearly detected inside microfilariae in the corneal stroma (arrows). Mf: microfilariae. (c, d) 18 h after injection microfilariae containing Wolbachia were surrounded by neutrophils (PMN). WSP labeled with gold particles (arrows) are present in the microfilariae adjacent to the neutrophils. Magnifications: (a) 4800; (b) 8400; (c) 5300; (d) 16 000. Reprinted with permission from Gillette-Ferguson, I., Hise, A. G., McGarry, H. F., et al. (2004). Wolbachia-induced neutrophil activation in a mouse model of ocular onchocerciasis (river blindness). Infection and Immunity 72: 5687. (e) Longitudinal section of adult female Brugia malayi immunostained with anti-WSP showing WSP positive microfilariae in the uterus (magnification 400). Reprinted from Science; photomicrograph by Amy Hise.

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containing Wolbachia, or isolated Wolbachia bacteria selectively activate TLR2 and TLR6, and adaptor molecules MyD88 and Mal. Figure 3 shows that corneal inflammation (neutrophil infiltration and increased corneal haze) is entirely dependent on activating TLR2. Moreover, studies using chimeric mice have shown that TLR2 expressed on bone-marrow-derived cells have an important role in provoking corneal inflammation. Taken together, findings from our group and others indicate that Wolbachia induces TLR2 activation in resident macrophages in the corneal stroma, and produce proinflammatory cytokines and CXC chemokines, which mediate neutrophil recruitment from peripheral, limbal vessels into the corneal stroma. Neutrophil responses to Wolbachia are also dependent on TLR2/MyD88, which mediate cytokine production by these cells, and may contribute to degranulation and secretion of reactive oxygen species and matrix metalloproteinases, resulting in cell death and loss of corneal clarity. In chronically infected, untreated individuals, there is also an ongoing adaptive immune response, due to repeated invasion of microfilariae into the corneal stroma, and prolonged worm degeneration and release of Wolbachia. Infiltrating eosinophils and macrophages also contribute to tissue damage, manifesting as corneal opacification, loss of vision, and blindness. We found that a third role for the TLR2 is filaria/Wolbachia activation of dendritic cells and T-cell production of IFN-g but not IL-4 or IL-5. IFN-g also has an indirect role in enhancing pro-inflammatory and chemotactic cytokine production, thereby increasing neutrophil recruitment to the corneal stroma (Figure 4). Together, these findings demonstrate that TLR2 governs the host response to Wolbachia at several levels, including systemic and corneal responses, and may be a target for blocking corneal inflammation.

Identification of a Wolbachia TLR2/TLR6 Ligand The TLR2/TLR6 heterodimer is activated by diacylated lipoproteins. To identify possible Wolbachia lipoproteins that activate TLR2/TLR6, Taylor and colleagues searched the lipoprotein databases, and identified Brugia malayi Wolbachia peptidoglycan-associated lipoprotein PAL (wBmPAL). Synthetic diacylated wBmPAL was shown to selectively induce IFN-g production, to induce systemic TNF-a in a murine model of lymphatic filariasis, and to induce corneal inflammation in a TLR2/TLR6–dependent manner. These data indicate that the interaction between these (and likely other) lipoproteins and TLR2/6 in the cornea is essential for the development of O. volvulus keratitis.


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Figure 3 O. volvulus /Wolbachia keratitis is dependent on TLR2. O. volvulus extract containing Wolbachia (OvAg) was injected into the corneal stroma of C57BL/6, TLR2/, TLR4/ and TLR2/4/ mice. After 18 h, mice were sacrificed and corneas were examined as described. (a) Single cell suspension was prepared from the corneal stroma, and total neutrophils were detected by flow cytometry using MAb NIMP-R14. (b, c) Bone marrow cells from donor C57BL/6 / eGFP+ and TLR2/ mice were used to reconstitute sublethally irradiated recipient C57BL/6 or TLR2/ mice. After 2 weeks, chimeric mice were injected intrastromally with O. volvulus extract (OvAg) or saline (HBSS), and 18 h later, 5 mm corneal sections were examined by fluorescence microscopy. Corneal sections were examined for neutrophils by immunohistochemistry. (b) Representative corneas from irradiated C57BL/6 mice reconstituted with C57BL/6 / eGFPþ bone marrow cells. After 2 weeks, mice were either untreated (naive), or examined 24 h after injection with either saline (HBSS) or O. volvulus extract (OvAg). (c) Total neutrophils per corneal section. Data points represent individual corneas; these data points are combined from three repeat experiments. Reprinted from Gillette-Ferguson, I., Daehnel, K., Hise, A. G., et al. (2007). Toll-like receptor 2 regulates CXC chemokine production and neutrophil recruitment to the cornea in Onchocerca volvulus/Wolbachia-induced keratitis. Infection and Immunity 75: 5908–5915.

Conclusion River blindness appears to be mostly under control due to mass distribution of ivermectin (Mectizan), which kills microfilariae and has led to reduced prevalence of disease. Recent studies targeting Wolbachia for antibiotic treatment now demonstrate that doxycycline can also be used to treat infected individuals. This is not only a paradigm shift in treating filarial-infected individuals, but given the risk for ivermectin resistance, also provides a critical second approach to treatment, although this is still at the clinical trial stage. The Bill and Melinda Gates Foundation recently provided funds to develop novel antibiotics as an adjunct treatment for river blindness and lymphatic filariasis, and are described in the antiWolbachia website. Overall, the increased understanding

of the pathogenesis of this disease and targeting Wolbachia in particular bring these devastating diseases closer to being controlled.

Acknowledgments We gratefully acknowledge the contribution of our colleagues in these studies, including Illona Gillette-Ferguson, Amy Hise, Achim Hoerauf and Mark Taylor. This work was supported by NIH grants EY10320 and EY11373, by the Research to Prevent Blindness Foundation and the Ohio Lions Eye Research Foundation. See also: Innate Immune System and the Eye; Pathogenesis of Fungal Keratitis.


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Figure 4 IFN-g production is dependent on TLR2 and regulates neutrophil recruitment to the corneal stroma. (a, b) Mice were immunized three times with soluble O. volvulus extract containing Wolbachia (OvAg). After the final immunization, mice were sacrificed and spleens removed for in vitro recall response with soluble OvAg. As a positive control, splenocytes were incubated with stimulatory anti-CD3. Cytokine production by splenocytes after 72 h of culture was measured by ELISA. Note TLR2-dependent IFN-g, but not IL-5 production. (c, d) C57BL/6 and IFNg/ mice were immunized 3 times subcutaneously with OvAg, and injected into the corneal stroma with OvAg. After 24 h, 5 mm corneal sections were immunostained for neutrophils or eosinophils, and the number of positive cells in the corneal stroma was assessed by fluorescence microscopy. Each data point represents an individual cornea, and the experiment was repeated 3 times. Note IFN-g-dependent recruitment of neutrophils, but not eosinophils to the cornea. (a, b) Daehnel, K., GilletteFerguson, I., Hise, A. G., et al. (2007). Filaria/Wolbachia activation of dendritic cells and development of Th1-associated responses is dependent on Toll-like receptor 2 in a mouse model of ocular onchocerciasis (river blindness). Parasite Immunology 29: 455–465. (c, d) Gentil, K. and Pearlman, E. (2009). IFN-g and IL-1R1 regulate neutrophil recruitment to the corneal stroma in a murine model of Onchocerca volvulus keratitis (river blindness). Infection and Immunity 77(4): 1606–1612.

Further Reading Boatin, B. A. and Richards, F. O., Jr. (2006). Control of onchocerciasis. Advances in Parasitology 61: 349–394. Brattig, N. W. (2004). Pathogenesis and host responses in human onchocerciasis: Impact of Onchocerca filariae and Wolbachia endobacteria. Microbes and Infection/Institut Pasteur 6: 113–128. Daehnel, K., Gillette-Ferguson, I., Hise, A. G., et al. (2007). Filaria/ Wolbachia activation of dendritic cells and development of Th1associated responses is dependent on Toll-like receptor 2 in a mouse model of ocular onchocerciasis (river blindness). Parasite Immunology 29: 455–465. Gentil, K. and Pearlman, E. (2009). IFN-g and IL-1R1 regulate neutrophil recruitment to the corneal stroma in a murine model of Onchocerca volvulus keratitis (river blindness). Infection and Immunity 77(4): 1606–1612. Gillette-Ferguson, I., Daehnel, K., Hise, A. G., et al. (2007). Toll-like receptor 2 regulates CXC chemokine production and neutrophil recruitment to the cornea in Onchocerca volvulus/Wolbachiainduced keratitis. Infection and Immunity 75: 5908–5915. Gillette-Ferguson, I., Hise, A. G., McGarry, H. F., et al. (2004). Wolbachia-induced neutrophil activation in a mouse model of ocular onchocerciasis (river blindness). Infection and Immunity 72: 5687–5692. Higazi, T. B., Filiano, A., Katholi, C. R., et al. (2005). Wolbachia endosymbiont levels in severe and mild strains of Onchocerca volvulus. Molecular and Biochemical Parasitology 141: 109–112. Hise, A. G., Daehnel, K., Gillette-Ferguson, I., et al. (2007). Innate immune responses to endosymbiotic Wolbachia bacteria in Brugia

malayi and Onchocerca volvulus are dependent on TLR2, TLR6, MyD88, and Mal, but not TLR4, TRIF, or TRAM. Journal of Immunology 178: 1068–1076. Hoerauf, A. (2008). Filariasis: New drugs and new opportunities for lymphatic filariasis and onchocerciasis. Current Opinion in Infectious Diseases 21: 673–681. Hoerauf, A., Mand, S., Adjei, O., Fleischer, B., and Buttner, D. W. (2001). Depletion of Wolbachia endobacteria in Onchocerca volvulus by doxycycline and microfilaridermia after ivermectin treatment. Lancet 357: 1415–1416. Pearlman, E., Johnson, A., Adhikary, G., et al. (2008). Toll-like receptors at the ocular surface. Ocular Surface 6: 108–116. Saint Andre, A., Blackwell, N. M., Hall, L. R., et al. (2002). The role of endosymbiotic Wolbachia bacteria in the pathogenesis of river blindness. Science 295: 1892–1895. Taylor, M. J., Bandi, C., and Hoerauf, A. (2005). Wolbachia bacterial endosymbionts of filarial nematodes. Advances in Parasitology 60: 245–284. Turner, J., Langley, R. S., Johnston, K. L., et al. (2009). Filarial Wolbachia lipoprotein stimulates innate and adaptive inflammatory responses through TLR2 and TLR6 and induce disease manifestations of lymphatic filariasis and river blindness. Journal of Biological Chemistry 284: 22364–22378.

Relevant Websites http://www.a-wol.net – A-WOL – Anti-Wolbachia Consortium.

Immunopathogenesis of Pseudomonas Keratitis L D Hazlett, Wayne State University School of Medicine, Detroit, MI, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Akt1 – Also known as Akt or protein kinase B, it is an important molecule in mammalian cellular signaling and inhibits apoptotic processes. Eicosanoids – Signaling molecules made by oxygenation of 20-carbon essential fatty acids. There are four families of eicosanoids: the prostaglandins, prostacyclins, thromboxanes, and the leukotrienes. IRF1 (interferon regulatory factor 1) – A member of the interferon regulatory transcription factor family. IRF1 serves as an activator of interferons alpha and beta transcription, and in mouse it has been shown to be required for double-stranded RNA induction of these genes. IRF1 also functions as a transcription activator of genes induced by interferons alpha, beta, and gamma. Further, IRF1 has been shown to play roles in regulating apoptosis and tumor-suppression. Keratitis – A condition in which the cornea becomes inflamed. The condition is often marked by moderate to intense pain and usually involves impaired eyesight. LPS (lipopolysaccharide, endotoxin) – Major outer membrane component of Gram-negative bacteria comprising a lipid A core (endotoxin) and polysaccharide of varying length and composition. Toll-like receptor 4 (TLR4) and MD2 (myeloid differentiation 2) bind to the lipid A moiety. MAPK (mitogen-activated protein kinase) – Serine/threonine-specific protein kinases that respond to extracellular stimuli (mitogens) and regulate various cellular activities, such as gene expression, mitosis, differentiation, and cell survival/ apoptosis. Matrix metalloproteinase-9 (MMP-9) – Gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase is a biological enzyme. Proteins of the MMP family are involved in the breakdown of extracellular matrix in normal physiological processes, as well as in disease processes. Most matrix metalloproteinases (MMPs) are secreted as inactive pro-proteins which are activated when cleaved by extracellular proteinases. The enzyme encoded by this gene degrades type IV and V collagens. NF-kB (nuclear factor-kappa B) – A protein complex that is a transcription factor. NF-kB is found in almost all animal cell types and is involved in cellular responses to stimuli such as stress,

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cytokines, free radicals, ultraviolet irradiation, and bacterial or viral antigens. Peroxynitrite – An anion, with the formula ONOO , it is an unstable valence isomer of nitrate, NO3 , which has the same formula but a different structure. Although peroxynitrous acid is highly reactive, its conjugate base, peroxynitrite is stable in basic solution. SIGIRR (single immunoglobulin interleukin 1 receptor (IL-1R)-related protein) – An inhibitory member of this receptor superfamily. SIGIRR seems to temper cellular activation by IL-1, LPS, and probably other activators of receptors in the TLR–IL1R superfamily, such that the biological outcome will be the result of a balance between activation by a receptor and dampening by SIGIRR. SIGIRR therefore acts as a brake on the TLR system, which may be essential for regulating the detrimental effects of innate immunity, as occurs in sepsis and chronic inflammation. siRNA (short interfering RNA or silencing RNA) – A class of 20–25 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference pathway, where it interferes with the expression of a specific gene. T helper (Th) 1 (e.g., predominant IL-12 and interferon gamma production) and Th2 (predominant IL-4, 5, 10, 13) response – Response involving effector T cells that play an important role in establishing and maximizing the capabilities of the immune system.

Introduction In the United States, microbial keratitis is frequently associated with complications resulting from use of extended wear contact lenses with an incidence of about 30 000 cases annually. The cost of treatment is estimated at between $15 and $30 million, representing a considerable medical and economic impact. Of the bacterial organisms able to induce keratitis, Pseudomonas aeruginosa remains an important Gram-negative pathogen. The bacteria is often referred to as opportunistic, as it is capable of inducing keratitis not only in extended wear contact lens users, but

Immunopathogenesis of Pseudomonas Keratitis

also in more tropical climates, and in patients that are either debilitated or hospitalized. Most of the complications of bacterial keratitis are structural alterations of the cornea, but other sight-threatening problems include development of secondary glaucoma and cataract. These consequences are largely caused by the host’s inflammatory response, but the influence of bacterial toxins, exoproducts, and toxicity from antibiotic treatment cannot be overlooked. There is also a recent increase in the incidence of reported antibiotic resistant strains and failure of an initially promising treatment using antimicrobial peptides to manage keratitis, suggesting that better understanding of the pathogenic mechanisms of disease induction by this pathogen will be critical to development of improved therapeutic strategies. P. aeruginosa, like most other microorganisms, typically requires surface injury to permit corneal invasion. Because it has few nutritional requirements, it can adapt to a variety of ecological conditions and niches, such as preserved ophthalmic solutions and the hospital environment. Pseudomonal and other Gram-negative bacterial infections often present as a rapidly progressing, suppurative stromal infiltrate with a marked mucopurulent exudate. Yellowish coagulative necrosis surrounded by inflammatory epithelial edema is distinctive and stromal ulceration can lead to stromal tissue destruction and vision loss. A ring infiltrate may appear in the surrounding paracentral cornea and hypopyon (a dense inflammatory coagulum) is usually present; in addition, descemetocele (ulcer penetrated through cornea) formation or corneal perforation are not uncommon. Animal models of bacterial keratitis continue to be of value in the study of this disease and are produced by topical bacterial application after abrading the epithelium, by intrastromal inoculation, or by placing a contaminated suture or contact lens on the cornea. These approaches and models have led to increased understanding of the mechanisms of corneal inflammation and innate immunity that are operative in bacterial keratitis. Bacterial eradication by neutrophils (PMN) involves phagocytosis, lysosomal degranulation and bacterial killing within the acidic lysosomal compartment of the cell. Phagocytosis and intracellular degranulation by PMN also involve oxidative attack, production of toxic oxygen metabolites, triggering of the respiratory burst, biosynthesis of superoxide anions, and other oxidizing agents such as hydrogen peroxide and formation of peroxynitrite. Phagocytic secretion and lysis result in release of extracellular lysosomal enzymes, including, but not limited to, elastase, collagenase and myeloperoxidase (MPO). These enzymes and the oxygen-derived free radicals cause stromal destruction by breaking down collagen, digesting glycosaminoglycans, and disrupting stromal keratocytes. Nitric oxide (NO) also mediates vasodilation and can be important in bacterial killing as well as in bystander tissue

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damage. These and other substances released from activated PMN and other inflammatory cells (e.g., macrophages, Mf) contribute to stromal necrosis and corneal edema during bacterial disease. Bacterial endotoxin and exotoxins also stimulate Mf to release biologically active substances including, but not limited to, interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-a, cytokines that synergize to elicit inflammatory events. Selected cytokines, certain eicosanoids, and other molecular mediators are also involved in ulceration and angiogenesis during bacterial keratitis. Maintenance of leukocyte recruitment during inflammation requires intercellular communication between infiltrating leukocytes, the epithelium, neuropeptides such as substance P (SP) and vasoactive intestinal peptide (VIP), vascular endothelium, and resident stromal cells. These events are mediated by generation of early response cytokines (e.g., IL-1), the expression of adhesion molecules such as intercellular adhesion molecule-1, and the production of chemotactic cytokines and chemokines.

PMN, Cytokines, and Chemokines If leukocytes such as PMN persist within the cornea, destructive pathology ensues, including stromal scarring and perforation, potentially requiring corneal transplantation. PMN infiltration into inflamed tissue is controlled largely by local production of inflammatory mediators. In the mouse, two members of the CXC family of chemokines, MIP-2 (functional homolog of human IL-8) and KC, are potent chemoattractants and activators of PMN. In corneal infections, MIP-2 has been shown to be the major chemokine that attracts PMN into the P. aeruginosa infected cornea and persistence of PMN in the cornea of susceptible (cornea perforates) C57BL/6 versus resistant BALB/c (no corneal perforation) mice (Table 1) was found to correlate with higher MIP-2 chemokine levels (both mRNA and protein). IL-1, produced by Mf, monocytes, and resident corneal cells also influences PMN infiltration into tissues. When tested, IL-1a and b (mRNA and protein) were elevated in the infected cornea of C57BL/6 over BALB/c mice. Furthermore, after infection, MMP-9 was shown to upregulate chemotactic cytokines/chemokines (IL-1b and MIP-2), contribute to degradation of collagen IV, and overall, enhanced P. aeruginosa keratitis. In contrast, neutralization of IL-1b in infected C57BL/6 mice reduced Table 1

Response to P. aeruginosa corneal infection in mice

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Th1 responder Corneal perforation IFN-g, IL-12

Th2 responder Corneal healing IFN-g, IL-10

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disease severity, evidenced by reduction of PMN in cornea (MPO assay), decreased bacterial load, and decreased levels of MIP-2 at both the mRNA and protein levels. The use of caspase 1 (enzyme that processes IL-1b to generate the mature cytokine) inhibitor treatment in C57BL/6 mice confirmed these data, even when inhibitor treatment was initiated 18 h after disease onset. In addition, improvement was augmented when the caspase 1 inhibitor was given after infection together with the antibiotic ciprofloxacin. A live attenuated P. aeruginosa vaccine also has been tested and found to elicit outer membrane protein-specific active and passive protection against corneal infection.

T Cells and IL-12 The role of other cells, such as T cells in P. aeruginosa corneal infection, was first studied in inbred C57BL/6 wild type and cytotoxic, CD8(+) T deficient, b2 microglobulin knockout mice (on the C57BL/6 background). Corneas of both groups of mice perforated by 7 days post-infection (p.i.) and histopathology was similar, with infiltration of PMN within 24 h p.i. In contrast, corneas of wild-type mice antibody depleted of helper, CD4(+) T cells and infected with P. aeruginosa did not perforate at 7 days p.i., versus mice depleted of CD8(+) T cells or treated with an irrelevant antibody. Antibody neutralization of IFN-g before infecting C57BL/6 mice also prevented corneal perforation and was associated with a lower delayed type hypersensitivity response when compared with C57BL/6 mice similarly treated with an irrelevant antibody. These data support that a CD4(+) T cell T helper type 1 (Th1) dominant response following P. aeruginosa infection is associated with genetic susceptibility and corneal perforation in C57BL/6 mice and provided the first evidence that CD4(+) T cells are important in development of severe keratitis and eventual corneal perforation. In addition, use of gene array studies confirmed a Th1 versus Th2 bias of C57BL/6 versus BALB/c mice to infection with Pseudomonas. Other studies investigated whether IL-12 (IL-12 p40) was associated with IFN-g production and the susceptibility response of C57BL/6 mice after P. aeruginosa challenge. IL-12 p40 knockout mice (C57BL/6 background) versus wild-type mice were tested to examine disease progression in endogenous absence of the cytokine. When tested, both groups of mice were susceptible to corneal challenge with P. aeruginosa and corneal perforation was observed at 5–7 days p.i. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay analyses confirmed that IL-12 p40 message and protein levels were elevated after infection in the wild type over the expected absence of IL-12 p40 in the knockout mouse cornea. Immunostaining for IL-12 in wildtype C57BL/6 mice revealed that stromal PMN were at least one source of the cytokine.

IL-18 and IFN-g The role of IL-18 and IFN-g production in the resistance response of the predominantly Th2 responding BALB/c mouse was also tested. Semi-quantitative RT-PCR detected IFN-g mRNA expression levels in the cornea of infected mice at 1–7 days p.i. Cytokine levels were significantly upregulated when compared with control, uninfected normal mouse corneas. Using RT-PCR, IL-18 mRNA expression was detected constitutively in the normal, uninfected cornea, but levels were significantly elevated 1–7 days p.i. To test whether IL-18 regulated IFN-g production, BALB/c mice were injected with an anti-IL-18 monoclonal antibody. Treatment decreased corneal IFN-g mRNA levels and both bacterial load and disease severity increased when compared to immunoglobulin G injected control mice. Data provided evidence that IL-18 is critical to the resistance response of BALB/c mice by induction of IFN-g and that IFN-g is required for bacterial killing/ stasis in the cornea. Another separate study showed that the killing effect of IFN-g was indirect, through regulation of NO levels in the infected cornea. IFN-g and SP Further study of the resistance response in BALB/c mice examined the role of the pro-inflammatory neuropeptide, SP in IFN-g production. This study provided evidence that natural killer (NK) cells were required to produce IFN-g; that the cells expressed the neurokinin-1 receptor (NK1R, the major SP receptor); and that they directly and tightly regulated IFN-g production through SP interaction with this receptor, suggesting a unique link between the nervous system and development of innate immunity in the cornea. On the other hand, a disparate distribution of SP in the infected cornea of susceptible C57BL/6 (higher levels) versus resistant BALB/c (lower levels) mice also has been documented and blocking the interaction of SP with its major receptor (NK1R) in C57BL/6 mice improved disease outcome, supporting a role for SP in development of the susceptible phenotype after P. aeruginosa corneal infection. Thus, the amount of SP and its interaction with available NK1R sites after infection contributes either to resistance or susceptibility, and appears an important component of keratitis outcome in these murine models.

Neuropeptides SP In this regard, SP involvement in the pathophysiology of inflammatory disease generally has been evidenced by aberrant levels of SP and SP containing nerve fibers,


as well as NK1R, in diseased tissues. SP has been shown to elicit cytokine secretion (IL-2, IL-4, IL-10, and IFN-g) from mouse T cells. In addition, it was demonstrated that human bronchial epithelial cells produce IL-6, IL-8, and TNF-a after SP treatment. SP-induced cytokine production and secretion by leukocytes, including T cells, Mf, and dendritic cells leads to the release of a number of inflammatory mediators such as additional cytokines, oxygen radicals, arachidonic acid derivatives, and histamine, all of which further amplify the inflammatory response. VIP Recent studies have also provided ample evidence for another neuropeptide, VIP, functioning as a potent endogenous anti-inflammatory molecule affecting the immune response antithetically when compared to SP. VIP regulates inflammatory mediators via several transduction pathways and transcription factors essential for gene activation, such as nuclear factor-kappa B, interferon regulatory factor-1 (IRF-1), mitogen-activated protein kinase (MAPK), and cAMP response element. VIP downregulates the production of several pro-inflammatory cytokines, including: TNF-a, IL-1, IL-6, IL-12, and IFN-g, while stimulating production of anti-inflammatory cytokines IL-10, IL-1 receptor (R) antagonist, and TGF-b. Investigation of the effect of VIP in a murine endotoxin challenge model showed that after treatment with VIP, levels of TNF-a and IL-6 in serum and peritoneal fluid were reduced by almost 50%. Regarding the eye, VIP treatment converted the susceptible phenotype (corneal perforation) to resistant (no perforation) in a mouse model of P. aeruginosa-induced infection via downregulation of pro-inflammatory mediators, upregulation of anti-inflammatory molecules, and modulation of host inflammatory cell activation. Thus VIP, a 28-amino-acid peptide, delivered by several types of neurons to immune organs and lymphoid tissues in the heart, gastrointestinal tract, lungs, kidney, cornea and skin, is anti-inflammatory. In fact, evidence indicated a differential response to VIP between infected BALB/c (more) and C57BL/6 (less) mice, due to disparate VIPR1 expression by Mf (which can be induced in a dose-dependent manner by VIP itself). Mf are known to play a key role in regulating/ balancing pro- and anti-inflammatory activity in the resistant (BALB/c) and susceptible, C57BL/6 murine models; therefore, evidence that VIP influences the functional behavior of these cells further supports a more salient role for this neuropeptide in regulating the inflammatory response.

TLR Mouse eye infection models have also been used to study the role of Toll receptors in disease. The Toll family of

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receptors, conserved throughout evolution from flies to humans, is central in initiating innate immune responses. This family of receptors, composed of trans-membrane molecules, links the extracellular compartment where contact and recognition of microbial pathogens occurs and the intracellular compartment, where signaling cascades leading to cellular responses are initiated. Gene array data showed that the expression of TLRs and related molecules including CD14, soluble IL-1 receptor antagonist, TLR-6, and IL-18R-accessory-protein were significantly elevated in susceptible (C57BL/6) versus resistant (BALB/c) mice following challenge with P. aeruginosa. In another model system involving induction of sterile keratitis, when C3H/HeJ (Toll-like receptor 4, TLR4, point mutation) versus control mice were treated with lipopolysaccharide (LPS) from P. aeruginosa, a significant increase in stromal thickness and haze was seen in the cornea of TLR4 sufficient control, but not in TLR4 deficient mutant mice. The severity of disease coincided with PMN stromal infiltration, indicating that TLR4 signaling enhances corneal disease. In contrast, in a bacterial keratitis model, TLR4 mRNA expression was markedly increased in the cornea of resistant BALB/c mice after bacterial infection. These data led to testing corneas from TLR4-deficient and wild-type control (BALB/c) mice after challenge with live P. aeruginosa to determine the role of TLR4 in bacterial keratitis. Given that TLR4 deficiency was suggested to be protective in sterile keratitis, we might predict that corneas of TLR4-deficient mice would be less susceptible and exhibit a decreased inflammatory response to bacterial infection. In marked contrast, TLR4-deficient versus control mice exhibited significantly increased inflammation and corneal perforation instead of healing after infection. Furthermore, data from clinical score, slit lamp, and histopathology confirmed that TLR4-deficient versus wild-type control mice exhibited significantly increased corneal disease with more opacity and more severe stromal swelling and destruction. In addition, bacterial load (more than 10-fold higher) and PMN recruitment (MPO activity) were markedly upregulated in the infected cornea of TLR4-deficient versus TLR4-sufficient control mice. The data provide strong evidence that TLR4 is required for the resistance response of BALB/c mice to P. aeruginosa challenge and, unlike the sterile keratitis model, TLR4 is required for disease resolution in bacterial keratitis. Overall, it appears conflicting that TLR4 is critical in the pathology of corneal disease in sterile keratitis, while it is protective in bacterial keratitis and required for host resistance. In fact, these data illustrate the characteristic double-edge sword activity of TLR4 activation. On the one hand, in the keratitis model, TLR4 recognized LPS, a component of P. aeruginosa, and initiated an innate immune response that was important for bacterial clearance. TLR4 deficiency impaired bacterial clearance, led

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to overgrowth of bacteria, an overwhelming PMN infiltrate and excessive pro-inflammatory cytokine production. This in turn contributed to corneal destruction and perforation. In the sterile keratitis model, activation of LPS-TLR4 signaling in TLR4 sufficient mice, lead to pro-inflammatory cytokine production and PMN infiltration, increased stromal thickness and contributed to haze production which increased, albeit transiently, corneal perturbation when compared with TLR4 mutant mice. Negative regulators of TLR are also of importance and recent evidence showed that one of them, single immunoglobulin IL-1R related molecule is differentially expressed in BALB/c (resistant) versus C57BL/6 (susceptible) mice. This Toll receptor is critical in resistance to P. aeruginosa infection in BALB/c mice, functioning to downregulate type 1 immunity and negatively regulating sustained IL-1 and TLR4 signaling. siRNA treatment to knockdown TLR9 was also found to influence the outcome of bacterial keratitis, and lead to reduced inflammation, but with the unwanted effect of decreased bacterial killing.

Apoptosis Delay in apoptosis in bacterial keratitis, as evidenced by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, also contributes to corneal perforation in C57BL/6 mice. Consistent with this finding, Bcl-2, an anti-apoptotic gene, was significantly upregulated in C57BL/6 mouse cornea at 18 h p.i., suggesting that the delayed onset of apoptosis in C57BL/6 mouse cornea may be, in part, due to upregulation and signaling of this gene. These data are also consistent with previous studies showing that over-expression of Bcl-2 reduces lymphocyte apoptosis in P. aeruginosa-induced pneumonia. In the process of apoptosis, execution of cells largely depends on proteolytic cleavage and activation of caspase 3. In this regard, BALB/c versus C57BL/6 mouse cornea expressed more intense staining for activated caspase 3 at 1 day p.i. compared to the delayed peak intensity in C57BL/6 mice. The two mouse groups were also disparate for expression of caspase 9, with significantly more mRNA expression in BALB/c over C57BL/6 mice. Although only hypothetical, these data suggest that different pathways of apoptosis may be operative in the infected cornea of the two groups of mice. Our studies, using a combination of TUNEL and immunostaining, as well as PMN depletion, also provided evidence that the corneal apoptotic cells identified in both groups of mice were predominantly PMN and confirmed that apoptosis of these cells is delayed in C57BL/6 mice. We hypothesize that earlier apoptosis of PMN in resistant BALB/c mice is consistent with effective elimination of invading bacteria, while inducing minimal tissue damage due to unresolved and persistent inflammation.

Neuropeptides The balance between apoptosis and cell survival, as well as the tissue milieu and timing of apoptosis, is critical in immune defense. In this regard, the neuropeptide SP, mentioned above, is a potent anti-apoptotic regulator and can exacerbate inflammation. SP has been shown to stimulate phosphorylation of the anti-apoptotic molecule Akt in colonic mucosa both in vivo and in vitro, preventing apoptosis in humans with colitis. In another in vitro study, SP induced p53, Bcl-2, and NO expression in peritoneal Mf, blocking apoptotic signals. SP delays spontaneous apoptosis of PMN in a dose-dependent fashion by its interaction with the NK1R in vitro, and this effect could be inhibited by application of the NK1R antagonist GR82334. In this regard, C57BL/6 mice treated with another NK1R antagonist, Spantide I, showed significantly improved disease outcome and an earlier onset of apoptosis, similar to the pattern observed in naturally resistant BALB/c mice. Consistent with earlier onset of apoptosis, mRNA expression of caspase 3 was also significantly upregulated earlier in the cornea of Spantide I, versus control treated animals. The data suggest that the protective mechanism of Spantide I treatment in C57BL/6 mice involves induction of earlier PMN apoptosis in the infected cornea, with less bystander tissue damage. Whether in C57BL/6 mice, the effects of Spantide I are directly mediated via the PMN or indirectly through Mf regulation of PMN also required resolution. Clodronate depletion of Mf with or without Spantide I treatment revealed that in the absence of the Mf, apoptosis was reduced/absent in the cornea. To further explore the role of this cell in susceptibility and resistance, Mf from both groups of mice (C57BL/6 and BALB/c) were incubated in the presence of SP together with LPS or with LPS alone. Significantly fewer apoptotic cells were detected in cells from C57BL/6 mice in the presence of SP and LPS versus LPS alone, while the same combined treatment (SP and LPS) did not decrease the number of LPS induced apoptotic Mf from BALB/c mice. To determine the mechanism for the disparate response to SP treatment between Mf from the two mouse groups, expression levels of the NK1R were comparatively tested. Although cells from both groups expressed the receptor, with a slightly weaker signal in BALB/c cells, after LPS stimulation, mRNA expression for the NK1R was only detected in cells from C57BL/6 mice. These data suggest that the possible mechanism for the absence of the anti-apoptotic effects observed in BALB/c Mf after SP treatment may involve a low level of NK1R expression on the cells and possible rapid depletion of the receptor upon LPS stimulation. In this regard, VIPR1, the major receptor for the neuropeptide VIP, was also reported to be expressed disparately in Mf from C57BL/6 (less) versus BALB/c (more) mice.


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initiated and progresses similar to the human pattern. A photograph of the new animal model is provided in Figure 1. This model will allow us to explore treatment paradigms that have been efficacious in the mouse and other rodent species and may more quickly result in clinical trial of successful candidates. See also: Corneal Epithelium: Response to Infection.

Figure 1 P. aeruginosa infection in Marmoset monkey model.

Mf also may provide distinct activation signals for Th1/Th2 differentiation. In this regard, others have reported that Leishmania major infected Mf enhanced the proliferation and IL-4 secretion of Th2 T cells, but inhibited the response of Th1 T cells. When testing for this possibility, we detected that Mf from C57BL/6 mice expressed significantly more IL-12, while BALB/c Mf expressed more IL-10 after LPS stimulation. IL-10 appears protective in the BALB/c infected cornea, as after subconjunctival injection of clodronate-containing liposomes to deplete these cells, higher levels of IFN-g and lower levels of IL-10 were detected and resistant mice were converted to the susceptible phenotype. It was also reported that VIP treated C57BL/6 mice showed improved disease outcome and increased IL-10 expression after P. aeruginosa corneal infection. Furthermore, the data suggest that SP, which acts in an anti-apoptotic manner toward activated C57BL/6 mouse Mf, may also enhance an IL-12 driven, Th1 type immune response and thus further contribute to the susceptibility of this mouse strain to P. aeruginosa infection.

New Animal Model Without doubt, experimental animal models of bacterial infection with P. aeruginosa have provided us with important insights into mechanisms underlying ocular infection and inflammation and our understanding of the effector and regulatory mechanisms involved in disease continues to grow. However, our understanding and knowledge of the precise mechanisms operative in human cases of keratitis (sterile and infectious) remains much more limited. In this regard, development of a new primate model of keratitis would be a timely and important translational approach to facilitate eventual human application of what has been learned in rodent and other species. The Marmoset monkey, with 98% homology to the human genome, provides such a model. Initial studies have been undertaken in the monkey and show that with scarification of the cornea (similar to that done in the mouse), infection can be

Further Reading Delgado, M., Pozo, D., and Ganea, D. (2004). The significance of vasoactive intestinal peptide in immunomodulation. Pharmacological Reviews 56(2): 249–290. DeVane, L. The fibromyalgia community, substance P: A new era, a new role. http://fmscommunity.org/subp.htm (accessed June 2009). Dinarello, C. A. (2005). Blocking IL-1 in systemic inflammation. Journal of Experimental Medicine 201(9): 1355–1359. Ferguson, T. A. and Griffith, T. S. (2007). The role of Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL) in the ocular immune response. Chemical Immunology and Allergy 92: 140–154. Harrison, S. and Geppetti, P. (2001). Substance P. International Journal of Biochemistry and Cell Biology 33(6): 555–576. Hazlett, L. D. (2004). Corneal response to Pseudomonas aeruginosa infection. Progress in Retinal and Eye Research 23(1): 1–30. Janeway, C. A., Jr. and Medzhitov, R. (2002). Innate immune recognition. Annual Review of Immunology 20: 197–216. Kernacki, K. A., Barrett, R. P., Hobden, J. A., and Hazlett, L. D. (2000). Macrophage inflammatory protein-2 is a mediator of polymorphonuclear neutrophil influx in ocular bacterial infection. Journal of Immunology 164(2): 1037–1045. Lighvani, S., Huang, X., Trivedi, P. P., Swanborg, R. H., and Hazlett, L. D. (2005). Substance P regulates natural killer cell interferon-gamma production and resistance to Pseudomonas aeruginosa infection. European Journal of Immunology 35(5): 1567–1575. McClellan, S. A., Huang, X., Barrett, R. P., van Rooijen, N., and Hazlett, L. D. (2003). Macrophages restrict Pseudomonas aeruginosa growth, regulate polymorphonuclear neutrophil influx, and balance pro- and anti-inflammatory cytokines in BALB/c mice. Journal of Immunology 170(10): 5219–5227. Meek, B., Speijer, D., de Jong, P. T., de Smet, M. D., and Peek, R. (2003). The ocular humoral immune response in health and disease. Progress in Retinal and Eye Research 22(3): 391–415. Rudner, X. L., Kernacki, K. A., Barrett, R. P., and Hazlett, L. D. (2000). Prolonged elevation of IL-1 in Pseudomonas aeruginosa ocular infection regulates macrophage-inflammatory protein-2 production, polymorphonuclear neutrophil persistence, and corneal perforation. Journal of Immunology 164(12): 6576–6582. Strand, F. L. (1999). Neuropeptides: Regulators of Physiological Processes. Cambridge, MA: MIT Press. Substance P: A modulator of inflammation. (1998). http://www. woongbee.com/Cytokine/Cytokine%20bulletin/Spring%201998/ spring1998-3.htm (accessed June 2009). Szliter, E. A., Lighvani, S., Barrett, R. P., and Hazlett, L. D. (2007). Vasoactive intestinal peptide balances pro- and anti-inflammatory cytokines in the Pseudomonas aeruginosa-infected cornea and protects against corneal perforation. Journal of Immunology 178(2): 1105–1114. Taylor, P. R., Martinez-Pomares, L., Stacey, M., et al. (2005). Macrophage receptors and immune recognition. Annual Review of Immunology 23: 901–944. Todar, K. (2008). Todar’s Online Textbook of Bacteriology: Immune Defense against Bacterial Pathogens: Innate Immunity. http:// textbookofbacteriology.net/innate.html (accessed June 2009). Winkler, J. D. (ed.) (1999). Apoptosis and Inflammation. Basel: Birkhauser Verlag.

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Relevant Websites http://www.diseasesdatabase.com – Diseases Database Ver 1.8; Medical lists and links Diseases Database, Vasoactive Intestinal Peptide.

http://www.macrophages.com – Macrophages.com. http://users.rcn.com – RCN Corporation: Apoptosis. http://users.rcn.com – RCN Corporation: Innate Immunity. http://www.ResearchApoptosis.com – Research Apoptosis.

Immunobiology of Acanthamoeba Keratitis J Y Niederkorn, University of Texas Southwestern Medical Center, Dallas, TX, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Clodronate – Drug that preferentially kills macrophages, but has no known deleterious effects on other cells of the innate or adaptive immune responses. Interferon-g (IFN-g) – Cytokine produced by T cells that activates macrophages and enhances their capacity to kill Acanthamoeba trophozoites. Mannose-binding protein (MBP) – Lectin receptor that is expressed on Acanthamoeba trophozoites and facilitates their adherence to mannosylated proteins that are expressed on corneal epithelial cells. Major histocompatibility complex (MHC) class-I antigens – Antigens that allow cytotoxic T lymphocytes to recognize and kill cells infected with viruses and some protozoal parasites. Mannose-induced protease 133 (MIP-133) – The 133-kDa protease that is induced when Acanthamoeba trophozoites engage mannose in the cell walls of bacteria or upon mannosylated proteins on corneal epithelial cells. This protease facilitates invasion of Acanthamoeba trophozoites by degrading basement membranes and it also induces apoptosis of corneal cells. Mucosal immunity – Immune responses that are primarily in the form of secretory immunoglobulin A (IgA) antibodies, which preferentially accumulate in the milk, tears, and in mucosal secretions. This form of T-cell-dependent immunity acts primarily to prevent pathogens from entering the body via mucosal surfaces and rarely directly kills microorganisms. Trophozoite – Amoebic phase of Acanthamoeba spp. Acanthamoeba spp. can exist as either dormant cysts or as the active trophozoite (amoebic) phase. Unlike cysts, trophozoites are invasive, produce pathogenic proteases, and directly kill host cells by apoptosis and direct cytolysis.

Introduction Acanthamoeba spp. are the causative agents for Acanthamoeba keratitis (AK) and can be isolated from virtually any terrestrial, aquatic, and marine environment. Viable Acanthamoeba

spp. have even been isolated from eyewash stations, bottled water, and contact lens cases of asymptomatic contact lens wearers. Acanthamoeba can exist either as a dormant cyst or as the active amoebic stage called the trophozoite. Trophozoites are the active vegetative stage that normally exist as free-living amoebae and feed on bacteria and fungi. Trophozoites are approximately the size of a leukocyte (10–25 mm) and are readily identified by their spiny pseudopodia that give them a sea urchin-like appearance. Acanthamoeba cysts are the dormant stage and are approximately half the size of the trophozoite. The cyst wall is comprised primarily of protein and cellulose. Interestingly, the latter molecule is not normally found in animals, but is restricted to members of the plant kingdom including bacteria and fungi. The cyst is remarkably resistant to environmental agents and can remain viable even after 20 years of storage at room temperature or following treatment with over 250 000 rads of gamma irradiation or doses of ultraviolet B (UVB) irradiation that are known to kill every category of mammalian cells. Although AK is believed to be caused by corneal infections produced by trophozoites adhering to contact lenses, cysts can also adhere to contact lenses, and, under certain circumstances, can produce corneal infections in experimental animals. Cysts can persist in corneal tissue for up to 31 months following antiamoebic treatment and may be the underlying cause for recrudescence in patients, especially those who receive corneal transplants to restore the vision lost as a consequence of AK. Corticosteroids are often used to extinguish the inflammation that is provoked in AK. However, corticosteroids have been shown to induce cysts to excyst and transform into infectious trophozoites. Moreover, corticosteroids activate trophozoites and render them more pathogenic and invasive. Thus, corticosteroid treatment may unwittingly exacerbate AK and contribute to the recrudescence that has been reported in AK patients who are treated with topical corticosteroids to prevent immune rejection of their corneal transplants. In spite of the ubiquitous distribution of Acanthamoeba spp. in the environment and widespread contact lens wear, AK is remarkably rare. Moreover, environmental exposure to Acanthamoeba spp. is commonplace; up to 100% of the normal individuals with no history of AK possess serum antibodies to Acanthamoeba antigens, suggesting previous environmental exposure to Acanthamoeba spp. Viable Acanthamoebae can be isolated from the contact lens cases of individuals with no symptoms or history of AK. Collectively, these findings suggest that a large portion of the population is exposed to Acanthamoeba spp. and

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may express some degree of immunity against corneal Acanthamoeba infection. This proposition begs the obvious question as to which immune component provides protection and how does it do it.

infectious agents, thereby providing much-needed time for the development of the adaptive immune response.

Anatomical and Physiological Barriers to Corneal Infections with Acanthamoeba

In vitro studies have demonstrated that macrophages are capable of killing Acanthamoeba trophozoites. Moreover, depletion of periocular macrophages by subconjunctival injection of liposomes containing the macrophagicidal drug clodronate results in a dramatic exacerbation of AK in experimental animals. Likewise, in vitro or in vivo exposure to liposomes containing interferon-g (IFN-g) activates macrophages, increases their capacity to kill Acanthamoeba trophozoites, and mitigates AK in experimental animals. In animal models, AK is a self-limiting disease that resolves in 4–5 weeks. However, depletion of conjunctival macrophages results in progressive AK that does not resolve and mimics the human counterpart. The extraordinarily low incidence of AK is not commensurate with the enormous number of contact lens wearers and the ubiquitous distribution of Acanthamoeba spp. This suggests that the presence of an additional risk factor is involved in the development of AK. It is tempting to speculate that patients who develop AK represent a small population of individuals who have underlying deficiencies in their conjunctival macrophage population or altered macrophage function.

Clinicians have long suspected that contact lenses served as vectors for transmitting Acanthamoeba trophozoites to the corneal surface, and that antecedent injury to the corneal surface created epithelial defects that permitted trophozoites to gain a foothold in the cornea. Less than one-third of the cases of AK involve both eyes, even though contact lens wearers typically store their lenses in the same contact lens case, use the same contact lens solutions, and use the same finger for inserting their contact lenses. If a preexisting corneal epithelial barrier defect were not necessary, one would expect that the overwhelming majority of AK cases would be bilateral. Studies in animal models of AK confirm the importance of preexisting corneal epithelial defects in the development of AK that is produced by applying Acanthamoeba-laden contact lenses to the abraded corneal surface. However, other animal studies have shown that breaching the intact corneal epithelium by direct injection of trophozoites into the corneal stroma produces AK. Thus, the intact corneal epithelium provides a barrier for preventing the establishment of ocular infections with Acanthamoeba trophozoites. In addition to the physical barrier provided by an intact corneal epithelium, the ocular surface is bathed in tears, which contain multiple factors that inhibit trophozoite binding and cytopathic effects. Although never formally proven, it is suspected that the shear forces produced by the blinking eyelid interfere with trophozoite adherence to the corneal epithelium and reduce the likelihood of the trophozoites gaining a foothold in the cornea.

Innate Immune System and Resistance to Acanthamoeba Infections The immune system is divided into two functionally distinct components: (1) the innate immune apparatus – which is characterized by its nimble response, but lack of antigen specificity – and (2) the adaptive immune apparatus – which, although slower in its response, provides long-lasting immunity and memory. Elements of the innate immune apparatus are the first responders via their detection of pathogen-associated molecular patterns (PAMPS) that are widely and promiscuously expressed by many microorganisms. By utilizing PAMPS, macrophages and neutrophils are able to rapidly identify invading microorganisms and mount an initial response that restrains the

Role of Macrophages in the Resistance to Acanthamoeba Infections

Role of Neutrophils in the Resistance to Acanthamoeba Infections The neutrophil is another constituent of the innate immune system that plays an important role in the resistance and resolution of AK. Neutrophils are consistently found in AK lesions – both in patients and in experimental animals. Neutrophils are highly effective in detecting the presence of both trophozoites and cysts. They kill both cysts and trophozoites in a myeloperoxidase-dependent manner. Blocking chemotactic responses of conjunctival neutrophils or depleting neutrophils with anti-neutrophilic antiserum results in progressive AK. Likewise, intracorneal injection of macrophage inflammatory protein-2 (MIP-2) – a potent chemoattractant for neutrophils – results in a swift infiltration of neutrophils into the central cornea and in an accelerated resolution of AK in experimental animals. Ocular Acanthamoeba infections rarely progress beyond the cornea and are not known to produce endophthalmitis. Only three reports in the literature suggest that Acanthamoeba infections of the cornea progress to the posterior segment of the eye and involve the choroid or retina. Moreover, only one publication provides histopathological documentation of Acanthamoeba cysts in the posterior segment of the eye in a single AK patient. Moreover, the patient in this study had received four

Immunobiology of Acanthamoeba Keratitis

separate corneal transplants in the affected eye. Likewise, more than 16 years of experience with both the pig and Chinese hamster models of AK has failed to produce any evidence of Acanthamoeba infections progressing posterior from the cornea or involving the uveal tract or retina. In vitro studies have shown that trophozoites can penetrate Descemet’s membrane and are, theoretically, capable of entering the anterior chamber. Further studies showed that intraocular injection of 1 million trophozoites into the anterior chamber of the eye in Chinese hamsters did not produce intraocular infection. A swift neutrophilic infiltrate eliminated the injected trophozoites without inflicting collateral damage to the intraocular tissues. Humoral Factors of the Innate Immune System that Affect Resistance to Acanthamoeba Infections Tears contain a potpourri of antimicrobial factors that protect the ocular surface from pathogenic insults. Among these are lysozyme, lactoferrin, and complement components. Lysozyme is active against Gram-negative bacteria and some fungi, but is ineffective against Gram-positive bacteria. By contrast, lactoferrin and transferrin are effective in controlling Gram-positive bacteria. Complement is present in tears and can be activated by the alternate pathway via bacterial products or by the classical pathway by antibodies. Thus, the complement system straddles the innate and adaptive immune systems. Complement appears to have little or no effect in controlling AK, as pathogenic Acanthamoeba spp. express complement-regulatory proteins – including decay-accelerating factor – which disable the complement system. Tears and milk also contain a factor that inhibits trophozoite adherence and cytolysis of corneal cells. Interestingly, the factor in both milk and tears is not an Ig. The milk-borne factor is >100 kDa and is inactivated by proteinase K, indicating that it is a protein. The two major proteins in milk – a1-antitrypsin and a1antichemytrypsin – are not the milk-borne factors, as neither of these proteins blocks trophozoite adherence or cytolytic activity. Thus, both humoral and cellular elements of the innate immune system can contribute to the resistance to AK (Table 1). Table 1

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Adaptive Immune System and Resistance to Acanthamoeba Infections Elements of the innate immune system serve as the first responders to pathogens and are characterized by capacity to act immediately in response to microbial infections. However, the innate immune system acting alone cannot clear all microbial infections. Unlike the innate immune system, the adaptive immune apparatus needs a jump start to generate its effector elements, but – once engaged – is crucial for the recovery from microbial infections and the establishment of immunity to future infections. The innate and adaptive immune systems do not function in isolation, but communicate with each other in a coordinated immune response. Macrophages and dendritic cells present antigens to T and B cells, which process is facilitated by another innate immune system component – complement. T and B cells have the capacity to generate an endless array of antigen receptors that, when confronted with antigens expressed on antigen-presenting cells, culminates in the generation of antibodies and T cells that possess exquisite specificity and are used by antibodies to identify and kill bacteria and neutralize viruses. T cells utilize their antigen receptors and CD8 surface molecules to identify and kill virus-infected cells. The adaptive immune system is characterized by its exquisite specificity and memory. The efficacy of preventive immunization relies entirely on the capacity of the vaccine to activate crucial elements of the adaptive immune system. One has to look no further than the biology of acquired immune deficiency syndrome (AIDS) to recognize the importance of the adaptive immune system. With few exceptions, recovery and survival from microbial infections is contingent upon the effective activation of the adaptive immune system. However, AK is a notable exception to this rule. There is no evidence to date that patients with AIDS have an increased incidence of AK, suggesting that a disabled adaptive immune system does not increase the susceptibility to corneal infections with Acanthamoeba even though Acanthamoeba spp. express antigens that are capable of activating the adaptive immune system. Fifty to one hundred percent of the individuals with no past history of AK possess serum and tear antibodies specific for Acanthamoeba antigens – indicating that Acanthamoeba antigens can

Elements of the innate immune system that control ocular Acanthamoeba infections

Component

Function

Evidence for role in protection

Neutrophils

Kill trophozoites

Macrophages

Kill trophozoites

Nonimmunoglobulin tear-borne factor

Prevents trophozoite adherence to cornea

Treatment with anti-neutrophil serum exacerbates AK; stimulating neutrophil infiltration into the cornea mitigates AK; neutrophils kill trophozoites in vitro. Depletion of conjunctival macrophages exacerbates AK; activating conjunctival macrophages mitigates AK; macrophages kill trophozoites in vitro. In vitro assays demonstrate that tears from normal animals block adherence of trophozoites to corneal cells and prevent trophozoite-mediated cytolysis of corneal cells.

416


activate the adaptive immune system and that exposure to these antigens is remarkably widespread. The T-cell arm of the adaptive immune system is also activated by Acanthamoeba antigens, as 50% of the normal population with no known history of AK demonstrate T-cell lymphoproliferative responses to Acanthamoeba antigens. In spite of this, patients whose initial AK has been brought under control with antimicrobial therapy can experience recrudescence. This suggests that even if the adaptive immune system has been activated, it is ineffectual in controlling AK. This impression has been confirmed in animal models of AK. Both in Chinese hamsters and pigs, subcutaneous immunization with Acanthamoeba antigens elicits robust IgG antibody and T-cell responses, yet fails to protect the animals against ocular challenge with Acanthamoeba trophozoites. Moreover, resolution of AK does not render animals resistant to second ocular infections with trophozoites; repeat ocular Acanthamoeba infections are as severe as the primary infections.

Role of the Mucosal Immune System in Resistance to AK On first blush, it would appear that the adaptive immune system is utterly incapable of preventing or resolving AK. However, animal studies and serological studies on human AK patients suggest that secretory IgA antibodies in the tears may prevent initial corneal Acanthamoeba infections. It is widely accepted that corneal infection with Acanthamoeba spp. begins when trophozoites adhere to mannosylated proteins on the corneal epithelium. Mannosylated proteins appear to be key ligands for trophozoite attachment – which is facilitated by the expression of a 136-kDa mannose-binding protein (MBP) that is expressed on the trophozoite cell membrane. Mannose expression on the corneal epithelium is dramatically upregulated by contact lens wear or by mild trauma to the Table 2

ocular surface. In addition to promoting the adhesion of trophozoites, mannose stimulates the trophozoites to secrete a 133-kDa pathogenic protease (mannose-induced protease-133 (MIP-133)), which facilitates trophozoite invasion and cytolysis of corneal cells. Upregulation of mannosylated protein expression on the corneal epithelium is crucial for the establishment of AK. However, blocking the interaction between mannosylated glycoproteins on the corneal epithelium and the MBP on the trophozoite cell membrane is an effective strategy for preventing AK. Immunization through mucosal surfaces such as the gastrointestinal tract preferentially stimulates the generation of secretory IgA antibodies that appear in the tears and in mucosal secretions. Animals immunized orally with the Acanthamoeba MBP develop secretory IgA antibodies in the tears, which prevent the development of AK. Secretory IgA antibodies block the adhesion of Acanthamoeba trophozoites to corneal epithelial cells, and thereby, disrupt the first key step in the pathogenic cascade of AK. Anti-MBP secretory IgA antibodies are not toxic to trophozoites, even in the presence of complement and are ineffectual if they are generated after a corneal infection had been established. Thus, IgA antibodies in the tears are the only known component of the adaptive immune system that has an effect on the development of AK (Table 2).

Evading the Adaptive Immune Response Exposure to Acanthamoeba antigens is commonplace as evidenced by the high incidence of serum antibodies and T-cell responses to Acanthamoeba antigens in individuals with no previous history of Acanthamoeba infections. In experimental animals, subcutaneous and intramuscular immunization with Acanthamoeba antigens elicits high titers of IgG antibodies and T-cell activation, yet fails to protect against AK. Although the adaptive immune

Role of adaptive immunity in preventing ocular acanthamoeba infections

Immune element

Role in AK

Evidence for effect

Serum IgG

None

Delayed-type hypersensitivity Complement

None

Cytotoxic T lymphocytes

None

Secretory IgA

Prevents initial corneal infection

Animals and humans with anti-Acanthamoeba IgG antibodies can develop AC. AntiAcanthamoeba antiserum fails to kill trophozoites in vitro, even in the presence of complement. Subcutaneous immunization with Acanthamoeba antigens induces robust delayed-type hypersensitivity to Acanthamoeba but fails to prevent or mitigate AK. Trophozoites resist complement-mediated lysis due to their expression of complement decay accelerating factor. Trophozoites are extracellular pathogens that do not express the crucial major histocompatibility complex class I restricting elements that are necessary for cytotoxic T lymphocyte function. Mucosal immunization with surface antigens expressed on trophozoites induces production of secretory IgA in the tears; anti-Acanthamoeba IgA prevents trophozoite adherence to corneal cells and prevents trophozoite-mediated cytolysis of corneal cells; passive transfer of IgA monoclonal antibody against Acanthamoeba surface eptiotpes protects animals against ocular Acanthamoeba infections.

None

Immunobiology of Acanthamoeba Keratitis

system is capable of eliminating a wide range of microbial agents, it is ineffectual in preventing corneal infections with Acanthamoeba or recrudescence of previous corneal infections. Acanthamoeba spp. employ at least three strategies to evade immune elimination: (1) inactivating immune effector elements; (2) escaping detection by cytotoxic T lymphocytes; and (3) forming dormant cysts that escape immune detection (Table 3). Acanthamoeba trophozoites can activate the complement cascade via the alternate pathway or by the direct pathway by complement-fixing IgG antibodies that recognize surface determinants on the trophozoite cell membrane. Complement components are present in the tears and at the ocular surface, yet trophozoites escape complement-mediated cytolysis – both in vitro and in vivo. The ability of trophozoites to elude the ravages of complement is ostensibly due to their expression of complement-regulatory proteins (i.e., decay-accelerating factor) – which disable the complement cascade. Complement-regulatory proteins are also present on the corneal epithelium and may further interfere with complementmediated cytolysis of trophozoites at the ocular surface. Trophozoites secrete a variety of proteases that are known to degrade Igs, including secretory IgA antibodies in the tears. The importance of this evasive strategy is limited, as IgA antibodies in the tears can prevent the initial establishment of Acanthamoeba infections of the cornea. Acanthamoeba trophozoites are extracellular pathogens and thus, escape detection and elimination by cytotoxic T lymphocytes – which are only able to kill intracellular pathogens whose antigens are presented on host cells that are expressing major histocompatibility complex (MHC) class-I molecules. Table 3

Acanthamoeba immune escape mechanisms

Factor or mechanism Secretion of serine proteases Complement decay accelerating factor Encystment

Immune privilege of cornea

Extracellular infection

Potential effect in Acanthamoeba keratitis Degrade immunoglobulins; induce apoptosis of leukocytes Inactivate complement and disable complement-fixing antibodies Escape detection by inflammatory and immune cells; resistant to phagocytosis by non-activated macrophages; weakly immunogenic; do not produce chemoattractants for leukocytes Corneal infections fail to elicit IgG antibody or delayed-type hypersensitivity responses to Acanthamoeba antigens Trophozoites are extracellular pathogens and do not reside in host cells that express MHC class I antigens. Thus, they escape recognition and attack by cytotoxic T lymphocytes

417

Protozoal parasites have the capacity to encyst and, thereby, escape immune detection and elimination. Acanthamoeba spp. are one of the few protozoal parasites of humans that form cysts in tissues. Although most intestinal protozoal parasites can encyst, the cysts reside transiently within the lumen of the large intestine and are typically eliminated in the feces. Toxoplasma gondii can encyst within mammalian cells and not in extracellular sites. Acanthamoeba cysts occur in tissue and have been reported to persist in corneal tissue for up to 31 months. Acanthamoeba cysts are remarkably resistant to a variety of agents and can remain viable for decades. Moreover, Acanthamoeba cysts are poor immunogens, have only weak chemoattractive properties, and often reside in an immune-privileged site (i.e., the cornea), thereby enhancing their ability to escape immune detection.

Anti-Disease Vaccine for AK Effector elements of the adaptive immune system appear to be incapable of killing or inactivating Acanthamoeba trophozoites. Thus, immunotherapeutic strategies are limited to two potential targets: (1) preventing the initial adherence of trophozoites to the corneal surface, or (2) inactivating the trophozoite-borne pathogenic molecules that damage the cornea. As stated earlier, secretory IgA antibodies directed against the cell-membrane molecules on the trophozoite can prevent the establishment of corneal infections by blocking the adherence of trophozoites to the corneal epithelium, but only if the antibodies are present in the tears when trophozoites are first introduced to the corneal surface. IgA antibodies are unable to alter the course of disease once the trophozoites have gained a foothold in the cornea. Trophozoites elaborate a variety of proteases that produce extensive cytolysis and apoptosis of corneal cells, degradation of basement membranes, and melting of the stroma. One of the most important trophozoite-borne proteases is a 133-kDa serine protease – MIP-133 – which is secreted in response to trophozoites binding to mannosylated glycoproteins on the corneal epithelium. Mucosal immunization with MIP-133 elicits the generation of secretory IgA antibodies that inhibit the cytolysis of corneal cells and neutralize the enzymatic degradation of stromal proteins. Hosts mucosally immunized with MIP-133 have much milder disease compared to controls. An attractive feature of eliciting mucosal immunity against a pathogenic molecule is that the ocular surface is bathed in tears containing neutralizing antibodies which are continuously replenished. An anti-disease vaccine could be implemented at the time AK is diagnosed and used in combination with conventional anti-Acanthamoeba chemotherapy.

418


Conclusions The immunobiology of AK is puzzling. Acanthamoeba spp. are free-living amoebae that can be found in virtually any environmental niche, yet rarely cause disease. Environmental exposure to Acanthamoeba spp. is commonplace and results in the activation of the adaptive immune response as evidenced by the high percentage of individuals who display antibody and T-cell responses to Acanthamoeba antigens. The low incidence of AK combined with the high frequencies of anti-Acanthamoeba antibody and T-cell immunity suggest that the adaptive immune response protects against corneal infection with Acanthamoeba spp. However, animal studies suggest otherwise and indicate that – with the exception of secretory IgA antibodies – the adaptive immune system is incapable of preventing or controlling Acanthamoeba infections. By contrast, the innate immune apparatus appears to control ocular Acanthamoeba infections. This article offers the following theory to explain the immunobiology of AK. Environmental exposure to Acanthamoeba spp. is common and occurs via mucosal surfaces by breathing airborne Acanthamoebae that can readily be isolated from heating and cooling vents or by ingestion of Acanthamoebae that are found in a variety of vegetables and fruits. Most environmental isolates of Acanthamoeba spp. are nonpathogenic, but are immunogenic. The presence of anti-Acanthamoeba IgA antibodies in the tears protects against ocular infection, especially in individuals without corneal epithelial defects. Occasionally, Acanthamoeba trophozoites will reach the ocular surface and escape detection by IgA antibodies in the tears, but are eliminated by neutrophils and macrophages. The small number of individuals who develop AK might have underlying deficiencies in their tear antibody titers or in their periocular neutrophil and macrophage repertoire.

Such immunological blind spots might account for corneal Acanthamoeba infections. This theory remains to be confirmed, but is consistent with results from animal studies and with clinical findings from AK patients. See also: Adaptive Immune System and the Eye: Mucosal Immunity; Adaptive Immune System and the Eye: T Cell-Mediated Immunity; Innate Immune System and the Eye.

Further Reading Clarke, D. W. and Niederkorn, J. Y. (2006). The immunobiology of Acanthamoeba keratitis. Microbes and Infection 8: 1400–1405. Clarke, D. W. and Niederkorn, J. Y. (2006). The pathophysiology of Acanthamoeba keratitis. Trends in Parasitology 22: 175–180. Khan, N. A. (2003). Pathogenesis of Acanthamoeba infections. Microbial Pathogenesis 34: 277–285. Khan, N. A. (2006). Acanthamoeba: Biology and increasing importance in human health. FEMS Microbiology Reviews 30: 564–595. Kumar, R. and Lloyd, D. (2002). Recent advances in the treatment of Acanthamoeba keratitis. Clinical Infectious Diseases 35: 434–441. Leher, H., Silvany, R., Alizadeh, H., Huang, J., and Niederkorn, J. Y. (1998). Mannose induces the release of cytopathic factors from Acanthamoeba castellanii. Infection and Immunity 66: 5–10. Leher, H., Zaragoza, F., Taherzadeh, S., Alizadeh, H., and Niederkorn, J. Y. (1999). Monoclonal IgA antibodies protect against Acanthamoeba keratitis. Experimental Eye Research 69: 75–84. Li, L. and Sun, X. (2008). Impaired innate immunity of ocular surface is the key bridge between extended contact lens wearing and occurrence of Acanthamoeba keratitis. Medical Hypotheses 70: 260–264. Marciano-Cabral, F. and Cabral, G. (2003). Acanthamoeba spp. as agents of disease in humans. Clinical Microbiology Reviews 16: 273–307. McClellan, K., Howard, K., Niederkorn, J., and Alizadeh, H. (2001). The effect of corticosteroid on Acanthamoeba cysts and trophozoites. Invesigative Ophthalmology and Visual Science 42: 2885–2893. Niederkorn, J. Y., Alizadeh, H., Leher, H., and McCulley, J. P. (1999). The pathogenesis of Acanthamoeba keratitis. Microbes and Infection 1: 437–443.

Molecular and Cellular Mechanisms in Allergic Conjunctivitis V L Calder, UCL Institute of Ophthalmology, London, UK ã 2010 Elsevier Ltd. All rights reserved.

Glossary Allergen – A protein to which the immune system responds. Amblyopia – Vision disorder characterized by poor or indistinct vision in an eye that is otherwise physically normal. Atopy – Allergic response in an area not in contact with allergen. Conjunctiva – Tissue lining of the ocular surface comprised of epithelium and stromal layers. Bulbar conjunctiva covers the outer surface of the eye; tarsal (palpebral) conjunctiva lines the eyelids. HLA-DR – A transmembrane human major histocompatibility complex 2 family member. Hyperemia – Increase of blood flow to a portion of the body. Photophobia – An abnormal sensitivity to light. Rhinitis – Inflammation of some internal areas of the nose. The primary symptom of rhinitis is nasal dripping.

The symptoms of SAC include itching, watering, redness, and swelling of the eyes and lids, and there may be increased discharge. There is often an associated rhinitis and a history of atopy. During the pollen season, the ocular signs can be dramatic, usually affecting both eyes to a similar degree. Edema of the lids and conjunctiva can be mild and often outweighs the degree of hyperemia, giving a milky or pink appearance to the eye. In severe cases the swelling can be gross, with the inability to open the lids and a ballooning out of the conjunctiva termed chemosis, particularly after exposure to high aeroallergen concentrations or after rubbing of the eye. Eversion of the lids to reveal the tarsal conjunctiva demonstrates some hyperemia and mild infiltration of the conjunctiva, leading to a loss of transparency and thickening, with diffuse small inflammatory excrescences known as papillae. Since there is no serious limbal disease or conjunctival scarring, and the cornea is not involved, the visual acuity remains normal. Outside the pollen season the eye examination is normal.

Perennial Allergic Conjunctivitis The term ocular allergy describes a spectrum of clinical conditions, ranging from the common, milder conditions of seasonal and perennial allergic conjunctivitis (SAC, PAC), to the rare but more severe diseases, vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC). This article describes the clinically different subtypes of ocular allergy (classification summarized in Table 1) and our current understanding of the cellular and molecular pathways involved in the different forms of ocular allergy.

Ocular Allergies Seasonal Allergic Conjunctivitis SAC, or hay fever, is the most common form of ocular allergy and, in fact, the most common of all ocular disorders. SAC occurs only during the pollen season, the timing being dependent on which pollen is allergenic for that individual (e.g., tree, grass, weed). In countries where pollen seasons are extended, SAC can affect individuals for up to 10 months per year. It can occur at any age but is more frequently seen in children and young adults and the severity tends to lessen with age.

PAC is another common ocular allergy with many similarities to SAC but with a very different time course. Since the allergens in PAC are present for most or all of the year, PAC is not seasonal. The disorder is again most frequently and severely seen in children and young adults. House dust mite (Dermatophagoides pteronyssinus) is the most common sensitizing allergen but animal hair and dander, molds and other allergens can also induce PAC. The symptoms are perennial and include ocular itch, discomfort, watering, redness, and some discharge. Patients may be able to correlate symptoms with exposure to an allergen. House dust mite allergy sufferers have a history of symptoms worse in the morning. Approximately one-third have an associated rhinitis, and a family and/or personal atopic history is very common. The clinical appearance is of a mild conjunctival inflammation and clinical signs may be very slight. The bulbar conjunctiva may be slightly red and edematous and the tarsal conjunctiva shows mild to moderate hyperemia, infiltration, and fine papillae. Lid edema is usually mild. Similar to SAC, in PAC there is no conjunctival scarring, or corneal involvement, such that vision is not affected. Due to the continuing presence of the allergens, the resultant inflammation in PAC is more chronic and hence the immunopathology of PAC differs from that of SAC.

419

420 Table 1

Immune Regulation of the Cornea and Conjunctiva and Its Dysregulation in Disease Classification of ocular allergic diseases

Disease

Timing

Age group

Prevalance

Keratopathy

Sight threatening

SAC

Seasonal

No

Perennial Seasonal – Perennial if severe

Very common Common Uncommon

No

PAC VKC

Majority children and young adults Adult Children

Common Yes

No Yes

AKC

Perennial

Adult

Rare

Yes

Yes

Course Mild, nonprogressive, often resolves Not serious, nonprogressive Serious, but usually resolves in 2–10 years with good outcome if well managed. Can change into AKC Serious and progressive, vision often reduced

SAC, seasonal allergic conjunctivitis; PAC, perennial allergic conjunctivitis; VKC, vernal keratoconjunctivitis; AKC, atopic keratoconjunctivitis.

Vernal Keratoconjunctivitis VKC is a more serious ocular allergy of childhood. It makes up 0.1–0.5% of ocular disease in the developed world but is more common and much more severe in hot dry countries, especially the Middle East, West Africa, and the Mediterranean. It is a chronic form of conjunctivitis, usually with seasonal exacerbations. In the United Kingdom, VKC is a rare, self-limiting, often seasonal ocular allergy that affects children and young adults, most of which are male (85%) and many have a history of atopy. The symptoms are worse in the spring and summer but, in severe disease, will last all year. Patients complain of severe itching, discomfort or pain, photophobia, stringy discharge, blurred vision, and difficulty opening the eyes in the morning. The ocular signs can be very asymmetrical. Conjunctival signs are maximal in the superior tarsal conjunctiva and limbus and the heavily inflamed lid may droop (ptosis). The conjunctival surfaces are hyperemic, edematous, and infiltrated, and a stringy mucoid discharge is present. The tarsal conjunctival tissues are densely infiltrated, with papillae that are often giant (>1 mm in diameter, known as cobblestone papillae). The limbus can have discrete swellings or, less often, diffuse hyperemia and inflammation, and the presence of small white chalky deposits (Trantas’ dots) is typical of vernal limbitis. In the later stages, fine reticular white scarring may be seen, but this does not lead to significant shrinkage and distortion of the ocular surface as in some cicatrising conjunctival diseases such as AKC (see below). Visual acuity can be affected by involvement of the cornea (keratopathy), which is most marked in the upper third of the cornea as a result of greater exposure to toxic inflammatory mediators, not mechanical rubbing by the papillae. At its mildest, there is a punctate disturbance of the epithelium, which may coalesce to form a discrete epithelial defect (macroerosion). Deposition of mucus, fibrin, and inflammatory debris can then result in the formation of a shallow oval plaque (or shield) ulcer, which repels the hydrophilic tears and the epithelial-healing

response. Herpetic and bacterial corneal infection may occur. In the later stages, scarring of the cornea may lead to permanent visual reduction. Steroid treatment-related complications, and (because of the young age group) sensorydeprivation amblyopia also contribute to the potential for long-term visual loss. Atopic Keratoconjunctivitis AKC is the least common but most serious of the ocular allergies. It is a life-long condition that affects adults who have systemic atopic disease, either atopic dermatitis or chronic asthma. The usual onset is in the late teens but, unlike VKC, the disease is persistent and may be relentlessly progressive; occasionally the disease can begin in childhood. AKC is a highly symptomatic disorder with severe itching, pain, watering, stickiness, and redness. There is usually facial atopic dermatitis involving the eyelids. The lid margins show severe blepharitis (chronic inflammation of the lash follicles and meibomian glands) and are thickened and hyperemic, posteriorly rounded, sometimes keratinized and the lid anatomy may be distorted with ectropion (outwardly turning eyelid), entropion (inwardly turning eyelid), trichiasis (inturning lashes), loss of lashes, and notching. The whole conjunctiva is affected and shows intense infiltration, papillae (which may be giant) and sometimes scarring with linear and reticular white scar tissue, lid to conjunctiva adhesions and shrinkage or loss of the conjunctival sac and secondary lid distortions. Marked limbal inflammation can develop and Trantas’ dots may occur. The disease may never affect the cornea, in which case it is sometimes referred to as atopic blepharo-conjunctivitis (ABC); in this situation, the overall inflammation is generally less severe. The cornea can be affected as a direct effect of the inflammatory process or may be damaged secondarily following extensive changes to the usually protective ocular surface by processes such as continual mechanical trauma, reduced lid protection, or severe loss of conjunctival tear

Molecular and Cellular Mechanisms in Allergic Conjunctivitis

production. Significant visual acuity reduction due to corneal involvement occurs in 40–70% of the cases. Keratopathy may consist of punctate and macroscopic epithelial defects, filamentary keratitis, plaque ulcer, progressive scarring, neovascularization (with or without lipid deposition), thinning, and secondary corneal infections (herpetic, bacterial, and fungal). Associations between AKC and eye rubbing, keratoconus, atopic cataract, and retinal detachment have been reported.

Cellular Mechanisms in Ocular Allergy The predominating immune mechanism occurring in SAC is an immediate (type 1) hypersensitivity response whereby conjunctival mast cells (MCs) (Table 2) and their secreted products primarily orchestrate the inflammatory response. In contrast, the cellular responses in PAC involve MC to a certain extent, although neutrophils and some T cells have also been detected in the conjunctival tissues, probably recruited as a result of the release of chemokines which attract these cells to the site of inflammation during the persistent allergen-driven inflammatory response. During VKC, studies have identified cells of both innate and adaptive immune responses becoming activated with T lymphocytes and eosinophils predominating, as well as MCs, neutrophils, and other cells infiltrating the conjunctival epithelium and stroma. In AKC, the predominant cell types infiltrating the conjunctival tissues are T cells, eosinophils, and neutrophils. In both VKC and AKC there are alterations to the epithelium and evidence of tissue remodeling and collagen deposition. Conjunctival MCs MCs are important effector cells at all mucosal sites including the conjunctiva, where rapid responses are necessary. During SAC, conjunctival MC become activated as a direct result of allergen cross-linking of surface IgE receptors (FceR1), resulting in degranulation and release Table 2 Summary of cell types in conjunctival tissues in ocular allergy

Disease SAC PAC VKC AKC

Predominant inflammatory cells

Involvement of tissueresident cells

MCCT MCCT and MCCT, neutrophils, T cells Eosinophils, CD4+T cells, neutrophils, MC CD4+T cells, eosinophils, neutrophils, MC

None None

421

of histamine, leukotrienes, proteases, prostaglandins, cytokines, and chemokines (Figure 1). This rapid MC histamine response causes the itching characteristic of SAC. Upon binding to its receptors (H1 and H2), histamine induces vascular leakage, resulting in further cellular infiltration of eosinophils and neutrophils from the blood, leading to chemosis. Relative increases in mucosal MC (MCT) were identified in tarsal conjunctival tissue specimens in SAC whereas increased numbers of both MCT and connective tissue type (MCCT) MC phenotypes have been detected in both tarsal conjunctival epithelial and substantia propria layers in PAC. The different pattern of MC subset activation occurring in PAC probably reflects the more persistent response to allergen. Therapeutic intervention in SAC initially focused on the use of topical antihistamines in the form of eye drops, to neutralize the localized effects of the histamine secreted by the degranulating MC. Another therapeutic approach has used MC stabilizing drugs (e.g., sodium chromoglycate) to inhibit release of histamine and other secretagogues from the cells. Combinations of antihistamines with chromolyns have been used to treat the signs and symptoms of SAC for many years. However, more recent topical antiallergic drugs for treating SAC (e.g., azelastine, epinastine, ketotifen, olopatadine) combine antihistamine action with MC stabilization. The additional benefits of these drugs are in their ability to selectively prevent MC secretion of various inflammatory mediators including histamine, as well as cytokines and chemokines. Due to the presence of other cell types in the more chronic forms of ocular allergy, these antiallergic drugs are most effective in SAC and are not effective for the more severe forms of ocular allergy when used alone, but can be of some benefit when given in combination with other anti-inflammatory drugs such as steroids or cyclosporine A (see below). While specific allergen immunotherapy has been successfully used for treating other forms of allergy where the specific allergens are known, it has not been widely used for treating SAC since there is a wide variation among individuals affected with SAC in terms of their allergen responsiveness, with many

Fibroblasts, epithelium Epithelium, fibroblasts

SAC, seasonal allergic conjunctivitis; PAC, perennial allergic conjunctivitis; VKC, vernal keratoconjunctivitis; AKC, atopic keratoconjunctivitis; MC, mast cells; MCCT, mucosal mast cells; MCCT, connective tissue mast cells.

(a)

(b)

Figure 1 Conjunctival mast cells in vitro : (a) Unstimulated mast cells (small arrows); (b) mast cells stimulated via FecR crosslinking (large arrows). Stimulated mast cells form clusters prior to degranulating.

422


responding to more than one allergen. In the chronic forms of allergic eye disease (VKC, AKC), the conjunctivitis is due less to a direct allergen-specific response, but more an immune-mediated response involving local tissue resident cells, and other nonallergen-specific inflammatory cells. In those severe forms of ocular allergy, specific allergen immunotherapy would not be appropriate. Conjunctival Eosinophils and Neutrophils MCs are predominately responsible for SAC, but in more chronic forms of conjunctivitis other cell types have been identified as playing an important role. Some neutrophils and eosinophils have been found in less than half of symptomatic patients with SAC but little or no T cell infiltration was observed. In SAC and PAC, neutrophils and eosinophils have been observed at the site of inflammation, and both of these cell types are able to secrete a wide range of proinflammatory cytokines (interleukin (IL)-3, IL-4, IL-5, IL-6, transforming growth factor alpha (TGF-a), tumor necrosis factor alpha (TNF-a)), chemokines (IL-8, RANTES), and multiple mediators (granule proteins: eosinophil cationic protein, major basic protein, and eosinophil-derived cationic protein) to amplify the inflammatory response. In PAC, due to the presence of eosinophils, neutrophils, and some T cells, several cellmediated processes are likely to be involved. MC-targeted therapy alone is not an effective treatment for PAC, supporting the hypothesis that there is a complex network of cells contributing to the chronic inflammation in this condition. Recently, intranasal corticosteroids have demonstrated a promising therapeutic effect in relieving the ocular symptoms associated with perennial allergic rhinitis. In the more severe forms of ocular allergy (VKC and AKC), increased numbers of eosinophils have been detected in the conjunctival tissues, although it has also been found that it is the extent of eosinophil activation expressing intracellular adhesion molecule (ICAM)-1 or a transmembrane human major histocompatibility complex 2 family member (HLA-DR) that correlates more with disease severity than the overall numbers of eosinophils. There are differences between VKC and AKC in the patterns of cytokines which colocalize to conjunctival eosinophils, with those from VKC mainly expressing IL-3, IL-5, IL-6, and granulocyte/macrophage colony stimulating factor (GM-CSF) whereas, in AKC, eosinophils express mainly IL-4, IL-8, and GM-CSF. Although the specific cellular interactions are as yet unclear, these different cytokine profiles point to different eosinophilmediated pathways being involved in each severe form of ocular allergy. Conjunctival Lymphocytes There are very few T cells detected in normal and in SAC conjunctival tissue specimens, which are mainly situated

in the epithelial layer. There are a few T cells detectable in PAC but their phenotypes remain unknown. In contrast, immunostaining of tarsal conjunctival tissue specimens from VKC patients have found significantly increased numbers of lymphocytes which are mainly activated CD4+ T cells, localized to the subepithelial layers of the affected tissue. There is also an increased HLA-DR expression within the epithelium and stromal layers of the conjunctival tissues as compared with normal subjects and increased numbers of Langerhans’ cells and activated macrophages (CD68+) were also observed. T cell clones, derived from VKC conjunctival tissues, were functionally characterized as Th2-type, since in situ hybridization staining demonstrated an increased Th2 cytokine (IL-3, IL-4, and IL-5) mRNA expression in VKC in areas of maximum T cell infiltration (Figure 2). In support of these studies, VKC tear samples were found to have increased intracellular T cell expression of IL-4 in more than 60% of the specimens. Further analysis of tear specimens using multiplex bead cytokine arrays, found that IL-4, interferon-gamma (IFN-g), and IL-10 were all elevated in SAC and VKC in comparison with nonatopic controls. Although such studies do not identify the cellular source of the cytokines, nevertheless they illustrate the differentially activated cytokine pathways in each form of ocular allergy, perhaps due to the different cell types involved in each form of ocular allergy. Similar to VKC, conjunctival biopsy specimens in AKC were found to have increased numbers of activated CD4+T cells, HLA-DR expression, and cells of the monocyte/macrophage lineage as well as mRNA expression of the Th2 cytokines (IL-3, IL-4, and IL-5) in the stromal tissues. However, in contrast to VKC, there was also a significant increase in the expression of IL-2 mRNA, and in numbers of IFN-g expressing T cells, suggesting a more Th1-type T cell response in the most severe of the ocular allergic diseases. In support of this, conjunctival biopsy specimen-derived T cell lines from AKC were found to secrete significantly increased levels of IFN-g, indicative of Th1-T cells. It has thus been proposed that AKC is an

(a)

(b)

Figure 2 Light microscopy immunostaining of conjunctival biopsy tissue sections for IL-13 expression (brown): (a) weak staining for IL-13 localizing to goblet cells within the epithelial layer in SAC biopsy specimen; (b) intense staining for IL-13 (brown) localizing to mononuclear cells within the subepithelial and epithelial layers in VKC biopsy specimen (magnification 200).


423

Mast cell Neutrophil

Immunopathogenesis in ocular allergy Clinical severity

Eosinophil

Dendritic cells

SAC

PAC

VKC

AKC B cells

Th1 T cell

Th2 T cell

Fibroblast activation

Collagen deposition

IL-4 IgE

Fibroblast

IL-4

Allergen Th2 cytokines

IL-8, RANTES

Th2/Th1 cytokines

Blood vessel

IFNγ IgE Histamine Blood vessel vasodilation

IgE

IL-5 IL-5 ECP, EDN, EPO

IgE

ECP, EDN, EPO

Figure 3 Schematic comparing the different types of immunopathogenis in ocular allergy. SAC, seasonal allergic conjunctivitis; PAC, perennial allergic conjunctivitis; VKC, vernal keratoconjunctivitis; AKC, atopic keratoconjunctivitis.

immune-mediated response involving Th1 T cells, whereas VKC involves a predominant Th2-type T cell response (Figure 3). Due to the severity of the inflammation in VKC and AKC, immunosuppressive drugs (steroids) are used to dampen the immune response. However, long-term steroid treatment can have serious side effects in the eye, causing raised intraocular pressure which can lead to glaucoma, and cataract formation. Following the identification of T cells and their cytokines within the conjunctival tissues in VKC and AKC, cyclosporine A (CsA; 2% in maize oil) was tested and found to be an effective steroidsparing treatment for VKC and AKC if administered locally as eye drops. Conjunctival Epithelial Cells As a consequence of chronic inflammation at the ocular surface, in particular in AKC, the epithelium can become

thickened. Immunostaining of conjunctival epithelial cells from conjunctival biopsies have demonstrated an increased expression of ICAM-1 and HLA-DR, but only in the most severe forms of allergic eye disease (VKC and AKC) and almost no expression of these costimulatory molecules in noninflamed control conjunctival tissues. The ability of conjunctival epithelial cells to express ICAM-1 might allow greater adhesion and recruitment of leukocytes, while the expression of HLA-DR molecules could simply reflect the activation status of the cells, although the possibility of conjunctival epithelial cells presenting antigen to T cells is still to be confirmed. Several in vitro studies have used conjunctival epithelial cells, either as primary cultures of cells isolated from biopsy specimens, or as immortalized epithelial cell lines. Upon activation of conjunctival epithelial cells in vitro, there is an upregulation of costimulatory molecules, including ICAM-1 and HLA-DR and secretion of various cytokines such as IL-6, CCL8 (IL-8), a potent

424 Table 3

Control VKC AKC

Immune Regulation of the Cornea and Conjunctiva and Its Dysregulation in Disease Summary of cytokines and molecules present in conjunctival tissues IL-2

IL-4

IL-5

MMP-1, -3, -9

IFN g

HLA-DR + epithelium

ICAM-1 + eosinophils

+

+ +++ ++

/+ +++ +++

+ ++ nd

+ ++ +++

++ ++

++ ++

/+ +++

VKC, Vernal keratoconjunctivitis; AKC, atopic keratoconjunctivitis; IL, interleukin; MMP, matrix metalloproteinases; IFN, interferon; HLA-DR, a transmembrane human major histocompatibility complex 2 family member; ICAM, intracellular adhesion molecule.

chemokine for neutrophils. The ability of conjunctival epithelial cells to secrete proinflammatory cytokines and chemokines suggests an important proinflammatory role for these cells (Table 3).

Conjunctival Fibroblasts In the severe forms of chronic ocular allergy, there is significant tissue remodeling involving collagen deposition and subepithelial fibrosis. Conjunctival biopsyderived fibroblasts in vitro have been found to secrete cytokines (e.g., IL-6), chemokines (e.g., MCP-1), as well as matrix metalloproteinase (MMP)-1 and -9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1. These cells were also found to respond to the Th2 cytokines IL-4 and IL-13, by secreting increased levels of eotaxin-1, IL-6, and RANTES, whereas there was a significant reduction of MMPs. Immunostaining of conjunctival biopsies from noninflamed controls and VKC identified a significant increase in MMP-1, -3, -9, and -13 expression in VKC tissues. Tear levels of MMP-1 and MMP-9 have also been found to be increased in VKC in comparison with controls, which is probably a reflection of the ongoing fibrotic tissue response.

Molecular Mechanisms The ocular surface of the eye is a mucosal site and plays an important role in protecting the eye from infection through a mucosal barrier as well as supporting both innate and adaptive immune responses as described elsewhere in this encyclopedia. The ocular surface is protected by the presence and continuous production of tears and mucins to prevent binding of antigens, and within the tear fluid, antibodies can be found which will bind to, and activate opsonization of antigens by phagocytes. In addition, the expression of toll-like receptors (TLRs) provides another mechanism at the ocular surface, whereby binding of evolutionarily conserved microbial proteins (pathogen-associated molecular patterns, PAMPs) to these receptors induces innate and adaptive immune responses to break down and remove the invading pathogen. Immunostaining has been used to demonstrate expression of

TLR-2, -4, and -9 in healthy conjunctival tissues, with expression mainly in the stromal layers. In comparison, in VKC, there is an increased expression of TLR-4 on both epithelium and stroma, together with a decrease in TLR-9. In AKC, TLR-2 expression has been found to be increased on human primary cultures of conjunctival epithelial cells following exposure to Staphylococcus aureus, as well as increases in ICAM-1 and HLA-DR expression and secretion of TNF-a and IL-8. This has been proposed as a mechanism whereby S. aureus infection at the ocular surface in AKC could activate a host epithelial cell response. In AKC, S. aureus colonization can occur at the ocular surface, probably due to a compromised mucosal barrier during this severe form of ocular allergy.

Costimulatory Molecules in Ocular Allergy Immunohistochemical studies of tarsal and bulbar conjunctival biopsy specimens demonstrated expression of adhesion molecules, ICAM-1 and e-selectin, to be increased in SAC in comparison with controls. However, this increased expression was only detected during the pollen season and outside the pollen season, the levels returned to those of controls. This pattern of expression correlated with the degree of neutrophil or eosinophil infiltration in the bulbar tissue, suggesting an MC-mediated cell recruitment process. Expression of HLA-DR and ICAM-1 molecules has been used as markers of cell activation. In AKC, there is an upregulation of HLA-DR expression and ICAM-1, localized to the epithelial cells, suggesting epithelial cell activation, probably as a result of exposure to proinflammatory cytokines as well as TLR activation. In both VKC and AKC there is an upregulation of HLA-DR expression and ICAM-1, localized to the eosinophils which correlated with an enhanced activation of these cells. IgE in ocular allergy Total serum IgE levels are significantly increased in VKC than in controls. However, IgE levels are variable among those with ocular allergy and cannot be used as a reliable indicator of disease activity or severity. Studies investigating allergen-specific serum IgE levels have detected a range of allergen specificities. A greater percentage of


VKC patients have specific serum IgE against D. pteronyssinus and Dermatophagoides farinae, whereas in SAC the specific serum IgE is against grass pollens. Allergenspecific IgE is also increased in tear specimens and there is a highly significant correlation with ocular allergy symptoms, supporting a diagnostic value for specific tear IgE, although limited tear volume restricts its use in routine immunoassays.

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side effects and there is an urgent need for improved treatments which can be given topically to reduce the impact of potential side effects. See also: Adaptive Immune System and the Eye: Mucosal Immunity; Adaptive Immune System and the Eye: T CellMediated Immunity; Conjunctiva Immune Surveillance; Defense Mechanisms of Tears and Ocular Surface; Innate Immune System and the Eye; Overview of Electrolyte and Fluid Transport Across the Conjunctiva.

Cytokines and Chemokines in Ocular Allergy Throughout this article, cytokines and chemokines have been discussed in relation to particular cell types. However, cell-free tear specimens have also been studied for the presence of cytokines and chemokines in the various forms of ocular allergy. Studies of VKC tear specimens have detected increased levels of IL-4, IL-10, IFN-g, eotaxin, and TNF-a in comparison with noninflamed control tears. This correlates well with the enhanced expression of Th2-cytokines detected in isolated T cell clones from conjunctival biopsy specimens from VKC patients, as well as the increased percentages of intracellular IL-4-expressing T cells from tear specimens in VKC. In severe forms of AKC, increased tear levels of eotaxin-1 were found to correlate with increased numbers of eosinophils in tears, although the cellular source of the eotaxin was not identified. The production of proinflammatory cytokines and chemokines by infiltrating conjunctival T cells could provide a mechanism whereby local tissue resident cells such as conjunctival fibroblasts become involved, since collagen deposition and conjunctival tissue remodeling is considerable in chronic allergic eye disease. Comparing conjunctival biopsy specimens from VKC patients with controls, increased expression of RANTES, eotaxin, monocyte chemotactic protein (MCP)-1, and MCP-3 was detected, reflecting the range of inflammatory cells present. VKC conjunctival tissue expression of the chemokine receptor CXCR3 was found to be specifically localized to T cells, and the CXC chemokine Mig was highly expressed, suggesting an important role for this ligand in recruitment of activated T cells. In conclusion, tissue-based studies, combined with in vitro and in vivo models (not discussed in this article), have identified discrete cellular and molecular pathways in each form of ocular allergy, and this knowledge has allowed a more selective therapeutic approach. Nevertheless, currently available therapies for the more chronic forms of ocular allergic disease are limited by their

Further Reading Abelson, M. B. and Granet, D. (2006). Ocular allergy in pediatric practice. Current Allergy and Asthma Reports 6(4): 306–311. Blaiss, M. S. (2008). Evolving paradigm in the management of allergic rhinitis-associated ocular symptoms: Role of intranasal corticosteroids. Current Medical Research and Opinion 24(3): 821–836. Bonini, S., Gramiccioni, C., Bonini, M., and Bresciani, M. (2007). Practical approach to diagnosis and treatment of ocular allergy: A 1-year systematic review. Current Opinion in Allergy and Clinical Immunology 7(5): 446–449. Bonini, S., Sacchetti, M., Mantelli, F., and Lambiase, A. (2007). Clinical grading of vernal keratoconjunctivitis. Current Opinion in Allergy and Clinical Immunology 7(5): 436–441. Calonge, M. and Enrı´quez-de-Salamanca, A. (2005). The role of the conjunctival epithelium in ocular allergy. Current Opinion in Allergy and Clinical Immunology 5(5): 441–445. Calonge, M. and Herreras, J. M. (2007). Clinical grading of atopic keratoconjunctivitis. Current Opinion in Allergy and Clinical Immunology 7(5): 442–445. Dogru, M., Okada, N., Asano-Kato, N., et al. (2005). Atopic ocular surface disease: Implications on tear function and ocular surface mucins. Cornea 24(8 supplement): S18–S23. Fukuda, K., Kumagai, N., Fujitsu, Y., and Nishida, T. (2006). Fibroblasts as local immune modulators in ocular allergic disease. Allergology International 55(2): 121–129. Kumagai, N., Fukuda, K., Fujitsu, Y., Yamamoto, K., and Nishida, T. (2006). Role of structural cells of the cornea and conjunctiva in the pathogenesis of vernal keratoconjunctivitis. Progress in Retinal and Eye Research 25(2): 165–187. Leonardi, A., De Dominicis, C., and Motterle, L. (2007). Immunopathogenesis of ocular allergy: A schematic approach to different clinical entities. Current Opinion in Allergy and Clinical Immunology 7(5): 429–435. Leonardi, A., Motterle, L., and Bortolotti, M. (2008). Allergy and the eye. Clinical and Experimental Immunology 153(supplement 1): 17–21. Mantelli, F. and Arguëso, P. (2008). Functions of ocular surface mucins in health and disease. Current Opinion in Allergy and Clinical Immunology 8(5): 477–483. Micera, A., Stampachiacchiere, B., Aronni, S., dos Santos, M. S., and Lambiase, A. (2005). Toll-like receptors and the eye. Current Opinion in Allergy and Clinical Immunology 5(5): 451–458. Schultz, B. L. (2006). Pharmacology of ocular allergy. Current Opinion in Allergy and Clinical Immunology 6(5): 383–389. Stern, M. E., Siemasko, K. F., and Niederkorn, J. Y. (2005). The Th1/Th2 paradigm in ocular allergy. Current Opinion in Allergy and Clinical Immunology 5(5): 446–450.

Pathogenesis of Fungal Keratitis E Pearlman, S Leal, A Tarabishy, Y Sun, L Szczotka-Flynn, Y Imamura, P Mukherjee, and J Chandra, Case Western Reserve University, Cleveland, OH, USA M Momany and S Hastings-Cowden, University of Athens, Athens, GA, USA M Ghannoum, Case Western Reserve University, Cleveland, OH, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Biofilm – The microbial secretion of an extracellular matrix surrounding the organisms. Conidia – These are fungal spores. Matrix metalloproteinases (MMPs) – The proteases that participate in tissue remodeling, wound healing, and inflammation. Multipurpose solution (MPS) – The contact lens care products that are used to disinfect daily-wear contact lenses. Toll-like receptor (TLR) – A family of surface and endosomal receptors that recognize microbial products. TLR signaling induces production of proinflammatory and chemotactic cytokines and antimicrobial peptides.

Contact-Lens-Associated Fungal Keratitis In June 2006, the Centers for Disease Control and Prevention (CDC) Fusarium investigation team (Chang and colleagues) reported 318 cases of Fusarium keratitis, with 164 confirmed cases in 33 states and one US territory, although smaller outbreaks were reported in Singapore, Hong Kong, and France. The age group was between 12 and 83, with a median age of 41; 94% wore soft contact lenses; and keratoplasty was needed for 34%. (Examples of contact-lens-associated Fusarium keratitis are shown by Alfonso and colleagues.) The CDC study demonstrated a clear relation to the use of Bausch and Lomb Renu with MoistureLock multipurpose solution (MPS), and the number of cases of Fusarium keratitis dropped shortly after withdrawal of this product. As unopened bottles were sterile, and Fusarium can be readily isolated from sink and shower drains, the CDC report concluded that the source of infection was in the patients’ homes. However, although the report implies that poor lens care habits were involved, it became clear that Fusarium clinical isolates were more resistant to disinfectants in the lens care solution than CDC strains that were used for comparison. Moreover, resistance was related to the ability of the microorganism’s capacity to form biofilm (see below). Reports from several regions of

426

the USA, including Florida and San Francisco, described cases of contact-lens-associated Fusarium keratitis demonstrating severe corneal opacification and descemetocele formation (hernia of Descemet’s membrane), most of which required keratoplasty. The CDC also reported that the number of cases of Fusarium keratitis dropped after Renu with Moisture Lock was voluntarily withdrawn from the market. However, several cases have been reported subsequently that were not due to this lens care product and were most likely due to increased awareness of Fusarium, although the causes were not always apparent.

Biofilm Formation in Contact-LensAssociated Keratitis Biofilm is defined as microbial secretion of an extracellular matrix surrounding the organisms. Biofilm formation allows the organisms to resist antibiotics (20–1000 times more resistant than planktonic forms), and to host immune responses. The CDC report on the contact-lens-associated outbreak of Fusarium keratitis also suggested that biofilm formation contributes to the resistance phenotype, as bacterial biofilm can form on contact lenses and lens cases. Bacterial biofilms can be generated rapidly on contact lenses and may therefore contribute to the pathogenesis of keratitis and endophthalmitis. Imamura and co-workers showed that Fusarium forms a biofilm on silicone hydrogel contact lenses; furthermore, the architecture, thickness, and composition of the biofilm differ according to the contact lens type. It is likely that the conidia germinate on the contact lens surface, and favorable conditions allow biofilm development. Once the biofilm is formed, the organisms are more resistant to antifungal agents, including those in multipurpose lens care solutions. Consistent with this notion, the Fusarium strain used to test lens care solutions did not form a biofilm and was more sensitive to lens care solutions (Figure 1).

Keratitis Caused by Candida Candida species are the most common pathogenic yeast associated with keratitis. Candida albicans is part of the normal commensal flora; however, these organisms can cause opportunistic corneal infections in immunosuppressed

Pathogenesis of Fungal Keratitis

Etafilcon A

Galyfilcon A

Lotrafilcon A

A

B

C

A

B

C

(a)

(b) Figure 1 Fusarium biofilm formed on different soft contact lenses. (a) Fusarium conidia were incubated with each contact lens for 2 h, after which time the lenses were removed and incubated a further 48 h. Biofilms were formed by FSSC 1-b isolate MRL8609 on soft contact lenses, and their gross morphologies were imaged using a digital camera. All lenses tested supported biofilm formation by strain MRL8609. (b) The Fusarium FSSC 1-b strain MRL8609 was allowed to form mature biofilms on Etafilcon A silicon hydrogel contact lenses and then was stained with ConA and FUN1 dyes to show extracellular matrix (red) and live organisms (green). Stained lens-containing biofilms were analyzed by confocal scanning laser microscopy. Etafilcon A (A), galyfilcon A (B), lotrafilcon A (C), balafilcon A. Arrows indicate extracellular matrix in the biofilms. Similar results were found for C. albicans (not shown). Reprinted from Imamura, Y., Chandra, J., Mukherjee, P. K., et al. (2008). Fusarium and Candida albicans biofilms on soft contact lenses: Model development, influence of lens type, and susceptibility to lens care solutions. Antimicrobial Agents and Chemotherapy 52: 171–182.

individuals or following trauma or surgery. In contrast to filamentous fungi in which trauma is the major predisposing condition, Candida is primarily associated with therapeutic contact lenses, steroid use or immunosuppressive disease, and corneal surgery. In these dimorphic organisms, the yeast stage initially infects the cornea, and then germinates to form pseudohyphae in the corneal stroma. Candida produces a number of proteases and phospholipases (particularly phospholipase B) that facilitate their penetration through the cornea and contribute to tissue destruction. Using C. albicans mutants in a murine model of keratitis, Jackson and colleagues showed that C. albicans virulence depends on expression of genes encoding or regulating hyphal formation, but not genes regulating adherence.

Fungal Keratitis Associated with Trauma Although relatively rare in North America and Europe, filamentous fungi are among the most common causes of microbial keratitis and corneal ulcers in India, China, and Ghana. In the southern USA, Fusarium solani and Fusarium

427

oxysporum are the most common causes of mycotic keratitis, with Aspergillus species being the second most common cause, including A. fumigatus, A. niger, and A. nidulans. Corneal trauma is the main predisposing factor, and the incidence of fungal keratitis increases during harvest season, which is consistent with the majority of cases associated with agricultural work, where it affects more males than females of working age. Trauma can be caused by numerous factors, such as airborne soil and plant material. Filamentous fungi are ubiquitous in the environment, especially on plants, and the fungal spores (conidia) can penetrate the corneal epithelium, germinate in the stroma, and if unchecked by the host response or effective antifungal therapy, fungal hyphae will grow in the corneal stroma, penetrate Descemet’s membrane, and invade the anterior chamber. Role of Matrix Metalloproteinases in Fungal Keratitis Matrix metalloproteinases (MMPs) have an important role in tissue remodeling, wound healing, and inflammation. Rohini and co-workers examined human tears from fungal keratitis patients and corneal sections after keratoplasty and detected elevated collagenases MMP-2 and MMP-8, and the MMP-9 gelatinase, which is consistent with the activation and degranulation of infiltrating neutrophils. In addition to microbial killing, which is primarily mediated by oxygen radicals, neutrophils also prevent microbial dissemination by releasing MMPs and causing local tissue damage. Dong and co-workers as well as Mitchell and co-workers showed that MMP-2 and MMP-9 were also elevated in rabbit and mouse models of Fusarium and Candida keratitis, and Lin and colleagues demonstrated a role for MMP-8 in corneal inflammation by mediating breakdown of collagen and release of chemotactic Pro–Gly–Pro peptides, which then facilitate neutrophil migration through the cornea. Although differences between Aspergillus and Fusarium growth in the stroma have been reported by Xie and colleagues, it is not clear at present how this relates to MMP activity, or if there is a difference in protease production by these organisms. Role of Innate Immunity in Fungal Keratitis Since the 1960s, it has been apparent that the host immune response regulates fungal growth and the outcome of the infection. In rabbit and murine models of Fusarium and Candida keratitis in which either conidia or yeast is applied topically to the abraded epithelium, or is injected intrastromally, a neutrophil-rich cellular infiltrate into the corneal stroma ultimately clears the infection. However, subverting the host response by systemic


treatment with cyclophosphamide leads to increased hyphal penetration of the corneal stroma, decreased cellular infiltration, (especially neutrophils), and the unchecked growth of hyphae, causing corneal perforation. These results indicate that the host response plays a critical role in restricting fungal growth in the cornea. To characterize the host response to fungal challenge, Candida-infected corneas were processed for microarray analysis, which demonstrated that the proinflammatory cytokines, interleukin-1 (IL-1) and tumor necrosis factoralpha (TNF-a), were upregulated. To characterize the innate immune response, Tarabishy and colleagues injected Fusarium conidia into the corneal stroma of immunocompetent C57BL/6 and mice on the same genetic background in which genes related to the Toll-Like Receptor (TLR) family of pathogen recognition molecules were knocked out. Figure 2 shows that 6 h after intrastromal injection of 10 000 conidia, hyphae were detected in the corneal stroma of C57BL/6 mice and in mice in which the gene for the MyD88 adaptor molecule common to most TLRs and IL1R1 is mutated. Although a cellular infiltrate was detected in the peripheral cornea of C57BL/6 mice, this infiltrate was absent in MyD88–/– mice. Figure 3 shows that whereas C57BL/6 mice rapidly develop corneal opacification associated with a pronounced infiltrate and clear the organisms, MyD88–/– mice had delayed cellular infiltration, and even though neutrophils were recruited to the corneal stroma,

Control DEB

Murine Model of Aspergillus Keratitis Aspergillus species, the second most common cause of fungal keratitis after Fusarium, are also ubiquitous in the environment, and most people inhale hundreds of conidia daily. Although pulmonary aspergillosis occurs primarily in immunosuppressed individuals, this is not the case in keratitis, where the risk factors are similar to those of Fusarium, that is, the highest incidence is associated with agriculture and trauma. In addition, Aspergillus conidia are smaller than Fusarium conidia, can therefore penetrate deeper into the lungs, and likely also penetrate deeper into the corneal stroma. We generated a strain of Aspergillus fumigatus expressing a red fluorescent protein, and injected conidia into the corneal stroma of C57BL/6 mice. Figure 6 shows that after 24 h, the cornea is opaque. However, Figure 6(b) also shows that the presence of corneal opacities coincides with the presence of Aspergillus. Figure 6(c) shows higher magnification of hyphae in the corneal stroma. Ongoing studies are examining the role of the host response and Aspergillus virulence factors in the pathogenesis of this disease.

0.20

Central

Peripheral

C57BI/6

Metabolic activity (XTT OD 492 nm, mean ± SD)

0.25

they were unable to clear the organisms (Figures 4 and 5). Fusarium hyphae are detected throughout the cornea (Figure 5), which perforated within 4 days. Subsequent experiments showed that IL-1R1 is required for neutrophil recruitment to the cornea, whereas TLR4 is important for fungal killing.

0.15 0.10 0.05

0.00 MRL8609

MRL8996 ATCC36031 Fusarium isolates

SC5314

MyD88–/–

428

C. albicans

Fungal isolate Figure 2 Fusarium strain differences in biofilm formation on lotrafilcon A lenses. Biofilms were formed in the absence or presence of DEB (which inhibits biofilm formation) on a lotrafilcon A lens using the FSSC 2-c ATCC 36031 reference isolate or clinical isolate FSSC 1-b (MRL8609), FOSC 3-a (MRL8996). Biofilms were quantified using the XTT metabolic activity assay. Data represent means (+/– SDs) calculated from three separate experiments. Reprinted from Imamura, Y., Chandra, J., Mukherjee, P. K., et al. (2008). Fusarium and Candida albicans biofilms on soft contact lenses: Model development, influence of lens type, and susceptibility to lens care solutions. Antimicrobial Agents and Chemotherapy 52: 171–182.

Figure 3 In vivo confocal microscopy of Fusarium keratitis in C57BL/6 and MyD88–/– corneas. C57BL/6 and MyD88–/– mice were injected intrastromally with 1 104 conidia from a clinical isolate of F. oxysporum. After 6 h, mice were examined by in vivo confocal microscopy (Confoscan). Representative images from the central and peripheral corneal stroma are shown. Note the presence of hyphae in the central corneal stroma of C57BL/6 and MyD88–/– mice (a, c); however, a cellular infiltrate is present in the peripheral cornea of C57BL/6, but not MyD88–/– mice (b, d). Reprinted from Tarabishy, A. B., Aldabagh, B., Sun, Y., et al. (2008). MyD88 regulation of Fusarium keratitis is dependent on TLR4 and IL-1R1 but not TLR2. Journal of Immunology 181: 593–600.

Pathogenesis of Fungal Keratitis

d1

d2

C57BL/6

6h

429

MyD88–/–

CFU/eye

10000

1000

(a) 100 Clinical score

4

p = 0.0006

3

B6 (c)

MyD88

B6

6h

MyD88 24 h

B6

MyD88 48 h

2 1 0

(b)

MyD88–/–

C57BL/6

Figure 4 Fungal keratitis in C57BL/6 and MyD88–/– mice. Mice were injected intrastromally with 1 104 conidia from a clinical isolate of F. oxysporum as described above. Corneal opacification, CFU, and histology were examined by standard methods. (a, b) Corneal opacification in MyD88–/– mice was impaired at 24 h, but increased until 72 h after which time MyD88–/– corneas perforated, whereas C57BL/6 corneas eventually resolved. (c) CFU decreased in C57BL/6 mice over time, whereas Fusarium replicated in the corneas of MyD88–/– mice. Reprinted from Tarabishy, A. B., Aldabagh, B., Sun, Y., et al. (2008). MyD88 regulation of Fusarium keratitis is dependent on TLR4 and IL-1R1 but not TLR2. Journal of Immunology 181: 593–600.

Epi Str a/c (a)

(c)

(b)

(d)

Figure 5 Impaired cellular infiltration in MyD88–/– mice (a, b) Histological analysis in MyD88–/– corneas after PAS staining shows Fusarium hyphae penetrating Descemet’s membrane after 24 h, and growing in the stroma and anterior chamber after 48 h despite the presence of a cellular infiltrate. In contrast, there was an early and pronounced cellular infiltrate in C57BL/6 mice (c, d) comprised mostly of neutrophils. Reprinted from Tarabishy, A. B., Aldabagh, B., Sun, Y., et al. (2008). MyD88 regulation of Fusarium keratitis is dependent on TLR4 and IL-1R1 but not TLR2. Journal of Immunology 181: 593–600.

(a)

(b)

(c)

Figure 6 Aspergillus fumigatus in murine cornea. Aspergillus fumigatus was transfected with a plasmid expressing m-cherry. Conidia were injected into the corneal stroma of C57BL/6 mice, and after 24 h, corneas were examined by light (a) and fluorescence (b) microscopy. (c) Whole mount cornea examined by confocal microscopy.

require a combination of human and animal studies to determine the course of events leading to fungal killing and resolution of infection. Human disease correlates are particularly difficult to study; however, Bochud and co-workers showed that polymorphisms in TLR4 are associated with susceptibility to systemic aspergillosis; therefore, it is possible that TLR4 also mediates susceptibility to Aspergillus keratitis.

Acknowledgments Conclusions The pathogenesis of fungal keratitis depends on the balance between the host response and expression of fungal virulence factors. Although some mediators of innate immunity and fungal virulence factors have been identified, it will

Studies presented in this article were supported by NIH grant EY18362 (EP) and EY11373 (EP), by DE017486-01A1 (MAG) and R01DE 13932 (MAG), and by the Research to Prevent Blindness Foundation and the Ohio Lions Eye Research Foundation.

430


See also: Contact Lenses; Corneal Epithelium: Response to Infection; Innate Immune System and the Eye.

Further Reading Alfonso, E. C., Cantu-Dibildox, J., Munir, W. M., et al. (2006). Insurgence of Fusarium keratitis associated with contact lens wear. Archives of Ophthalmology 124: 941–947. Bharathi, M. J., Ramakrishnan, R., Meenakshi, R., et al. (2007). Microbial keratitis in South India: Influence of risk factors, climate, and geographical variation. Ophthalmic Epidemiology 14: 61–69. Bochud, P. Y., Chien, J. W., Marr, K. A., et al. (2008). Toll-like receptor 4 polymorphisms and aspergillosis in stem-cell transplantation. New England Journal of Medicine 359: 1766–1777. Chang, D. C., Grant, G. B., O’Donnell, K., et al. (2006). Multistate outbreak of Fusarium keratitis associated with use of a contact lens solution. Journal of the American Medical Association 296: 953–963. Dong, X., Shi, W., Zeng, Q., and Xie, L. (2005). Roles of adherence and matrix metalloproteinases in growth patterns of fungal pathogens in cornea. Current Eye Research 30: 613–620. Grant, G. B., Fridkin, S., Chang, D. C., and Park, B. J. (2007). Postrecall surveillance following a multistate Fusarium keratitis outbreak, 2004 through 2006. Journal of the American Medical Association 298: 2867–2868.

Imamura, Y., Chandra, J., Mukherjee, P. K., et al. (2008). Fusarium and Candida albicans biofilms on soft contact lenses: Model development, influence of lens type, and susceptibility to lens care solutions. Antimicrobial Agents and Chemotherapy 52: 171–182. Jackson, B. E., Wilhelmus, K. R., and Mitchell, B. M. (2007). Genetically regulated filamentation contributes to Candida albicans virulence during corneal infection. Microbial Pathogenesis 42: 88–93. Mitchell, B. M., Wu, T. G., Chong, E. M., Pate, J. C., and Wilhelmus, K. R. (2007). Expression of matrix metalloproteinases 2 and 9 in experimental corneal injury and fungal keratitis. Cornea 26: 589–593. Pearlman, E., Johnson, A., Adhikary, G., et al. (2008). Toll-like receptors at the ocular surface. Ocular Surface 6: 108–116. Rohini, G., Murugeswari, P., Prajna, N. V., Lalitha, P., and Muthukkaruppan, V. (2007). Matrix metalloproteinases (MMP-8, MMP-9) and the tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2) in patients with fungal keratitis. Cornea 26: 207–211. Tarabishy, A. B., Aldabagh, B., Sun, Y., et al. (2008). MyD88 regulation of Fusarium keratitis is dependent on TLR4 and IL-1R1 but not TLR2. Journal of Immunology 181: 593–600. Wu, T. G., Keasler, V. V., Mitchell, B. M., and Wilhelmus, K. R. (2004). Immunosuppression affects the severity of experimental Fusarium solani keratitis. Journal of Infectious Diseases 190: 192–198. Xie, L., Zhai, H., Shi, W., et al. (2008). Hyphal growth patterns and recurrence of fungal keratitis after lamellar keratoplasty. Ophthalmology 115: 983–987. Yuan, X., Mitchell, B. M., and Wilhelmus, K. R. (2008). Gene profiling and signaling pathways of Candida albicans keratitis. Molecular Vision 14: 1792–1798.

Conjunctiva Immune Surveillance E Knop, Charite´ – Universita¨tsmedizin Berlin, Berlin, Germany N Knop, Hannover Medical School, Hannover, Germany ã 2010 Elsevier Ltd. All rights reserved.

Glossary Conjunctiva-associated lymphoid tissue (CALT) – It is the physiological protective mucosal immune tissue of the conjunctiva. It consists of lymphoid cells and accessory cells inside the mucosal tissue and can be divided into the epithelial and underlying connective tissue (lamina propria) compartments. It is arranged as a diffuse lymphoid effector tissue along the whole extension of the conjunctiva and has interspersed organized lymphoid follicles for afferent antigen uptake and effector cell generation. Dendric cells (DCs) – They are a special class of professional antigen-presenting cells (APC, together with macrophages and B-cells). They take up external antigens, degrade them into small fragments (epitopes), present them on MHC-class-II to T-helper cells, and hence, induce immune reactions. Depending on their maturation status which is influenced by the presence of inflammatory signals, they modulate between the inductions of tolerance versus inflammation. They also link the unspecific innate to the induced specific immune system and are hence key modulators of the immune reaction. Eye-associated lymphoid tissue (EALT) – This tissue summarizes all the lymphoid tissues of the extended mucosal ocular surface, that is, of ocular surface proper (conjunctiva and cornea) along with its mucosal adnexa (the lacrimal-drainageassociated lymphoid tissue, LDALT, and the lymphoid cells inside the lacrimal gland). EALT is in line with the mucosal immune system in other parts of the body (e.g., gut-associated lymphoid tissue (GALT) in the gut and bronchus-associated lymphoid tissue (BALT) in the airways). High endothelial venules (HEVs) – Specialized postcapillary venules that have an endothelium of bright roundish cells compared to the ordinary flat dense ones. They are located in lymphoid tissues and have tissue-specific adhesion molecules (vascular addressins) on the cell surface that specifically interact with homing receptors on circulating lymphocytes in order to maintain a regulated immigration of lymphocytes into the tissue. Human leukocyte antigen (HLA) – A system of the major histocompatibility complex () MHC)

in humans. It contains of a number of genes and their respective encoded proteins (that can act as antigens). The term HLA is frequently used to describe immunological self and nonself in the context of transplant rejection. Intercellular adhesion molecule 1 (ICAM-1) – An adhesion molecule (CD54 according to the immunological cluster of differentiation, CD, nomenclature) mainly on vascular endothelial cells which is upregulated in inflammation and promotes the increased immigration of leukocytes, that carry corresponding integrin receptors, into the tissue. Membraneous cells (M-cells) – Also called microfolded cells, they are a special type of cells in the modified epithelium overlying organized lymphoid follicles, the so-called follicle-associated epithelium (FAE). Their name refers to the fact that they have a different, usually smooth, surface ultrastructure compared to the ordinary epithelial cells. They form cellular pockets populated by groups of leukocytes which are separated from the lumen by a thin luminal cytoplasmic sheet. M-cells actively transcytose luminal antigens for uptake by the leukocytes and their subsequent presentation to and activation of T- and B-cells in order to generate antigen-specific effector cells. Major histocompatibility complex (MHC) – It is differentiated mainly into class-I and class-II. Their encoded proteins on the surface of cells perform the presentation of protein antigen fragments (epitopes) to immune cells. MHC-class-I is found on all nucleated cells and presents antigens produced inside the cell (either own or viral proteins after infection) to cytotoxic CD8 lymphocytes and natural killer cells. MHC-class-II, in contrast, occurs physiologically only on specialized antigenpresenting cells and presents foreign antigens to the CD4 receptor of T-helper cells. In inflammation, it can be upregulated by other cells. Lipopolysaccharide (LPS) – A component of the outer cell membrane of the wall of Gram-negative bacteria that acts as an endotoxine. The presence of LPS, that is detected for example, by toll-like receptors, signals the pathogenic nature of antigens to the immune system and elicits a strong inflammatory reaction.

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Tolerance – Immune tolerance is a status in which the immune system is in a state of nonreactivity to an antigen in order to prevent inflammatory tissue-destructive reactions. Tolerance is actively generated and directed not only against the bodies’ own cellular self-antigens but also against nonpathogenic external antigens. If tolerance fails, autoimmunological disease or allergy may occur. Tolerance is the default mode of the mucosal immune system, including CALT and EALT, in order to preserve tissue integrity.

lymphocytes (IELs) that reside mainly in the basal layers (Figure 2(b) and Figure 3(a)) as well as dendritic cells (DCs), that have long narrow extensions, for uptake of antigens from the surface. The conjunctival epithelial surface is covered by small cytoplasmic protrusions (microvilli and microplicae) with a well-developed surface coat of filamentous projections (glycocalyx) that form a meshwork (Figure 1(c) and Figure 2(a)). Epithelial Immune Surveillance Takes Care of Environmental Antigens Physical and physicochemical barriers keep antigens outside

Conjunctival Morphology and Function Are Closely Interacting for Immune Surveillance Epithelial Defense Mechanisms Epithelial morphology and function

The conjunctiva is a moist mucous organ that consists of a surface epithelium and an underlying loose connective tissue (lamina propria), separated by the epithelial basement membrane. The epithelium of the human conjunctiva has, in contrast to small rodents (e.g., rat and mouse), a stratified nonsquamous morphology and consists of two to three cell layers of cubical cells in most parts. It becomes multilayered and assumes prismatic morphology at the fornix whereas it tends to become squamous toward the limbus (Figure 1(a) and 1(b)). Interspersed mucus-secreting goblet cells occur inside the epithelium as single cells or in small groups as well as intraepithelial

The structure of the conjunctival epithelium already contributes to basic protective mechanisms which can be considered as part of the innate defense. Epithelial cells are mechanically connected by desmosomes and have an apical belt of intercellular junctions including tight junctions that seal the intercellular space and limit the passive para-cellular leakage of antigens in and out of the tissue (Figure 2(a)). This physical cellular barrier is supplemented by the physicochemical barrier of the epithelial mucins, that consist of cell membrane-spanning mucins (glycocalyx) produced by the ordinary epithelial cells and of soluble mucins secreted by the goblet cells which mix with the aqueous phase. Together they form a layer in the range of few micrometers thickness, that is, a sticky gel to which microbes adhere and can hence be cleared by the constant renewal of the preocular tear film. Soluble protective factors, including secretory immunoglobin A (SIgA), are fixed to the mucin layer in order to make it an almost impenetrable and lethal barrier to antigens and in particular to microbes.

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Figure 1 Structure of the human conjunctival epithelium. (a) The epithelium of the human conjunctiva is stratified cuboidal in most regions and assumes more layers with prismatic surface cells toward the fornix. Interspersed goblet cells (BZ) release mucus (M) tufts onto the surface. (b) Goblet cells contain densely packed mucin granules and a flat or triangular nucleus. They may be slightly inclined if located in the relatively flat bulbar epithelium close to the limbus. The surface of the conjunctival epithelial cells shows numerous microprotrusions that result in a rough surface in low-magnification transmission electron microscopy. (c) In higher enlargement, microvilli (MV) and microplicae (MP) are seen which have a dense glycocalyx of fine molecular antennae (arrows) that project into the lumen and form a meshwork, as better seen in cross section (inset, 2). Reproduced from Knop, E. and Brewitt, H. (1992). Morphology of the Conjunctival Epithelium in Spectacle and Contact Lens Wearers – A Light and Electron Microscopic Study. Contactologia, Stuttgart: Enke Verlag.


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Figure 2 Defense systems of the human conjunctiva. (a) The conjunctival epithelium has an array of defense systems consisting of the integrity of the surface epithelial cells (provided with pattern recognition receptors, TLR) that are sealed by apical tight junctions (tj), of the attached mucin layer that is enforced by adhering antimicrobial proteins and peptides (AMPs) including specific secretory IgA (SIgA) and of the overlying tear film (shaded blue) that contains similar protective molecules and provides a washing effect. (b) A diffuse effector tissue is formed by lymphoid cells of the specific adaptive immune system and by innate cells such as macrophages (mø), mast cells (mc), neutrophilic granulocytes (n), and dendritic cells (dc). They functionally interact with stromal fibrocytes (fi). Lymphoid cells consist of CD4þ and CD8þ T-cells (black circles) that constitute intraepithelial and lamina propria lymphocytes. Differentiated B-cells (plasma cells (pc), large blue) produce dimeric IgA, which is transported through the epithelium as SIgA. (c) Interspersed solitary lymphoid follicles consist of B-cells (small blue circles), frequently have a bright germinal center due to cell proliferation, have an apical follicle-associated epithelium (FAE) with M-cells for antigen transport but without goblet cells (gc) and have para-follicular T-cell (small black circles) zones with lymph vessels (yellow) and high endothelial venules (HEVs); small arrows indicate the direction of cell migration. The mechanisms for conjunctival immune surveillance are explained topographically in this figure from the epithelium (a) over the diffusely interspersed effector cells (b) toward the organized lymphoid follicles that generate the effector cells (c). Functionally, it is reverse because the effector cells generated in lymphoid follicles after antigen uptake and presentation recirculate via the blood circulation (symbol of heart and blood flow between (c) and (b)) to and migrate into the diffuse effector sites to exert their protective function by cell contact or by soluble mediators. The drawing is not to scale.

The mechanical washing effect of the tear film wipes away antigens and detritus

The tear film is an important functional component of the ocular surface mucosal protection system. Apart from providing the necessary moisture, the constant flow of tears over the ocular surface and in particular, over the cornea, together with the wiping effect of the lid margin with every blink, provides a constant mechanical washing. This discharges antigens and removes dust and cell detritus. Other parts of the ocular surface along the retropalpebral tear film are not so rapidly cleared so that antigens can stay in longer contact with the epithelium. Therefore, the tear film contains a large number of antimicrobial factors that contribute more specifically to the innate immune defense. Epithelial innate immune defense factors

The innate immune system uses pattern-related receptors (PRRs) that mainly detect conserved pathogen-associated molecular patterns (PAMPs) but also host antigens from destroyed cells. It reacts via effectors, which consist of soluble antimicrobial proteins and peptides (AMPs)

which bind to the microbial cell wall in order to destroy it or which interfere with the microbial metabolism. The innate immune system also employs production of soluble mediators, such as inflammatory cytokines and chemotactic cytokines (chemokines) that functionally couple the innate and adaptive immune answer. PRRs on epithelial cells provide an external alarm system

As soon as microbial antigens have breached the physicochemical barrier, they get in touch with epithelial PRRs (Figure 2(a), the most prominent of which is presently the diverse family of toll-like receptors (TLRs). Binding of their ligands causes activation of the host cell via a MyD88-dependent signaling pathway that activates a nuclear transcription factor, nuclear factor kappa B (NFkB), and results in production of inflammatory cytokines such as interleukin 6 (IL-6), interferon gamma (IFN-g), or tumor necrosis factor alpha (TNF-a). Subsequently, these induce the production of chemokines, adhesion molecules, and inducible AMP. Altogether this represents an inflammatory cascade with activation, first

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Figure 3 Characteristics of diffuse CALT. (a) Tarso-orbital conjunctiva. Plasma cells (P) and lymphocytes (l) form a diffuse lymphoid cell layer in the lamina propria covered by an epithelium with intraepithelial lymphocytes (arrowhead). A high endothelial venule (HEV) underneath has typical roundish endothelial cells (E) and contains lymphocytes within and around the wall (arrows). (b) TEM shows an intraepithelial lymphocyte (l) between epithelial cells (E) on the basement membrane (arrowheads). (c) Immunostaining indicates T-cells inside the epithelium (arrowhead), in the lymphoid layer (l) and around or in the wall (arrows) of a HEV (asterisk). (d) Ultrastructurally, a HEV shows large bright endothelial cells (E), a contractile pericyte layer (PE), and adjacent (l) or intramural (arrow) lymphocytes. (e) The vast majority of plasma cells in the lymphoid layer are IgA positive as also deposits in the epithelium (arrowheads), while IgM (f) is rare. (g) The epithelium is positive for the transporter SC. (h) A plasma cell lying in the loose collageneous (C) tissue has extended rough endoplasmic reticulum (RER), mitochondria (M), large nucleolus (N), and radial heterochromatin; (b, d, h: bar ¼ 1 mm; a, c, e–g: bar ¼ 10 mm). (i (1–3)) Lacrimal gland, LG, with lymphocytes (arrow), and plasma cells (arrowead) between the roundish acini. (2) IgA is found strongly in plasma cells and as weaker patchy staining in acinar epithelial cells, which more strongly express SC (3). (j (1–3)) Excretory lacrimal ducts that connect the LG to the conjunctiva have similar characteristics but in the epithelium IgA (2) and SC (3) are mainly expressed in the luminal layer; the duct has two cell layers but appears wider to the left due to oblique plane of section. (k (1,2)) Multiple-fluorescent staining for IgA (green), SC (red), and cell nuclei (blue) shows that the components of the secretory immune system are similarly arranged in the LG (1) and the conjunctiva (2, here orbital zone); bm level indicated by fine lines. IgA-positive plasma cells are diffusely interspersed in the LP of both tissues; in the LG frequently in groups. Epithelium (E) shows strong staining for SC;


of the epithelial cells and later also of lamina propria leukocytes and vascular endothelial cells. It induces leukocyte recruitment into the epithelium and their immigration from the blood stream into the tissue. The normal conjunctival epithelium expresses a number of different TLRs, similar to the cornea. TLR1, 2, 3, 5, and 6 were found in all conjunctival and limbal epithelial cell samples, TLR4 and 9 only inconstantly, but not TLR7, 8, and 10. TLR2 may only occur upon stimulation by IFN-g and bacterial cell wall extract, for example, in patients with ocular allergy. This results in upregulation of inflammatory markers, such as the intercellular adhesion molecule 1 (ICAM-1), human leukocyte antigen (HLA), TNF-a, and IL-8, in a dose-dependent manner. Bacterial-specific TLRs are of interest in ocular allergy because colonization by bacteria is common there. The activation of TLRs represents an important co-factor in ocular allergy and their blockade can significantly inhibit release of inflammatory mediators which may turn out as a promising new therapy option for ocular allergy. The conjunctival epithelium secretes diverse AMPs

The spectrum of epithelial derived AMPs is distinct for cornea and conjunctiva but overlapping. Conjunctival epithelium produces not only the human b-defensins (hBD)-1,2,3 and further AMPs such as liver-expressed antimicrobial peptides (LEAPs) 1 and 2 and cathelicidin (LL-37) but also macrophage inflammatory protein 3alpha (MIP-3a) and thymosin beta 4 (Tb-4). Some of the AMPs are constitutively produced, whereas others are inducible. hBD-2 is induced by inflammatory cytokines in ocular surface inflammation and by presence of bacterial LPS, while hBD3 is induced by infection and LL-37 by epithelial wounding. Conjunctival AMPs such as LL-37 are active against bacterial (Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis) and viral (Herpes simplex virus 1, adenovirus) infection. They can act as multifunctional factors in wound healing and signaling pathways. Interestingly, the antimicrobial activity of some of these AMPs (e.g., hBD-1, hBD-2, and Tbeta-4) is almost completely inhibited in the presence of tear fluid. This may indicate that not all epithelial AMPs are produced in order to act as tear film factors but rather play a major role for local protection inside the conjunctival epithelium itself. Apart from AMPs, there is a plethora of other protective proteins. AMPs continue downstream in the lacrimal drainage system.

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Conventional antibacterial factors are surprisingly versatile defense tools

Even the ‘old fashioned’ established antimicrobial proteins in the tear film, such as lysozyme and lactoferrin, have surprising newly detected functions. Apart from being bactericidal, either by lysis of components in the Gram-positive bacterial cell wall (lysozyme) or by interfering with their iron metabolism (lactoferrin), they are also antifungal and antiviral. Through the absorption of the strongly inflammatory bacterial endotoxin lipopolysaccharide (LPS), which is a surface molecule on Gram-negative bacteria, they also act anti-inflammatory. They have further anti-inflammatory functions by their influence on antigenpresenting cells (APCs) and hence appear as key elements in host defense that link innate and adaptive immunity. Conjunctival Lamina Propria: Morphology and Function of the Diffuse Mucosal Immune System Diffusely arranged lymphoid and innate cells contribute to conjunctival immune surveillance

The lamina propria contains bone-marrow-derived cells and vessels of different types. Apart from capillaries and lymph vessels, specialized high endothelial venules (HEVs) occur. Vessels serve for the supply with nutrition and discharge of metabolites, for hormonal regulation of the tissue, and also for the migration of immune cells. Lymphocytes, together with accessory leukocyte populations (macrophages, granulocytes, mast cells, and DCs), form a diffuse lymphoid tissue (Figure 3(a)) which is regarded mainly as an effector site of CALT although antigen uptake via DCs can also occur here to a certain extent. The diffuse lymphoid tissue is located in the vast majority of the surface, except for the solitary lymphoid follicles. The thickness of this cell layer depends on the location along the conjunctiva, shows a certain topographical variation, and is frequently only one to two cells wide, which may be a reason why these cells have often been overlooked in the past. It also shows a certain interindividual variation that may depend on the immune status of the person. IEL also functionally belong to the diffuse effector cells (Figure 3(b)). Different subtypes of lymphocytes occur in the conjunctiva

Diffuse conjunctival lymphocytes are mainly CD3þ T-cells (Figure 3(c)) (whereas CD20þ B-cells are largely restricted to the solitary lymphoid follicles). They are activated (CD45Roþ, CD25þ) and express the human

goblet cells (asterisks) are negative for SC. Mixed color indicating both proteins (¼SIgA) is seen in the tubuloacinar lumina (LU) of the LG and frequently delineates the luminal cell surface. (a–h) Adapted from Knop, N. and Knop, E. (2000). Conjunctiva-associated lymphoid tissue in the human eye. Investigative Ophthalmology and Visual Science 41: 1270–1279. (i–k) Knop, E., Knop, N., and Claus, P. (2008). Local production of secretory IgA in the eye-associated lymphoid tissue (EALT) of the normal human ocular surface. Investigative Ophthalmology and Visual Science 49: 2322–2329.

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mucosa lymphocyte antigen (HML-1, integrin aEb7) which substantiates the integration of CALT into the mucosal immune system. In the epithelium CD8þ, cytotoxic/suppressor cells prevail that have been proposed to act mainly in the suppressor mode and may hence provide a component of the immune tolerance at the ocular surface. Lamina propria lymphocytes, in contrast, consist of equal or prevailing amounts of CD4þ T-helper (Th) cells compared to CD8þ T-cells. All known types of T-cells exist in CALT and their immune regulation is considered below (see the section titled ‘Mechanisms of conjunctival immune regulation’). Plasma cells (see the section titled ‘The conjunctiva contributes actively to the secretory immune system’) account for about 20% of the conjunctival leukocytes in histology but their absolute number is in the range (2/3) of those in the lacrimal gland that was long regarded as the sole source of tear film SIgA. Conjunctival lymphocytes are even more abundant. This supports the concept that the conjunctiva considerably contributes to its own specific defense and it very well supports the recognition of a diffuse CALT along the whole extension of the human conjunctiva in tissue whole mounts. The conjunctiva contributes actively to the secretory immune system

Specific soluble antigen-receptors (immunoglobulins, Ig) produced by local mucosal differentiated B-lymphocytes (plasma cells) and transported through the overlying epithelium constitute the secretory immune system. This is a major mucosal defense mechanism and is also present in the conjunctiva. Mucosal Ig mainly consist of polymeric (p)IgA that also forms the predominant Ig in the tear film, besides small amounts of the other polymeric Ig (IgM) and trace amounts of IgG. During eye closure, overnight IgA is the predominant protein in the closed eye tear film, but only recently the components of the conjunctival secretory immune system could be consistently verified by immunohistochemistry and polymerase-chain reaction. Local conjunctival plasma cells produce mainly the antiinflammatory IgA

The vast majority of conjunctival plasma cells produce IgA and hence stain positive for it in immunohistochemistry (Figure 3(e)). IgM, which performs the initial acute secretory immune answer, is rarely observed (Figure 3(f)) and hence indicates that the physiological conjunctival diffuse lymphoid cells do not reflect any kind of reaction to an acute insult. The epithelial transporter molecule for both of them (pIgR, represented by its extracellular domain secretory component, SC) is strongly expressed throughout the human conjunctival epithelium (Figure 3(g)). After transport, SC remains linked to pIgA which together constitute secretory IgA (SIgA). Conjunctival plasma cells show a typical ultrastructure in transmission electron microscopy (Figure 3(h)).

IgA-positive plasma cells in the lamina propria and SC in the overlying epithelium are continuously expressed from the lacrimal gland (Figure 3(i) 1–3) along its excretory ducts (Figure 3(j) 1–3) into the conjunctiva and further within the lacrimal drainage system. In multifluorescent immune staining, the secretory immune system of the lacrimal gland and the conjunctiva show the same characteristics (Figure 3(k) 1,2). SIgA performs diverse protective and antiinflammatory functions at the ocular surface

SIgA is deposited onto the epithelial surface and into the tear film (Figure 2(a)). It contributes to the binding of specific antigens and to their immobilization and discharge. It binds to the surface of microbes and viruses and thereby limits their binding to and entrance into the tissue. It binds and thereby neutralizes bacterial toxins such as LPS. SIgA antibodies occur naturally to the physiological commensal ocular flora and are induced by the presence of pathological microbes, such as Acanthamoeba and Pseudomonas. IgA does not only exert immune functions at the luminal ocular surface but also locally inside the tissue. IgA has a low complement-binding activity and hence acts in an antiinflammatory fashion. Bound antigens are opsonized to phagocytes which facilitates microbe uptake and destruction. IgA can bind to pathogens that have already penetrated into the tissue including intracellular viral particles. During the vectorial transport of pIgA toward the lumen, the bound pathogens are cleared from the tissue. IgA-bound antigens have an antiinflammatory effect on signaling networks and immune regulation inside the tissue by induction of the tolerogenic cytokines TGF-b and IL-10 and by limiting the activation of DC. Lamina Propria Leukocytes Provide Immediate Innate Response against Invading Pathogens and can Orchestrate an Inflammatory Reaction Apart from the lymphocytes, various other types of bonemarrow-derived leukocytes exist in the diffuse conjunctival effector tissue that are all not purely pathogenic, but exert important protective immune functions. Macrophages are reportedly frequent in immunohistochemistry but less obvious in conventional histology (Figure 3(a)). Phagocytes, such as macrophages, granulocytes, and DCs, can engulf and devour the invader but in addition, they act in different ways. Macrophages mainly destroy pathogens by internal digestion but are also capable to present epitopes of the pathogen on the MHC-class-II molecule (MHC-II) to CD4þ T helper-cells for their subsequent activation. DCs digest pathogens mainly for this purpose. Granulocytes, also known as polymorphonuclear (PMN) cells, are usually the first cells that arrive at the site of acute defense reactions.


Protective functions of conjunctival lamina propria leukocytes

Neutrophile granulocytes are regularly observed in the normal human conjunctiva. They account for about 5% of the leukocytes and occur massively in the closed-eye tear film. In addition to their phagocytic capacity, they secrete several soluble factors such as antimicrobial proteins (e.g., lactoferrin, alpha-defensins, and cathelicidin) for immediate destruction of microbes. Furthermore, they secrete proteases (e.g., cathepsins, gelatinase, and neutrophile elastase) that lead to digestion of the extracellular matrix in order to provide space for accommodating the plethora of cells necessary to mount an effective inflammatory response. Eosinophile granulocytes are reportedly not observed in a normal conjunctiva but immigrate in parasitic infection. They produce chemokines and cytokines (e.g., RANTES [CCL5], TGF-b, TNF-a) for the activation and recruitment of other leukocytes including T-cells, as found in ocular allergy. Mast cells, apart from their potential physiological function, are mainly known for their inflammatory activity during IgE-mediated allergic disease where they release a variety of vasoactive mediators (e.g., histamine and heparin) and Th1 and Th2 cytokines (IL-4, IL-5, IL-6, and TNF-a) that can orchestrate an inflammatory response. HEVs Provide the Regulated Immigration of Bone-Marrow-Derived Cells into the Tissue The bone-marrow-derived cells that populate the conjunctiva all arrive here via the blood stream. Most of them stay here but lymphocytes, after being primed, and DCs, after antigen uptake, can also leave the tissue again via lymphatics. Although lymphocytes can emigrate through ordinary capillaries and venules, they do so with higher efficiency through conjunctival HEV via their tissuespecific adhesion molecules (lymphocyte homing molecules) that interact with vascular endothelial addressins. HEVs occur particularly in the para-follicular T-cell areas of lymphoid follicles (Figure 2(c)) but they are also found in the diffuse lymphoid effector tissue of the conjunctiva. Emigrated T-cells are frequently observed around HEV (Figures 3(c) and 3(d)). Conjunctival HEVs are a normal component of the lymphoid tissue and they have a characteristic ultrastructure similar to that in other mucosal organs.

Conjunctival Lymphoid Follicles Have a Typical Morphology and Function Solitary organized lymphoid follicles are interspersed into the diffuse effector tissue along the conjunctiva. They are relatively flat due to the limited space in the narrow conjunctival lamina propria but still show typical follicular characteristics. They consist of accumulations of

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B-cells (Figure 4(a)). The overlying epithelium changes its morphology toward the apex into a follicle-associated epithelium (FAE) by losing the goblet cells, by assuming a flat cell shape (Figures 4(a)–4(c)), and by a rarefied expression of SC (Figure 4(c)). Groups of lymphocytes, including CD20þ B-cells (Figure 4(a)) and CD3þ T-cells, occur inside the epithelium and are separated from the lumen just by a very narrow epithelial lining. Altogether, this is a conspicuous sign for the presence of specialized M-cells that form intraepithelial pockets populated by lymphoid cells. M-cells serve for the uptake and transport of luminal antigens toward the lymphoid cells in the pocket that can detect the antigen and present it to naive lymphocytes. The typical morphology of conjunctival M-cells and their antigen transport have been verified in a number of animal species, including guinea pig, turkey, chicken, rabbit, dog, and monkey. It has been shown that CALT is able, for example, to induce a tolerance against retinal antigens upon their topical conjunctival instillation. The number of lymphoid follicles in elderly humans is relatively low (about 10 follicles per eye) with an average diameter of about 0.25 mm. Their small size again offers an explanation for the fact that CALT has frequently been overlooked in the past. They are more frequent in the upper conjunctiva than in the lower one (Figure 5(a)) and show a bilateral symmetry. In younger individuals, however, lymphoid follicles are more frequent and before puberty they occur in every person. Therefore, CALT follicles show a similar involution with age as observed for other locations of the mucosal immune system in general. Mucosal lymphoid B-cell follicles and their associated para-follicular T-cell zones (Figure 2(c)) serve for the generation of B- and T-effector lymphocytes, respectively (Figure 7).

The Topographical Distribution of CALT is in the Right Place to Assist Corneal Immune Surveillance If the distribution of CALT is used to draw a topographical map (Figure 5(b)), it corresponds to the position of the cornea during eye closure. CALT in the tarso-orbital regions of the palpebral conjunctiva is then in the right position to support the immune protection of the cornea, which itself is largely devoid of lymphoid cells and other leukocytes. CALT may act during blinking as an immunological windscreen wiper and during sleep as an immunological cushion that covers the cornea (Figure 5(c)). CALT can provide the cornea with innate and specific antibacterial peptides and proteins including SIgA that are not produced there. This concerns the usual daytime setting when the conjunctiva regularly glides over the cornea and wipes it clean. Even more so, CALT may be

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Figure 4 Characteristics of follicular CALT. (a) Even smaller lenticular lymphocyte accumulations are primarily composed of B-cells in immunostaining (brown dots). Over the apex, the overlying epithelium becomes flatter ((b), this follicle is not exactly met at the apex) and changes into a follicle-associated epithelium (FAE, between arrowheads in (c)), where goblet cells are absent, immunostaining for secretory component is rarefied (c) and numerous intraepithelial lymphocytes (IEL) are present in groups, suspicious for M-cells. The IEL are arranged in groups, including B-cells (arrowhead in (a)). Immunostaining for CD20þ B-cells (a) and the IgA-transporter SC (c) of a small, almost flat, follicular accumulation that appears homogeneous, apart from disintegration at the location of the former germinal center, in HE staining (b); size bar in all figures ¼ 100 mm. From Knop, N. and Knop, E. (2000). Conjunctiva-associated lymphoid tissue in the human eye. Investigative Ophthalmology and Visual Science 41: 1270–1279.

relevant during nighttime when the eye is closed. Then, an upregulated level of proinflammatory factors from PMN cells is obtained as a temporary approach in order to dampen the growth of the entrapped microorganisms that enjoy a comfortable environment without disturbance. Due to the intimate contact, CALT can also detect corneal antigens and generate respective effectors.

recirculation of lymphocytes (Figure 2(b) and (c)) within the mucosal immune system in order to repopulate the ocular surface mucosal tissues and other mucosa-associated lymphoid tissue (MALT) locations and in return, EALTcan also share effector cells from other organs.

Mechanisms of Conjunctival Immune regulation CALT is a Part of the Complete Eye-Associated Lymphoid Tissue

CALT Is Physiologically Biased to Tolerogenic, Anti-Inflammatory Responses

The conjunctival mucosa is, at the temporal and nasal side (Figure 6), anatomically continuous, through the lacrimal excretory ducts, with the lacrimal gland and through the lacrimal canaliculi with the lacrimal drainage system, respectively. The histology clearly shows that a continuous mucosal immune system is also present from the periacinar tissue of the lacrimal gland throughout the conjunctiva into the lacrimal drainage system, that is, along the extended ocular surface. Together, this constitutes an eye-associated lymphoid tissue (EALT) and CALT is the regional part of it at the ocular surface proper (Figure 6). EALT is an undividable anatomical and functional unit and its different parts support each other in function. EALT is in line with the other parts of the mucosal immune system of the body, such as gut-associated lymphoid tissue (GALT) in the gut or bronchus-associated lymphoid tissue (BALT) in the bronchi. Therefore, primed effector cells from EALT can be distributed by the regulated

The mucosal immune regulation including CALT is maintained via the mode of antigen presentation by APC, on their MHC-class-II, to the T-cell-receptor (TCR) of naı¨ve CD4þ T-cells (To) and influenced by additional signals such as co-stimulatory molecules and the prevailing cytokine milieu within the tissue. This leads to the generation of different types of CD4þ Th cells which produce characteristic cytokine patterns and have different functions (Figure 7). Due to the prevalence of nonpathogenic antigens and the delicate tissue construction, CALT is biased toward anti-inflammatory immune answers. Tolerance is also necessary in order to avoid autoimmune reactions against own tissue constituents by self-reactive T-cells. Normally, CALT favors Th2 cells under the influence of cytokines such as IL-4. These interact with B-cells and produce cytokines (e.g., IL-4, IL-5, and IL-13) that promote B-cell Ig iso-type class switch to IgA and their differentiation into IgA-secreting plasma cell precursors


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Figure 5 Topography of CALT – in the right position to assist corneal immune protection. (a) Morphometrical analysis of lymphoid follicles in the different zones (tarsal, orbital, fornical, bulbar) of upper (left) and lower (right) human conjunctiva, in a flat preparation of a tissue whole mount, shows a main expression in the tarso-orbital zones. More follicles are present in the upper lid and the average total number of CALT follicles in an elderly population is about 10 follicles per eye. (b) The topographical distribution of lymphoid follicles is the same as that of the diffuse lymphoid effector tissue in which they are interspersed, as seen in a topographical map of CALT in a complete flat whole mount of a human conjunctiva with the lid margin to the top and the nasal zone in the middle. The map differentiates the previously mentioned zones (T, O, F, B) as well as temporal, medial, and nasal locations. Increasing density of diffuse lymphoid cells is indicated as increased shades of gray. Hatched lines indicate the location of conjunctival crypts of Stieda that are associated with CALT. (c) If the topographical distribution of CALT is projected onto the bulbar surface in a closed lid situation, it is obvious that CALT covers the cornea as seen in frontal en face view (middle) and sagittal section (right). The central portion is covered by the tarsal crypts of Henle (open circles in middle figure) that are associated with CALT but not indicated in the topographical map in (b). From Knop, E. and Knop, N. (2003) Eye-Associated Lymphoid Tissue (EALT) is Continuously Spread Throughout the Ocular Surface from the Lacrimal Gland to the Lacrimal Drainage System. Der Ophthalmologe, Heidelberg: Springer.

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Figure 6 Eye-associated lymphoid tissue (EALT). Eye-associated lymphoid tissue integrates the continuous mucosal immune system of the conjunctiva (CALT) and its mucosal adnexa, composed of the lacrimal gland and lacrimal drainage system, which together form the extended functional ocular surface. The ocular tissues belong together embryologically and functionally. They are connected by the flow of tears (yellow arrows) which lets them share protective immune factors as well as antigens and they are furthermore connected by the regulated recirculation of lymphoid cells in the body via efferent lymph vessels and blood vessels. Lymphoid cells enter the tissues via blood vessels, including high endothelial venules (represented by roundish endothelial cells), and leave them via lymphatics. EALT is continuous throughout these organs in the form of a diffuse lymphoid effector tissue composed of T-lymphocytes (represented by black cells in the drawing) and plasma cells (represented by large blue cells) together with accessory leukocytes (not indicated here, compare Figure 2(b)). Inductive sites, in the form of lymphoid follicles, composed of B-cells (small blue cells) with adjacent para-follicular T-cell areas (compare Figure 2(c)), are present in CALT and LDALT. They serve for the uptake and presentation of antigens and for the subsequent generation of respective effector cells specific for the ocular surface relevant antigens. Not only the effector cells generated in EALT, but also those from other mucosal sites, can, after recirculation in the lymph and blood system, populate the diffuse lymphoid effector tissue that is present along the whole extended ocular surface including the large mucosa-associated gland (lacrimal gland). Adapted from Knop, E. and Knop, N. (2007). Anatomy and immunology of the ocular surface. In: Niederkorn, J. Y. and Kaplan, H. J. (eds.) Immune Response and the Eye. Chemical Immunology and Allergy. Basel: Karger Verlag.

and mature plasma cells as observed in the conjunctiva. Under the influence of mainly IL-6, the well-known inflammatory Th1 cells are formed that produce inflammatory cytokines such as IFN-g and TNF-a which have the physiological function to activate cells, in particular phagocytes, to destroy intracellular pathogens. If inflammatory cytokines and other danger signals, such as bacterial LPS or components of dead cells occur in the tissue, they can bind to TLRs and mediate the secretion of excess inflammatory cytokines that skew CALT toward inflammatory immune answers. The antagonistic action of Th1 and Th2 cells led to the construction of the Th1–Th2 paradigm for explanation of immune regulation and phenomena at the ocular surface. In recent years, however,

other anti-inflammatory (regulatory T-cells, Treg) and inflammatory (Th17 cells) were also observed which indicated that immune regulation is more complex (Figure 7) and needs further investigation. Deregulation of EALT Is a Central Component of Inflammatory Ocular Surface Disease Various stress mechanisms, for example, mechanical alteration, hyperosmolar tears or exposure to inflammatory cytokines can pathologically activate the ocular surface epithelium that responds by secretion of (further) inflammatory cytokines and proteases (such as matrixmetalloproteinase, MMP) and upregulates surface


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Figure 7 Generation of T- and B-effector cells in CALT. Immune regulation in CALT is governed via the presentation of antigens by antigen-presenting cells (APC) on their MHC-class-II (MHC-II) to the T-cell-receptor (TCR) of naive CD4þ T-cells (To), and assisted and modulated by co-stimulatory molecules. This is influenced (indicated by uninterrupted arrows) by cytokines (interleukins, IL) and by microbial antigens that are recognized, for example, by TLR. This leads to the generation (interrupted arrows) of different types of CD4þ T-helper cells (Th) that produce characteristic cytokine patterns and perform different functions. CALT is naturally biased toward anti-inflammatory immune answers. It favors the generation of Th2 that promote B-cells to differentiate into IgA secreting plasma cells. It also favors Treg that produce anti-inflammatory TGF-beta. Binding of microbial antigens to TLR and presence of IL-6 represents danger signals to the immune system and induces inflammatory immune answers via Th17 and Th1, the latter of which normally assist phagocytic cells to clear intracellular pathogens.

markers, such as ICAM-1 and MHC-class-II. This promotes an inflammatory process and through MCH-classII the epithelial cells acquire the potential for presentation of antigens, including self-antigens, to resident conjunctival T-cells (Figure 8(a)) that can induce a loss of natural conjunctival immune tolerance. Similar events are also shown for the acinar epithelial cells in inflammatory disease of the lacrimal gland where diverse perturbations result in altered intracellular protein traffic, alter the lacrimal acinar cell autoantigenic spectra, and upregulate MHC-class-II. This results in a loss of tolerance to own cell constituents, such as the M3 receptor with a subsequent autoimmune process. This again indicates that the natural tolerogenic bias can be lost in inflammatory disease and may be the underlying reason for a self-perpetuating inflammatory process at the ocular surface and its associated gland. In fact, in inflammatory ocular surface diseases such as dry eye disease, autoreactive T-cells are generated that are specific to ocular surface tissue. They can be

transferred and lead to destruction of the same ocular tissues in a naive recipient that has never experienced the pathological condition. Respective Tregs can prevent the tissue destruction and offer therapeutic potentials. In addition, wounding can allow the entry of nonpathogenic antigens into the tissue and their presentation to Tand B-cells, as observed in ocular allergy. Downstream effects are the activation of conjunctival vascular endothelial cells that upregulate adhesion molecules (such as ICAM-1, VCAM-1, or E-selectin) with subsequent recruitment of further leukocytes from the vascular compartment and the activation of bystander cells including stromal fibroblasts. They contribute to the accumulation of MMPs that lead to tissue degradation. Altogether, this constitutes an immune-mediated conjunctival inflammatory process (Figure 8(a)), that can be compared with events in other mucosal organs, for example, in inflammatory bowel disease, and is based on a deregulation of the physiologically protective CALT and perpetuated by several vicious circles.

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Figure 8 Deregulation of CALT determines immune-mediated inflammatory ocular surface disease. (a) Compromised integrity leads to loss of mucosal immunological tolerance and to immune-mediated inflammation. Irritation of the surface epithelium, for example, by tear film (light blue) defects, its infection or its wounding results in activation of the epithelial cells. These may respond by secretion of inflammatory cytokines and proteases (such as matrix-metalloproteinase, MMP) and by expression of the antigen-presenting molecule MHC-class-II (MHC-II) resulting in potential presentation of self-antigens to resident conjunctival T-cells. Wounding with physical defects can, in addition, allow the entry of nonpathogenic antigens into the tissue and their presentation in the context of inflammatory cytokines as observed in ocular allergy. As described for immune-regulation (Figure 7) this represents danger signals that contribute to a further accumulation of inflammatory cytokines in the tissue and to the generation of inflammatory, potentially autoreactive, types of T-cells. All of which is shown in inflammatory ocular surface disease. Downstream effects are the activation of vascular endothelial cells that upregulate adhesion molecules with subsequent recruitment of further leukocytes from the vascular compartment and the activation of bystander cells, including stromal cells (fibroblasts). They contribute to the accumulation of MMPs that lead to tissue degradation which all together constitutes an immune-mediated conjunctival mucosal inflammatory process that is based on a deregulation of the physiologically protective CALT. (b) Immune-mediated inflammation is a core mechanism that results in several vicious circles in the pathogenesis and propagation of ocular surface disease. An immune-mediated inflammation represents an important common factor in the vicious circles of ocular surface disease, including the dry eye syndrome and ocular allergy, which is first subclinical but tends to amplify if it is not limited. Tear film deficiency results in epithelial defects and these in turn are an important primary factor for onset of an immune-mediated inflammatory conjunctival process that tends to selfpropagation via several vicious circles. These include disturbance of afferent innervation resulting in impaired secretion with further tear film deficiency and increase of epithelial damage and in impairment of mature ocular surface differentiation (leading to squamous metaplasia) that results in wetting defects and amplification of epithelial destruction. (a) From Knop E. and Knop N. (2005). Influence of the Eye-associated Lymphoid Tissue (EALT) on Inflammatory Ocular Surface Disease. The Ocular Surface, Ethis Communications. (b) Adapted from Knop E. et al. (2003). Dry Eye Disease as a Complex Dysregulation of the Functional Anatomy of the Ocular Surface. New Impulses to Understanding Dry Eye Disease. Der Ophthalmologe, Heidelberg: Springer.

See also: Adaptive Immune System and the Eye: Mucosal Immunity; Corneal Epithelium: Response to Infection; Defense Mechanisms of Tears and Ocular Surface; Dry Eye: An Immune-Based Inflammation; Immunopathogenesis of Onchocerciasis (River Blindness); Molecular and Cellular Mechanisms in Allergic Conjunctivitis; Pathogenesis of Fungal Keratitis; Tear Drainage.

Further Reading Argueso, P. and Gipson, I. K. (2001). Epithelial mucins of the ocular surface: Structure, biosynthesis and function. Experimental Eye Research 73: 281–289. Brandtzaeg, P. and Pabst, R. (2004). Let’s go mucosal: Communication on slippery ground. Trends in Immunology 25: 570–577. Chodosh, J., Nordquist, R. E., and Kennedy, R. C. (1998). Comparative anatomy of mammalian conjunctival lymphoid tissue: A putative

mucosal immune site. Developmental and Comparative Immunology 22: 621–630. Dua, H. S., Gomes, J. A., Jindal, V. K., et al. (1994). Mucosa specific lymphocytes in the human conjunctiva, corneoscleral limbus and lacrimal gland. Current Eye Research 13: 87–93. Hingorani, M., Metz, D., and Lightman, S. L. (1997). Characterisation of the normal conjunctival leukocyte population. Experimental Eye Research 64: 905–912. Knop, E. and Brewitt, H. (1992). Morphology of the conjunctival epithelium in spectacle and contact lens wearers – a light and electron microscopic study. Contactologia 14: 108–120. Knop, N. and Knop, E. (2000). Conjunctiva-associated lymphoid tissue in the human eye. Investigative Ophthalmology and Visual Science 41: 1270–1279. Knop, E. and Knop, N. (2003). Eye-associated lymphoid tissue (EALT) is continuously spread throughout the ocular surface from the lacrimal gland to the lacrimal drainage system. Ophthalmologe 100(11): 929–942. Knop, E. and Knop, N. (2005). Influence of the eye-associated lymphoid tissue (EALT) on inflammatory ocular surface disease. Ocular Surface 3(4): S180–S186. Knop, E. and Knop, N. (2005). The role of eye-associated lymphoid tissue in corneal immune protection. Journal Anatomy 206: 271–285.

Conjunctiva Immune Surveillance Knop, N. and Knop, E. (2005). Ultrastructural anatomy of CALT follicles in the rabbit reveals characteristics of M-cells, germinal centers and high endothelial venules. Journal of Anatomy 207: 409–426. Knop, E. and Knop, N. (2007). Anatomy and immunology of the ocular surface. In: Niederkorn, J. Y. and Kaplan, H. J. (eds.) Immune Response and the Eye. Chemical Immunology and Allergy, vol. 92, pp. 36–49. Basel: Karger Verlag. Knop, E., Knop, N., and Brewitt, H. (2003). Dry eye disease as a complex dysregulation of the functional anatomy of the ocular surface. New impulses to understanding dry eye disease. Ophthalmologe 100: 917–928. Knop, E., Knop, N., and Claus, P. (2008). Local production of secretory IgA in the eye-associated lymphoid tissue (EALT) of the normal human ocular surface. Investigative Ophthalmology and Visual Science 49: 2322–2329. Liu, H., Meagher, C. K., Moore, C. P., and Phillips, T. E. (2005). M cells in the follicle-associated epithelium of the rabbit conjunctiva

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preferentially bind and translocate latex beads. Investigative Ophthalmology and Visual Science 46: 4217–4223. McDermott, A. M., Perez, V., Huang, A. J., et al. (2005). Pathways of corneal and ocular surface inflammation: A perspective from the cullen symposium. Ocular Surface 3: S131–S138. Mircheff, A. K., Wang, Y., Jean, Mde S., et al. (2005). Mucosal immunity and self-tolerance in the ocular surface system. Ocular Surface 3: 182–192. Pflugfelder, S. C. and Stern, M. E. (2007). Future directions in therapeutic interventions for conjunctival inflammatory disorders. Current Opinion in Allergy and Clinical Immunology 7: 450–453. Sack, R. A., Beaton, A., Sathe, S., et al. (2000). Towards a closed eye model of the pre-ocular tear layer. Progress in Retinal and Eye Research 19: 649–668. Sullivan, D. A. (1999). Ocular mucosal immunity. In: Ogra, P. L., Mestecky, J., Lamm, M. E., et al. (eds.) Handbook of Mucosal Immunology, 2nd edn., pp. 1241–1281.

Defense Mechanisms of Tears and Ocular Surface A M McDermott, University of Houston, Houston, TX, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Antigen-presenting cell – Bone-marrow-derived cell that ingests antigen and presents it to T lymphocytes to trigger adaptive immune response. Antimicrobial peptide – Small protein, typically cationic, with broad spectrum antimicrobial activity. Pattern recognition receptor – Cell surface or intracellular protein receptor that binds conserved motifs (pathogen-associated molecular patterns) on microorganisms. Siderophore – Iron chelating compound secreted by bacteria and fungi to facilitate uptake of this essential nutrient.

the cornea is involved as this structure provides the majority of the refracting power of the eye and loss of visual acuity or even blindness may be the consequence. Given appropriate circumstances, a range of organisms can infect the ocular surface. The most common bacterial species are Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pneumoniae. Herpes simplex virus is the most common viral culprit. Fungal infection is less common than bacterial or viral infection, but is on the rise, with the most frequently isolated species in the USA being Aspergillus, Candida, and Fusarium. Some of the ocular surface and tear defenses that help prevent these infections are depicted in Figure 1 using the corneal epithelium as a model.

Defense Mechanisms of Tears Introduction The ocular surface is composed of the epithelia of the cornea, limbus, and conjunctiva, the anatomy and physiology of which are discussed in detail elsewhere in this encyclopedia. The tear film coats the epithelia and is a complex structure composed of an outer anterior-most lipid component that prevents evaporation, an aqueous component that has ions, soluble mucins, enzymes, and a range of other proteins and closest to the epithelial surface is a thick mucus, primarily composed of the gelforming mucin MUC5AC. Together, the ocular surface epithelia and tears: (1) create a formidable barrier that helps prevent microbial attachment in the first place, (2) bombard organisms with a plethora of chemicals to stop them dead in their tracks (or at least stop them proliferating), and (3) provide a detection system such that when an organism actually manages to circumvent the primary innate defenses, adaptive immunity can be activated to provide further help to eliminate the offending organism. The defense mechanisms are remarkably effective, as despite constant exposure to the external environment and frequent interaction with unwashed fingertips, the ocular surface rarely succumbs to infection. Generally only when there is physical disruption of the epithelial barrier such as occurs with contact lens wear or injury do the defenses encounter serious challenge and infections become a significant cause of morbidity. Infection can affect both the cornea (infectious keratitis) and conjunctiva (infectious conjunctivitis), but is most serious when

Tears provide both physical/mechanical and chemical defense to the ocular surface. The act of blinking moves tears toward the puncta and into the lacrimal sac, thus helping to wash away any potential pathogens before they have had time to interact with and invade the ocular surface epithelial cells. Furthermore, reflex tearing increases tear volume which helps to rapidly dilute harmful substances released by invading pathogens. Chemical entities with antimicrobial properties present in tears primarily originate from lacrimal and accessory gland secretions and the ocular surface epithelial cells. Other components include serum exudates and secreted products of neutrophils and other infiltrating cells. Several tear film components have been identified that have direct antimicrobial activity or which can otherwise limit pathogen entry and growth. The first identified was lysozyme which was shown to kill Gram-positive bacteria by Alexander Fleming (of penicillin fame) in 1922. This enzyme accounts for 20–40% of total tear protein and has the ability to catalyze the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine in the peptidoglycan backbone of bacterial cell walls. It is also able to cleave chitodextrins in fungal cell walls. The compromised cell wall is then no longer able to maintain a stable osmotic environment and lysis of the organism ensues. Secretory phospholipase A2 (sPLA2) has been identified as the major tear protein active against Gram-positive bacteria, although it has no activity against Gram-negatives in the normal ionic environment of tears and notably is some 50-fold less abundant in tears than lysozyme. Secretory PLA2 hydrolyzes the sn-2-fatty acyl

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Figure 1 Summary of defense mechanisms of tears and ocular surface. Schematic diagram of some of the various defenses used by tears and ocular surface epithelia to prevent infection (additional ones are discussed in the text). The corneal epithelium is depicted (note not all cell layers are shown) but as described in the text similar mechanisms are used by conjunctival epithelial cells. PRR, pattern recognition receptor (e.g., TLRs); SP-A, surfactant protein-A; SP-D, surfactant protein-D; sIgA, secretory IgA; sPLA2, secretory phospholipase A2.

moiety from phospholipids, in particular, phosphatidylglycerol, which is abundant on bacterial cell membranes. Other tear fluid components showing direct microbial killing include the cationic antimicrobial peptides (AMP; see the section entitled ‘Defense mechanisms of the ocular surface epithelia’) and the a-defensins human neutrophil peptide (HNP) 1, 2, and 3. As implicated by their names these peptides are produced by neutrophils and while present in only low concentrations under normal circumstances their level in tears rises after ocular surface injury. As depicted in Figure 2, these positively charged peptides exert their activity by interacting electrostatically with the negatively charged microbial cell membrane and form pores or otherwise disrupt the membrane leading to disturbances of respiration and metabolism, leakage of cell contents, and eventual death of the organism. A lytic effect of these peptides is also possible.

Beta-lysin and secretory leukocyte protease inhibitor (SLPI) are other examples of cationic proteins found in tears, which also interact with bacterial cell membranes and cause cell lysis. SLPI, which comes from lacrimal gland and ocular surface epithelial cells, has a defensinlike domain that confers antimicrobial activity and is also a potent inhibitor of neutrophil elastase. Thus, SLPI can both help prevent infection and protect host cells from the damaging effects of neutrophil enzymes. Elafin is another protein, which exerts similar dual functionality. Histatins, which are small histidine-rich AMPs with antifungal activity, have also been detected in the tears. A number of tear components limit bacterial growth rather than actually killing the invading organism. Lactoferrin represents some 21% of the total reflex tear protein and has a high capacity to bind divalent cations including iron, thus depriving many bacteria of this essential

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Figure 2 Mechanism of action of antimicrobial peptides. Positively charged peptides (shown in yellow) such as defensins and cathelicidin (LL-37) interact with negatively charged microbial membranes leading to disruption of the membrane, and possibly transient or stable pore formation, which results in leakage of intracellular contents, disturbance of metabolism, and death of the organism.

nutrient for growth. Furthermore, a highly basic sequence at the N-terminus (referred to as lactoferricin) allows lactoferrin to act as a cationic detergent and disrupt the cell membrane of some organisms. Tear lipocalin represents approximately 25% of the reflex tear proteins and was recently shown to be capable of binding siderophores produced by a range of bacteria and fungi. Siderophores are chelating compounds that transport iron into microorganisms. Thus, like lactoferrin, lipocalin exerts a bacteriostatic effect by interfering with the ability of pathogens to take up iron. The tear film also contains members of the collectin family of C-type lectins, surfactant proteins (SP)-A and -D. Collectins bind to carbohydrates on the surface of various microorganisms and to receptors on phagocytic cells, which in turn promotes their phagocytic activity. SP-A and SP-D are produced by lacrimal gland cells and also corneal and conjunctival epithelial cells. SP-D is known to exert growth inhibitory effects on some Gram-negative bacteria, to promote pathogen phagocytosis by mononuclear cells and through an interaction with lipopolysaccharide (LPS), which is present on the outer membrane of Gram-negative bacteria, can inhibit bacterial adhesion to target cells. SP-D inhibits corneal epithelial cell invasion by P. aeruginosa (a Gram-negative pathogen that commonly causes ulcers in contact lens wearers), possibly via an LPS-dependent mechanism. Secretory IgA (sIgA) is the predominant immunoglobulin in the tear film. This antibody is of great importance as it facilitates removal of pathogens right at the point of entry at the ocular surface. sIgA is not a particularly efficient activator of complement (important for preventing unwanted inflammation) or a good opsonin (although

neutrophils do have receptors for sIgA which when engaged could trigger phagocytosis). The major effector mechanism of sIgA is neutralization, which prevents attachment to host cells. sIgA can also bind to lectin-like adhesin molecules on pathogens causing them to aggregate and trapping them within the tear film. sIgA is produced by plasma cells (terminally differentiated B lymphocytes) residing in the lacrimal gland and in specialized areas of the conjunctiva referred to as conjunctival-associated lymphoid tissue. sIgA binds to specific receptors on lacrimal gland acinar cells, conjunctival epithelial cells, and is taken up by endocytosis, and then traverses the cell by transcytosis. This antibody is then released into the tears attached to a protein called secretory component, a fragment of the receptor to which the antibody was bound during its passage through the cells. Secretory component stabilizes the antibody and masks proteolytic sites so conferring resistance to host and pathogen proteases. Low levels of functionally active complement and complement regulatory proteins have also been detected in tears. An overview of the complement pathway is presented in Figure 3. The relative amounts of different components, namely abundant C3 and factor B, but less C1q, suggest that activation via the alternative pathway (i.e., spontaneous hydrolysis of C3) is the predominant mechanism. Possible sources of the various complement components are leakage of plasma through the conjunctival vessels during sleep, infiltrating neutrophils, and local synthesis by corneal and conjunctival epithelial cells. Activation of the complement pathway generates fragments involved in acute inflammatory responses, fragments that act as opsonins which facilitate target recognition by neutrophils and results in the formation of membrane attack complexes that can lyse pathogens (and host cells). The complement pathway is believed to be most active when the eyes are closed (see comments below on closed-eye tears). To prevent unnecessary activation and hence tissue damage, the complement pathway is regulated by a number of factors. This pathway is inhibited by molecules such as lactoferrin and vitronectin both of which are present in the tears and CD55 (decay accelerating factor) as well as CD59 which are membranebound molecules expressed by corneal and conjunctival epithelial cells. In immediate apposition to the superficial epithelial cells is a blanket of mucus, composed primarily of the gelforming mucin MUC5AC which is secreted by goblet cells in the conjunctiva in response to parasympathetic stimulation. This blanket interacts with the glycocalyx coating the superficial cells. Membrane spanning mucins MUC 1, 4, and 16 produced by the epithelial cells are important components of the glycocalyx and can be cleaved from the cell surface and released into the tear film. Mucins are known to help prevent bacteria from


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reaching epithelial surfaces. This function has been attributed to a number of mechanisms, for example, mucin can bind and trap the bacteria, which are then effectively removed from the ocular surface by blinking. It should be noted, however, that the ability to interact with mucins varies widely among different organisms. There is also evidence that sIgA and positively charged proteins such as lysozyme and SLPI accumulate in the mucous blanket, thus providing a reservoir of antimicrobial agents. Therefore, mucins may trap microbes, which are then killed by accumulated antimicrobials or aggregated by sIgA and then cleared by blinking. Thus, tears are equipped with a plethora of chemical entities capable of neutralizing invading pathogens. While many tear components have independent antimicrobial effects, several are thought to cooperate in a synergistic fashion to yield maximal effect. For example, sequestering of cations by lactoferrin destabilizes the cell wall of Gramnegative bacteria making the peptidoglycan layer more accessible to cleavage by lysozyme. Also, it should be brought to the reader’s attention that the composition of the tears changes during sleep. Open-eye and reflex tears have primarily lysozyme, lactoferrin, lipocalin, and sIgA, whereas closed-eye tears have increased amounts of sIgA (up to 80% of total tear protein), complement components, and of serum-derived proteins. There is also a large influx of neutrophils within 2–3 h of eye closure, which

provides additional defense factors in the guise of AMPs and reactive oxygen species, for example. Overall, these changes appear to represent a shift to a subclinical state of inflammation, which is believed to be necessary to protect the ocular surface from invasion by entrapped pathogens while the lids are closed. There is also an increase in proteins such as SLPI and elafin, which have potent antiprotease activity, and vitronectin, which inhibits complement, which serve to protect the ocular surface cells in this proinflammatory environment.

Defense Mechanisms of the Ocular Surface Epithelia Mechanical/Physical Defenses The outermost superficial epithelial cells are bound by tight junctions, which effectively seal two cells together forming a barrier against free diffusion of fluids, electrolytes, and macromolecules as well as microorganisms and their secreted products. Tight junctions are also important in establishing and maintaining cell polarity. Polarized cells are characterized by differences in the composition and distribution of proteins and other surface molecules between apical and basolateral surfaces. This arrangement is maintained by the aforementioned tight junctions that segregate the domains and targeted delivery

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that sends the molecules to their correct location. Disruption of polarity, such as occurs with corneal epithelial cells migrating to recover an injured area, has been shown to increase susceptibility to infection. This may be the result of a number of factors, for example, host cell receptors with which pathogens interact may be more abundant on basolateral surfaces. Also, loss of polarity disrupts the apical mucin-containing glycocalyx, which normally helps restrict bacterial attachment. Another important feature contributing to defense is the constant turnover of the ocular surface epithelial cells. Both corneal and conjunctival epithelia have a population of strategically located stem cells that provide new cells to replace those being shed into the tear film. In the absence of a penetrating injury, infection begins in the most superficial epithelial layers and so through the constant renewal of cells, outer infected cells may be sloughed off before there is time for the infection to spread to the lower epithelial layers. Pathogen Recognition While the tear film acts to prevent pathogens from reaching the ocular surface epithelial cells in the first place, it is very important that the cells have a system to recognize the presence of invading organisms if they do happen to conquer the outer defenses. Host cell proteins that mediate pathogen recognition are often referred to as pathogen recognition receptors (PRRs) and they recognize pathogen-associated molecular patterns present on bacteria, viruses, and fungi. The primary PRRs on epithelial cells are toll-like receptors (TLRs). These are type I transmembrane glycoproteins, which have an extracellular leucine-rich domain and a cytoplasmic domain homologous to the signaling domain of the interleukin (IL)-1 receptor. Of the 10 functional human TLRs that have been identified all have been reported to be expressed by corneal and conjunctival epithelial cells. Figure 4 shows a simplified diagram of the distribution of TLRs, their ligands, and signaling pathways. TLR1, 2, 4, 5, 6, and 10 are typically located at the cell surface. TLR2 forms heterodimers with TLR1 and with TLR6 and so can recognize a large variety of microbial products. For example, TLR2/6 heterodimers recognize lipoteichoic acid from Gram-positive bacteria and TLR2/1 heterodimers recognize triacyl lipoprotein/peptides of bacterial cell walls. TLR4 forms a complex with MD2 (also known as lymphocyte antigen 96) and cluster of differentiation 14 (CD14) protein and recognizes LPS from Gram-negative bacteria, while TLR5 recognizes flagellin, a component of bacterial flagella. TLR10, the ligand for which is unknown, is able to dimerize with TLR1 and TLR2. TLR3, 7, 8, and 9 are (typically) all located intracellularly, on endosomal membranes and recognize nucleic acids. TLR3 recognizes double-stranded RNA, a by-product of the replication of some viruses, whereas TLR7 and 8

recognize viral single-stranded RNA. TLR9 responds to unmethylated cytosine–phosphate–guanosine dinucleotide motifs found in both bacterial and viral DNA. Thus, by interacting with specific pathogen-derived molecules TLRs can detect the presence of a wide range of organisms, including those that replicate intracellularly. The engagement of TLRs with their specific microbial ligand results in activation of intracellular signaling pathways, leading to a variety of functional changes in the ocular surface epithelial cells. The latter include production of inflammatory cytokines such as IL-6 and chemokines such as IL-8 that will attract neutrophils for phagocytosis and AMPs such as human b-defensin-2 that can directly kill invading pathogens (see the section entitled ‘Antimicrobial peptides’). It is important that members of the normal ocular flora do not trigger TLR activation and hence cause unwanted inflammatory reactions at the ocular surface. To this end, it has been observed that flagellin from pathogenic, but not from nonpathogenic bacteria, can activate TLR5 in corneal epithelial cells. Expression of TLR5 (and possibly TLR4) appears to be restricted to basal and wing cells suggesting that TLR5 will only be activated when there is a breach in the corneal epithelium. Also, rather than being surface bound, TLR4 may be expressed intracellularly and so would not be available. Evidence for the expression of another class of PRRs, the cytoplasmic nucleotide-binding and oligomerization domain (NOD) proteins, has yet to be investigated for the human ocular surface. However, mouse anterior eye tissue expressed both NOD1 (which recognizes meso-DAP, a component of peptidoglycan in Gram-negative organisms) and NOD2 (which recognizes muramyl dipeptide found in both Gram-positive and -negative bacteria). Antimicrobial Peptides AMPs are small peptides, most less than 50 amino acids, that are amphipathic and typically carry an overall positive charge (+2 or greater) due to a relative excess of amino acids such as arginine and lysine. These peptides show a broad spectrum of antimicrobial activity and many have additional effects on mammalian cell behavior. The two major categories of mammalian AMPs are the defensins and cathelicidins. Human defensins are characterized by the presence of six cysteine residues that interact to form three disulfide bonds (the specific pattern of connectivity gives rise to two classes referred to as a and b). Both corneal and conjunctival epithelial cells express at least three b-defensins (hBDs). hBD-1 and hBD-3 are constitutively expressed, whereas the expression of hBD-2 is variable, being expressed by normal tissue only occasionally. Ocular surface hBD-2 expression is known to be inducible by exposure to both Gram-negative and -positive bacteria and


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Cytokines, chemokines, cell surface markers, antimicrobial peptides Figure 4 Toll-like receptor activation. The major TLRs expressed by ocular surface epithelial cells and their known bacterial and viral ligands are shown. For clarity the details of the several signaling pathways that lead to transcription factor activation and gene transcription have been omitted. LPS, lipopolysaccharide; CpG, cytosine–phosphate–guanosine dinucleotide; ssRNA, single stranded RNA, dsRNA, double stranded RNA, MyD88, myeloid differentiation protein 88; IRF 3/7, interferon regulatory factor 3/7; AP-1, activating protein-1; NF-kB, nuclear factor kB.

bacterial products such as LPS, peptidoglycan, and lipoproteins. This upregulation is chiefly mediated via the activation of TLRs such as TLR2. In a recent study, expression of a novel beta defensin gene DEFB 109 was detected in the ocular surface epithelia, and interestingly its expression was decreased in inflammation and infection. As noted earlier, a-defensins HNP-1 through -3 are produced by neutrophils and are present in the normal tear film. They can also be detected in the cornea and conjunctiva when neutrophils infiltrate in response to a specific stimulus. The cathelicidins have a highly conserved N-terminal cathelin domain and a variable antimicrobial domain. Only one, LL-37, is expressed in humans. LL-37 is expressed by both corneal and conjunctival epithelial cells and its expression is increased in response to corneal epithelial injury and bacterial challenge with P. aeruginosa and S. aureus. LL-37 is also a major component of neutrophil granules;

thus, its ocular surface levels are expected to rise in situations leading to infiltration of these and other inflammatory cells. While defensins and LL-37 represent the main AMPs present at the ocular surface, others have been reported including liver expressed AMP-1 and -2, statherin, CCL28 and CXCL-1 (two of many antimicrobial cytokines), MIP3a, and thymosin b-4. However, as most of these molecules have other recognized functions, it is unlikely that antimicrobial effects are the major facet of their action at the ocular surface. The primary site of AMP action is the microbial cell membrane, electrostatic disruption of which leads to permeabilization, loss of essential intracellular components, and death (see Figure 2). However, intracellular targets may also be utilized leading to inhibition of protein, peptidoglycan, and nucleic acid synthesis and interference with the activity of bacterial heat-shock proteins. Epithelial b-defensins and LL-37 are effective against

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common ocular surface bacterial pathogens in vitro with hBD-3 and LL-37 showing the broadest spectrum and most potent activity. Animal studies have revealed that experimental infection with P. aeruginosa in genetically modified mice unable to express cathelicidin causes much more severe disease and corneal damage than in normal wild-type mice, thus showing that AMPs are important for ocular surface defense in vivo. In addition to exerting direct antimicrobial effects, AMPs have other properties that help protect against pathogens and their destructive actions. For example, LL37 is known to bind and neutralize LPS. The latter is a product from Gram-negative bacteria and through activating TLR4 induces an inflammatory response and likely mediates much of the ocular surface damage that results from infections with pathogens such as P. aeruginosa. Thus, LL-37 may have a role in dampening LPS-mediated ocular surface inflammation and damage. Also, both defensins and LL-37 have been shown to be chemotactic for a variety of immune and inflammatory cells, including lymphocytes, monocytes, and immature dendritic cells thus may help draw these cells to a site of infection. AMPs also stimulate inflammatory and immune cell cytokine production, which in turn can modulate cellular functions. Additionally, hBD-3 has been shown to activate dendritic cells, raising the possibility that corneal/conjunctival epithelial hBD-3 may be able to activate epithelial Langerhans cells and stromal dendritic cells which in turn may initiate an adaptive immune response.

The Langerhans cells are typically found between basal epithelial cells and in the cornea their density is lowest in the central region and gradually increases toward the periphery. Also, most cells in central cornea appear to be small immature cells. In the periphery, larger mature major histocompatibility complex class II (MHC II) expressing cells with prominent dendritic processes are observed. Overall Langerhans cells perform a surveillance function, screening their environment for pathogens that have breached the defenses of the tear film and the epithelial barrier. If successful in their search they then activate adaptive immunity to help eliminate the invader. Sensory nerves, which are particularly abundant in the cornea, also provide an important contribution to ocular surface defense. When triggered nerves induce the production of reflex tears, which, by virtue of their increased volume, help wash pathogens from the ocular surface and dilute out their toxic products. Release of neuropeptides such as substance P from the nerve termini may also affect epithelial cell cytokine production that, as noted above, may modulate other aspects of host defense. The presence of a complement of nonpathogenic organisms also assists in preventing infection. Such commensals deplete the tears of nutrients, occupy attachment sites so preventing binding of pathogens, and produce bacteriocins that kill members of nonrelated species.

Other Contributions to Ocular Surface Epithelial Defense

In summary, the ocular surface and tears possess a wide range of chemicals and physical attributes that help prevent infection. Such redundancy is commonplace in biological systems, but is particularly important at the ocular surface where the inability to control and eliminate an infection can have dire consequences for visual function. Having multiple protective mechanisms is also necessary as pathogens are very adept at developing strategies to circumvent host defenses. While countering one specific mechanism is relatively easily achieved, developing multiple strategies is rather more challenging.

As noted earlier, the outermost superficial cells of both the cornea and conjunctiva are coated in a matrix of carbohydrate referred to as the glycocalyx. By projecting from the epithelial cell surface, membrane spanning mucins MUC 1, 4, and 16 of the glycocalyx physically prevent pathogens from reaching the cell membrane. Also, some organisms are repulsed by negatively charged glycosaminoglycans present on the mucin. The ocular surface epithelial cells also produce a variety of cytokines and chemokines that are important in protection from microbial invasion. IL-1 is an important cytokine released in response to trauma and injury and among a plethora of activities serves to regulate production of other molecules such as IL-6, growth regulated oncogene (GRO)-a, -b, -g, TNF-a, and IL-8, which in turn modulate inflammatory and immune cell infiltration and activation. Dispersed between the epithelial cells of the cornea and conjunctiva are bone-marrow-derived dendritic cells called Langerhans cells. These cells are highly potent antigen-presenting cells which capture antigen and when mature present it to T lymphocytes in nearby secondary lymphoid tissues so activating adaptive immunity.

Concluding Remarks

Acknowledgments The author acknowledges grant support from NIH, NSF, and the State of Texas for her work on ocular surface antimicrobial peptides and thanks Kimberly Thompson of the University of Houston College of Optometry audiovisual department for drawing the figures. See also: Antigen-Presenting Cells in the Eye and Ocular Surface; Conjunctiva Immune Surveillance; Corneal Epithelium: Cell Biology and Basic Science; Corneal Nerves: Anatomy; Corneal Nerves: Function; Immunopathogenesis of HSV Keratitis; Immunopathogenesis of

Defense Mechanisms of Tears and Ocular Surface Pseudomonas Keratitis; Overview of Electrolyte and Fluid Transport Across the Conjunctiva; Pathogenesis of Fungal Keratitis.

Further Reading Chen, G., Shaw, M. H., Kim, Y. G., and Nunez, G. (2009). Nod-like receptors: Role in innate immunity and inflammatory disease. Annual Review of Pathology 4: 365–398. Evans, D. J., McNamara, N. A., and Fleiszig, S. M. J. (2007). Life at the front: Dissecting bacterial–host interactions at the ocular surface. Ocular Surface 5: 213–227. Flanagan, J. L. and Willcox, M. D. P. (2009). Role of lactoferrin in the tear film. Biochimie 91: 35–43. Fleming, A. (1922). On a remarkable bacteriolytic element found in tissues and secretions. Proceedings of the Royal Society Series B 93: 306–317.

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Gupta, G. and Surolia, A. (2007). Collectins: Sentinels of innate immunity. BioEssays 29: 452–464. Hamrah, P. and Dana, M. R. (2007). Corneal antigen-presenting cells. Chemical Immunology and Allergy 92: 58–70. Hazlett, L. D. (2007). Bacterial infections of the cornea (Pseudomonas aeruginosa). Chemical Immunology and Allergy 92: 185–194. McDermott, A. M. (2009). The role of antimicrobial peptides at the ocular surface. Ophthalmic Research 41: 60–75. Paulsen, F. P. and Berry, M. S. (2006). Mucins and TFF peptides of the tear film and lacrimal apparatus. Progress in Histochemistry and Cytochemistry 41: 1–53. Sack, R. A., Nunes, I., Beaton, A., and Morris, C. (2001). Host-defense mechanisms of the ocular surfaces. Bioscience Reports 21: 463–480. Shafer, W. M. (ed.) (2006). Antimicrobial Peptides and Human Disease. Berlin: Springer. Yu, F.-S. X. and Hazlett, L. D. (2006). Toll-like receptors and the eye. Investigative Ophthalmology and Visual Science 47: 1255–1263.

Corneal Epithelium: Response to Infection Elizabeth A Szliter-Berger and L D Hazlett, Wayne State University School of Medicine, Detroit, MI, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Caspases – Humans express 11 different cysteineaspartic acid proteases classified as either initiator or effector caspases, which play essential roles in apoptosis, necrosis, and inflammation. CD44 – A type I transmembrane glycoprotein that regulates conformational changes of integrin heterodimers and their ability to microcluster and anchor to the actin cytoskeleton. Desmosomes – A junctional complex of adhesion molecules and linking proteins in the plasma membrane for cell-to-cell adhesion and contributes to the structural integrity through linkage of keratin cytoskeletal filaments of adjoining cells. Epidermal growth factor (EGF) – Expressed by corneal epithelial and stromal cells, it promotes cell migration and the attachment of corneal epithelial cells to fibronectin. Hemidesmosomes – Integral membrane protein complexes of integrin hetrodimers in the basal cell plasma membrane anchoring cells to the extracellular matrix. Langerhans cells – Unique subset of dendritic cells located in mucosal stratified squamous epithelium and skin epidermis; as professional antigenpresenting cells, they express toll-like receptors (TLRs) as well as C-type lectin receptors. Melanocytes – Pigment-producing cells located within the uvea of the eye. Nuclear factor-kappa B (NF-kB) – A protein complex that functions as a transcription factor; NF-kB is found in almost all animal cell types and is involved in cellular responses to stimuli such as stress, cytokines, free radicals, ultraviolet irradiation, and bacterial or viral antigens. Pathogen-associated molecular patterns (PAMPs) – Small, highly conserved molecular motifs present in bacteria, yeast, or viruses but not in mammalian cells and induce inflammatory signaling via interaction with receptors families, such as TLRs. Tritiated thymidine-labeling – Technique used to label cells actively undergoing DNA synthesis; it estimates the proportion of S-phase cells in a cell population.

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Trophic – It refers to a nutrition-derived function. Zonula occludens – Also known as tight junctions, these structures are primarily composed of occludins and claudins to form a virtual impermeable barrier between adjacent cell membranes.

Introduction Basic Structure and Function The corneal epithelium is a remarkably proficient tissue that combines structure and function to serve as the major refractive component of the eye and maintain ocular surface integrity; yet provide a critical barrier protecting the visual axis from the external environment. The corneal epithelium is the outermost layer of the cornea and is comprised of stratified, nonkeratinized, nonsecretory, squamous epithelial cells, intermixed with Langerhans’ cells and occasional dendritic melanocytes. This tissue layer is a highly organized structure that is avascular and almost perfectly transparent in order to preserve the optical properties of the cornea. It is 5–7 cell layers thick and contains three cell types (posterior to anterior): basal cells, wing cells, and superficial cells. The deepest layer is comprised of cuboidal basal cells. This single cell layer of progenitor cells undergoes mitosis at a rate of 10–15% per day, followed by intermediate differentiation of daughter cells into one to three layers of wing cells as they migrate toward the surface. Superficial to wing cells is a three- to four-cell thick layer of terminally differentiated squamous cells. These cells constantly degenerate and desquamate from the corneal surface in a continuous cycle of shedding of superficial cells and proliferation of cells in the basal layer resulting in complete renewal of the epithelium every 7 days, as has been demonstrated by mitotic rate measurements in basal cells and by tracking the migration of tritiated thymidine-labeled cells to the ocular surface. This regenerative process is further maintained by a constant renewal of basal cells from the limbal epithelium via stem cells located in the limbus that differentiate into basal cells, followed by centripetal migration into the cornea. Although the corneal epithelium is constructed as a highly effective, semipermeable membrane on the ocular surface,

Corneal Epithelium: Response to Infection

it is also well equipped to participate in the host response to invading pathogens and infection as described in greater detail below.

Corneal Infection The corneal epithelium, similar to other mucosal epithelial linings in the body, constitutes the eye’s first line of defense against microbial pathogens. The cornea has an immuneprivileged status, which includes features such as avascularity of the cornea and dearth of antigen-presenting cells (APCs) in the central region of the cornea in order to protect the visual axis. Although resident dendritic cells or Langerhans cells, known for their global antigen-presenting properties, are present in the corneal periphery, they can readily be recruited into the central cornea when necessary. In addition, the corneal epithelium plays an active role in host defense against invading pathogens. Cells of the corneal epithelium: (1) recognize pathogens and their byproducts and, as a result, (2) respond through expression and secretion of a network of proinflammatory cytokines and chemokines that recruit inflammatory cells into the cornea, (3) secrete antimicrobial products, and (4) promote wound healing and restoration of tissue homeostasis. Keratitis is a condition of corneal inflammation, which can be caused by a number of bacterial, viral, and fungal pathogens. Clinically, it is associated with symptoms such as redness, tearing, reduced visual clarity, corneal discharge, and severe pain. Bacterial keratitis is the leading cause of corneal infection. Staphylococci are the most commonly occurring organisms in bacterial conjunctivitis and keratitis, including Staphylococcus (S.) aureus, S. pneumoniae, S. intermedius, and a-hemolytic streptococcus. Pseudomonas aeruginosa (P. aeruginosa) is most frequently encountered in keratitis cases associated with extended contact lens wear and constitute 19–42% of bacterial keratitis cases. Other bacteria known to cause keratitis include Escherichia coli and Morganella morganii. Common pathogens associated with viral keratitis include, yet are not limited to, herpes simplex virus (HSV) and adenovirus. HSV infection is the most common cause of corneal blindness in the United States at present time. Approximately 400, 000 people in the United States have been infected with ocular herpes and 50, 000 initial and recurring cases of HSV keratitis are diagnosed annually. Fusarium, Aspergillus (both filamentary fungi), and Candida albicans (a yeast) constitute those fungi associated with the majority of fungal keratitis cases. Sterile keratitis also incites an immune response from the corneal epithelium; however, instead of bacteria adhering to and invading the

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ocular surface (requisite for bacterial keratitis), the epithelium responds to the presence of bacterial endotoxin (lipopolysaccharide or LPS). Sterile keratitis can also occur when bacteria colonize contact lenses and subsequently, release endotoxin onto the corneal surface, a condition known as CLARE or contact lens-induced acute red eye. The effects of both the invading pathogen on corneal tissue and the host immune response to the pathogen lead to many structural alterations that are otherwise essential to maintaining transparency of the cornea and, as a result, directly compromise visual acuity. As such, components of the corneal epithelium have evolved to respond to infection and these are described in greater detail below.

TLRs and TLR-Related Molecules Toll-like receptors (TLRs) are evolutionarily conserved type I transmembrane protein receptors that are expressed by corneal epithelial cells (as well as inflammatory cells) and function to respond to pathogens as the corneal epithelium’s first line of defense. These receptors initiate innate immunity and are essential for host defense against infection. TLRs recognize a broad spectrum of pathogen-associated molecular patterns (PAMPs), ranging from LPS (predominately recognized by TLR4), flagellin (TLR5), peptidoglycan (TLR2), single- or double-stranded RNA (TLR3,-7,-8), and unmethylated CpG DNA (TLR9). TLRs signal through several adaptor molecules, including the common adaptor protein myeloid differentiation factor (MyD)88 and MD-2; however, the reader is referred to Further Reading section for more information. It has been proposed that corneal epithelial cells play a central role in regulation of inflammatory responses by expression of functional TLRs and adaptor molecules. Epithelial cells are thought to intrinsically respond to the presence of pathogens through TLR recognition of PAMPs. Of the 13 TLRs identified to date, human corneal epithelial cells have been shown to express TLR-1,-2,-4,-5,-6, and -9 either intercellularly or at the cell surface (and has yet to be fully elucidated). TLRs primarily associate as homodimers, with exceptions for TLR-1,-2, and -6, which form heterodimers. Upon recognition of PAMPs expressed by invading pathogens on the ocular surface, TLRs produce downstream signaling events which induce translocation of nuclear factor kappa B (NF-kB), a major transcription factor of numerous genes important in both innate and acquired immune responses. These genes include proinflammatory cytokines and chemokines, leading to activation of adhesion molecules, and subsequently resulting in macrophage and polymorphonuclear neutrophilic leukocyte (PMN) recruitment into the

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cornea. Antimicrobial peptide expression by corneal epithelial cells is induced by TLR activation, as well. Although there is a common pathway for TLR activation (the myeloid differentiation protein–IL-1 receptor-associated kinase–tumor necrosis factor receptor-associated kinase (MyD88– IRAK–TRAF) pathway), individual TLRs most likely activate different, alternative, signaling pathways as well, and these remain under investigation (IL, interleukin).

TGF-b1, in particular, is a potent anti-inflammatory cytokine that modulates lymphocyte activation and promotes wound healing; regarding the cornea, it is further known to promote proliferation and lamellar differentiation of corneal epithelial cells via keratocyte-mediated stimulation.

Antimicrobial Molecules Cytokines/Chemokines The immune response of the corneal epithelium utilizes a variety of different substances to mount an attack upon organisms invading the ocular surface. Cytokines and chemokines are produced and endogenously released by corneal epithelial cells to directly and indirectly recruit and activate immune cells of both innate and adaptive immunity. Gram-negative bacterial endotoxin such as LPS, for example, elicit production of endogenous proinflammatory cytokines such as tumor necrosis factor (TNF)-a, interleukin (IL)-6, and IL-1. Protein levels for both IL-1a and IL-1b have been shown to be constitutively expressed in the normal human cornea. It has further been demonstrated that upon rupture of the epithelial cell membrane by infectious agents or trauma, IL-1a is passively released, contributing to increased vascular permeability, macrophage and lymphocyte infiltration and activation, angiogenesis, as well as regulatory effects on corneal fibroblasts. If left unchecked, these events would destroy the cornea; however, the epithelium also secretes both soluble and membrane-bound forms of IL-1RII, a receptor and natural antagonist of IL-1 in order to modulate the effects of this potent proinflammatory cytokine and consequently preserve visual function. In addition to IL-1, in vitro studies using primary human corneal epithelial cells (HCECs) and telomeraseimmortalized HCECs have revealed expression and secretion of IL-6, IL-8, and TNF-a following either P. aeruginosa or HSV-1 challenge. TNF-a is a major proinflammatory mediator that is known to promote apoptosis, inflammation, and regulate immune cells. IL-6 is both a pro- and anti-inflammatory cytokine that can potentiate the inflammatory response, yet also modulate inflammation through its inhibitory effects on TNF-a and IL-1, while activating IL-1 receptor antagonist and IL-10. IL-8 is a chemokine that is secreted by the epithelium under stress and functions as a strong chemoattractant for neutrophils (PMN); MIP-3a, also released by corneal epithelial cells after infection, promotes directed migration of leukocytes, such as immature dendritic cells and effector T cells. TGF-a,-b1, and -b2 are expressed also by corneal epithelial cells and contribute to re-epithelization, an essential step in resuming normal corneal function after infection.

The corneal epithelium also employs the action of small (100 amino acids or less), positively charged (arginineand lysine-rich) molecules known as antimicrobial peptides to further assist in combating invading pathogens. Over 500 naturally occurring antimicrobial peptides have been identified in mammals. Typically these molecules are cationic polypeptides that disrupt bacterial membranes through charge interactions and hydrophobic amino acids. Many antimicrobial peptides and their microbicidal effects are induced locally by inflammatory stimuli at the site of infection and act synergistically with other anti-inflammatory mechanisms (cytokines, inflammatory cells) in defending against microbial pathogenesis. Of the four distinct structural classes of antimicrobial molecules, recent studies have shown that corneal epithelial cells secrete peptides from the defensin and cathelicidin families, which are thought to help protect the eye through broad spectrum activity against microorganisms including Gram-positive and -negative bacteria, fungi, and certain enveloped viruses. These molecules and their roles in preventing microbial invasion and managing infection are discussed in greater detail below. b-Defensins The defensins include a- and b-defensin subfamilies, all of which are characterized by a b-sheet-rich fold and three disulfide bridges. A third class of y-defensins has been recently identified in rhesus macaque leukocytes. Although y-defensin mRNA is detectable in humans, these transcripts contain a premature stop codon preventing translation of functional protein. Leukocytes (PMN) and various types of epithelial cells have been shown to express both a- and b-defensins; however, the corneal epithelium has been demonstrated to produce and secrete only members of the b-defensin subfamily. Human b-defensins (hBDs) include 28 members, of which three have been associated with the corneal epithelium. Human b-defensin-1 (hBD-1) is constitutively expressed by corneal epithelial cells; while studies have shown that hBD-2 is induced by bacterial infection and bacterial products such as lipoprotein and lipotechoic acid, and is mediated by TLR2. In addition, cytokines TNF-a and IL-1 upregulate hBD-2 expression by corneal epithelial cells. Expression of hBD-3 is more variable,


whereby some studies have indicated solely constitutive expression by corneal epithelial cells; and others have demonstrated inducible expression by TNF-a and interferon-gamma (IFN-g). Regardless, hBD-3 was shown by McDermott and colleagues to exert most potent antibacterial activity against S. epidermidis, S. aureus, and P. aeruginosa using in vitro antimicrobial assays, followed by hBD-2; while hBD-1 showed moderate activity against Pseudomonas, but no activity against Staphylococcus strains. In vivo, it was recently demonstrated that murine (m) BD2, but not mBD1, was protective in the P. aeruginosa-infected mouse cornea. The importance of these antimicrobial peptides has been demonstrated by knocking out the mouse b-defensin-1 gene, which led to less effective clearance of Haemophilus influenzae from the lung and increased colonization of Staphylococcus in the bladder. Cathelicidins Of the numerous members of the cathelicidin family, only LL-37 has been described in humans. Cathelicidins express a highly conserved cathelin domain and a less conserved, more variable antimicrobial region. LL-37, as its name suggests, is a 37-amino acid linear peptide expressed by inflammatory/immune cells and epithelial tissue. It is derived by cleavage of the precursor, human cationic antimicrobial protein 18 (hCAP18) and appears to function as both an antibacterial peptide and immunomodulator. Low expression levels of LL-37 are detected constitutively and subsequently upregulated following injury, infection, and exposure to IL-1b, as well as heatkilled P. aeruginosa. Regarding antimicrobial properties, LL-37 is thought to function in a similar manner to that described for defensins and work synergistically with corneal epithelial proteins such as defensins, lactoferrin, and lysozyme (latter two present in tear film). LL-37 is able to bind and neutralize LPS and lipotechoic acid, thus reducing the inflammatory response associated with these molecules. In addition to eradicating ocular pathogens, studies have demonstrated that LL-37 enhances the innate and adaptive immune response in the corneal epithelium through modulation of cytokine/chemokine expression. Using an in vitro stimulation assay, LL-37 was shown to induce production of IL-1b, IL-6, IL-8, and TNF-a by human corneal epithelial cells. Furthermore, this molecule promotes wound repair through enhanced cell migration, including fibronectin-induced migration by stimulating corneal epithelial cells. In addition to microbicidal activities of b-defensins and LL-37, these molecules also wield effects over immune cells and influence wound healing. They have been demonstrated to recruit and activate immune cells through the induction of cytokine and chemokine production by epithelial cells, which further actuate the cellular components of the immune response to corneal infection. Regarding

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postinfection, both hBD-2 and LL-37 have been shown to be upregulated in an in vitro organ culture model of corneal epithelial wound healing suggesting roles for corneal epithelial cell migration and proliferation.

Complement System The complement system also contributes to the first line of innate immune defense against corneal infection. This critical system is composed of a series of effector and regulatory proteins that sequentially activate one another to generate biologically active molecules, such as opsonins and chemotaxins. The complement system is continuously activated at low levels in the eye under normal conditions, as supported by detection of soluble and membraneassociated complement regulatory proteins (e.g., rsCD59), which are also strongly expressed in the corneal epithelium for tight regulation of aberrant activation. Components of complement are also more heavily distributed in the peripheral versus the central cornea, potentially due to the diffusion of complement molecules from corneal limbal vessels. In response to infection activation of complement can occur via both the classical and alternate pathways.

Secretory IgA Although this molecule is not essential in ocular defense, secretory IgA (sIgA) does play a major role in the prevention of some corneal infections, including Pseudomonas and Acanthamoeba. In fact, over 75% of the general population contain anti-P. aeruginosa-specific IgAs in their tear film. sIgA protects the corneal epithelium by accumulating in the ocular mucin layer and displays an antigen–antibody clearance function. Aggregated sIgA opsonizes bacteria for PMN phagocytosis and processing via recognition by sIgA receptors expressed on the immune cell surface. IL-8, which is secreted by corneal epithelial cells during infection, further enhances the ability of sIgA to induce release of reactive oxygen species by PMN.

Adhesion Molecules Under normal conditions, integrity of the corneal epithelium depends upon a number of factors, including adhesion molecules. Corneal epithelial cells are interdigitated, particularly in the middle layers, and largely interconnected by desmosomes. Basal cells are firmly attached to the basement membrane, neighboring basal cells and overlying wing cells via hemidesmosomes. Tight junctional complexes, or zonula occludens, found only between superficial cells are

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of great importance regarding the barrier function of the corneal epithelium. As such, superficial cells form a highly effective, semipermeable membrane on the corneal surface. Not only does the tight junctional barrier prevent decreases in net fluid transport out from the stroma, but it also prevents corneal penetration by pathogens. In addition, gap junctions connect the cells of all layers of the corneal epithelium and function as communication conduits between cells. These tight anatomical arrangements with virtual absence of intercellular spaces contribute to the remarkable transparency of the epithelium, but have deleterious effects when breached after infection (or wounding) and must be restored forthwith in order to restore/preserve visual function. Upon infection of the corneal epithelium, breakdown of tight junctional integrity occurs due to the loss or disruption of the outermost layers of the epithelium by invading pathogens. This results in a collapse of cell membrane permeability and selectivity, in addition to creating an unrestricted portal for further pathogen intrusion into the cornea until the corneal epithelium and its adhesion molecules are regenerated. Additional adhesion molecules such as selectins, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), are modified after infection due to the secretion of epithelial-derived cytokines, such as TNF-a. As a result, these barriers are further breached to allow the transmigration of inflammatory cells into the injured or infected tissue site. Recruitment of activated leukocytes to sites of infection is essential to the function of both inflammation and innate immunity. However, the extent of leukocyte recruitment contributes largely to the intensity of the inflammatory response, and if this is not well balanced it can result in considerable tissue damage. However, the corneal epithelium does play an active role in regenerating and repairing itself after infection. CD44 is among the molecules that corneal epithelial cells express to mediate wound healing after infection. This molecule is thought to be involved in cell–cell interactions that provide adhesive strength for the epithelial sheet and in cell–matrix interactions that occur during cell migration and the re-epithelialization process. Basal cells of the corneal epithelium have been demonstrated to secrete APLP2, an amyloid precursor-like protein that is suggested to influence reorganization of the extracellular matrix and dynamic cell–matrix adhesion during re-epithelialization. It has been demonstrated that the corneal epithelium (as well as stromal and endothelial cells) produces and secretes epithelial growth factor (EGF), which is thought to promote cell migration and mitosis of epithelial cells. Corneal epithelial cells then resume expression of molecules such as connexins 45/43 and a6b4 integrin, which participate in the formation of gap junctions and hemidesmosomes/desmosomes, respectively, thus restoring homeostasis of an intact corneal epithelium.

Neuropeptides Innervation of the Corneal Epithelium The cornea is among the most densely innervated tissues in the body. In regards to the corneal epithelium, the subbasal epithelial nerve plexus originates from the ophthalmic division of the trigeminal nerve via the anterior ciliary nerves and provides innervation to the basal epithelial cell layer and terminates within the superficial epithelial layers. These nerve fibers are predominately sensory (most classified as nociceptors) and serve a protective role, typically responding to mechanical, thermal, and chemical stimuli. They are stimulated during corneal abrasions and ulcers, and are extremely painful. It is estimated that single corneal sensory neurons support approximately 200 and 3000 individual nerve endings in the mouse and rabbit, respectively, demonstrating the high density of innervation in the corneal epithelium. Corneal nerves in the epithelium have a trophic function, as well. In addition, neuropeptides have been associated with corneal nerves and include: substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP), and neuropeptide Y (NPY) among others. Nerve damage can lead to transient or chronic neurotrophic deficits following infection of the corneal epithelium. Corneal denervation also significantly impairs the ability of the corneal epithelium to heal itself and predisposes newly healed corneas to periodic, spontaneous epithelial erosions. Although the nerves coursing through the human corneal epithelium are known to produce a variety of neuropeptides, the following sections focus on the importance of two of these molecules: SP and VIP.

Substance P SP is an 11-amino acid neuropeptide that is largely associated with proinflammatory events during corneal infection, including upregulation of TLR4 and TLR9 mRNA and of cytokines/chemokines IL-1b, TNF-a, IFN-g, and MIP-2 as demonstrated in a murine model of P. aeruginosa-induced keratitis. Physiologically relevant concentrations of SP are present in the normal human (and mouse) cornea and corneal epithelial cells express the NK1 receptor, which is the major physiological receptor for SP. The neuropeptide also has been associated with wound healing properties and together with insulin-like growth factor-1 (IGF-1) has been demonstrated to have a stimulatory effect on corneal epithelial cell migration, adhesion, and wound closure.

Vasoactive intestinal peptide VIP is a 28-amino acid neuropeptide that exerts immunomodulatory properties in the cornea following infection.


It has been demonstrated in a murine model of P. aeruginosainduced ocular infection that VIP downregulates the production of several proinflammatory cytokines, including: TNF-a, IL-1, IL-6, IL-12, and IFN-g, while stimulating the production of anti-inflammatory cytokines IL-1 receptor antagonist, IL-10, and TGF-b. Macrophage and PMN activation was also shown to be influenced by the presence of VIP. Studies in the mouse further support a role for VIP in wound healing and restoration of immune homeostasis in the cornea; however, it has yet to be determined the extent of which is carried out by the epithelium versus stroma.

Thymosin-b4 As previously stated, after eradication of the invading pathogen, the corneal epithelium must heal and promptly resume normal activity. Thymosin (T)b-4 is a 43-amino acid protein produced by corneal epithelial cells that contributes to the resurfacing of the epithelium and regeneration of cell adhesion molecules to reconstitute the epithelial barrier. Studies have shown that Tb-4 levels are increased in murine corneas during re-epithelialization and also significantly enhance the migration of human corneal epithelial cells through upregulation of molecules associated with cell migration, including laminin-5 and matrix metalloproteinase 1 (MMP-1). Furthermore, Tb-4 modulates corneal cytokine production in an anti-inflammatory manner by downregulating levels of MIP-2 and TNF-a, as demonstrated in the murine cornea. Tb-4 has been demonstrated to inhibit apoptosis of human corneal epithelial cells through inhibition of caspases and suppression of Bcl-2

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(an antiapoptotic factor) release from mitochondria. Lastly, this molecule has been demonstrated to modulate matrix metalloproteinase expression and prevent PMN infiltration in an alkali injured mouse cornea model, further supporting a key role for Tb-4 in the repair and remodeling of the injured cornea.

Conclusion The cornea generates approximately 80% of the eye’s refractive power; therefore, it is imperative that the corneal epithelium possess the ability to effectively and appropriately activate an innate immune response when pathogens are encountered on the ocular surface. The protective mechanisms employed by the epithelium have evolved to balance recognition and elimination of pathogens while limiting corneal damage and preserving the visual axis. In order to do so, the corneal epithelium is able to recognize invading organisms through TLR signaling, generate a network of cytokines and chemokines to recruit and activate host inflammatory cells, produce antimicrobial molecules, and provide activation signals to the complement system, all of which coalesce into an effective and efficient immune response as depicted schematically in Figure 1. The corneal epithelium also possesses the ability to regenerate itself and promote wound healing through expression and secretion of adhesion molecules and Tb-4; this process is enhanced by the presence of corneal nerve fibers that release neuropeptides such as SP and VIP, molecules that further contribute to the healing process and restoration of tissue homeostasis.

LPS Bacterium

Cytokines/ chemokines

slgA TLR c9/rsCD59 c9/GPI anchor Antimicrobial peptides

Tear film

Epithelium

Stroma

Nerve

Figure 1 The corneal epithelium also possesses the ability to regenerate itself and promote wound healing through expression and secretion of adhesion molecules and Tb-4; this process is enhanced by the presence of corneal nerve fibers that release neuropeptides such as SP and VIP, molecules that further contribute to the healing process and restoration of tissue homeostasis.

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See also: Immunopathogenesis of Pseudomonas Keratitis.

Further Reading Akpek, E. K. and Gottsch, J. D. (2003). Immune defense at the ocular surface. Eye 17: 949–956. Bora, N. S., Jha, P., and Bora, P. S. (2008). The role of complement in ocular pathology. Seminars in Immunopathology 30: 85–90. Dohlman, C. H. (1971). The function of the corneal epithelium in health and disease. Investigative Ophthalmology 10(6): 383–407. Edelhauser, H. F. and Ubels, J. L. (2003). Adler’s Physiology of the Eye: The Cornea and the Sclera. St. Louis, MO: Mosby. Ganz, T. (2003). Defensins: Antimicrobial peptides of innate immunity. Nature Reviews Immunology 3: 710–720. Kumar, A., Zhang, J., and Yu, F. X. (2006). Toll-like receptor 2-mediated expression of b-defensins-2 in human corneal epithelial cells. Microbes and Infection 8: 380–389. Lu, L., Reinach, P. S., and Kao, W. W. (2001). Corneal epithelial wound healing. Experimental Biology and Medicine 226(7): 653–664. McDermott, A. M. (2004). Defensins and other antimicrobial peptides at the ocular surface. Ocular Surface 2(4): 229–247. McDermott, A. M. (2007). Ocular surface expression and in vitro activity of antimicrobial peptides. Current Eye Research 32(7–8): 595–609. Muller, L. J., Marfurt, C. F., Kruse, F., and Tervo, T. M. (2003). Corneal nerves: Structure, contents and function. Experimental Eye Research 76(5): 521–542.

Sack, R. A., Nunes, I., Beaton, A., and Morris, C. (2001). Host-defense mechanism of the ocular surfaces. Bioscience Reports 21(4): 463–480. Sosne, G., Qiu, P., and Kurpakus-Wheater, M. (2007). Thymosin b-4 and the eye: I can see clearly now the pain is gone. Annals of the New York Academy of Science 1112: 114–122. Szliter, E. A., Lighvani, S., Barrett, R. P., and Hazlett, L. D. (2007). Vasoactive intestinal peptide balances pro- and anti-inflammatory cytokines in the Pseudomonas aeruginosa-infected cornea and protects against corneal perforation. Journal of Immunology 178(2): 1105–1114. Uematsu, S. and Akira, S. (2008). Handbook of Experimental Pharmacology: Toll-like Receptors (TLRs) and Innate Immunity: Toll-Like Receptors (TLRs) and Their Ligands. Berlin: Springer. Wilson, S. E., Liu, J. J., and Mohan, R. R. (1999). Stromal–epithelial interactions in the cornea. Progress in Retinal and Eye Research 18 (3): 293–309. Zhang, J., Wu, X., and Yu, F. X. (2005). Inflammatory responses of corneal epithelial cells to Pseudomonas aeruginosa infection. Current Eye Research 30: 527–534.

Relevant Websites http://www.nei.nih.gov – Facts about the Cornea and Corneal Disease, National Eye Institute. http://www.revoptom.com – Handbook of Ocular Disease Management: Cornea.

Inflammation of the Conjunctiva T Nishida, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan ã 2010 Elsevier Ltd. All rights reserved.

Glossary Cell-adhesion molecule – Cell-adhesion molecules are cell surface proteins, usually glycoproteins, that mediate cell–cell adhesion. They play important roles in the assembly and maintenance of tissues, wound healing, morphogenic cellular movements, cell migration, and metastasis. Intercellular adhesion molecule-1 (ICAM-1) functions in leukocyte adhesion and inflammation. Its expression is induced in various cell types by interferon-g (IFN-g), and it mediates interactions with neutrophils in inflamed tissue. Vascular cell-adhesion molecule-1 (VCAM-1) is presented on the surface of various cell types, including endothelial cells, tissue macrophages, fibroblasts, and dendritic cells. Its expression is induced by cytokines, and it plays a key role in the recruitment of eosinophils to sites of inflammation. Collagenase (microbial) – Microbial collagenase is a metalloproteinase produced by bacteria that degrades helical regions of native collagen to yield small protein fragments. The preferred cleavage site is immediately upstream of the glycine residue in the sequence – proline–X–glycine–proline – where X is any amino acid. Six forms (or two classes) of microbial collagenase have been isolated from Clostridium histolyticum; these proteins are immunologically cross-reactive but possess different amino acid sequences and different specificities. Other variants have been isolated from Bacillus cereus, Empedobacter collagenolyticum, Pseudomonas marinoglutinosa, and species of Vibrio and Streptomyces. Helper T cell – Helper T cells constitute a specific subpopulation of CD4+ T cells that provides help to other cells of the immune system in mounting an immune response either through direct cell–cell interaction or the secretion of cytokines. They are also referred to as effector T cells. Several distinct subtypes of helper T cells, designated Th1, Th2, Th3, and Th17, have been identified. Matrix metalloproteinase – Matrix metalloproteinases (MMPs) constitute an important family of enzymes that regulate composition of the extracellular matrix. They are synthesized as inactive precursor proteins that consist of propeptide, catalytic, and hemopexin domains; proteolytic removal of the propeptide domain results in MMP activation. MMPs are zinc-dependent

endopeptidases that cleave one or several constituents of the extracellular matrix as well as nonmatrix proteins, and they play an important role in cleaving fibrillar collagen types I, II, and III into characteristic three-fourths and one-fourth fragments. Some MMPs are associated with the cell membrane, either through a transmembrane domain or through glycosylphosphatidylinositol anchor; such MMPs may act within the pericellular environment to influence cell migration. MMP-1, MMP-8, and MMP13 are also known as collagenase 1, collagenase 2 (neutrophil collagenase), and collagenase 3, respectively. Th1 cytokine – Th1 cytokines include interleukin (IL)-2, IFN-g, IL-12, and tumor necrosis factor-b. They are secreted by Th1 cells and play an important role in cell-mediated immunity and chronic inflammation. In general, Th1 responses are stimulated by intracellular pathogens, including viruses as well as certain mycobacteria, yeasts, and parasitic protozoans. Th2 cytokine – Th2 cytokines include IL-4, IL-5, IL-6, IL-10, and IL-13. They are secreted by Th2 cells and play a key role in the initiation of allergic responses. Th2 responses are also elicited by free-living bacteria and other parasites.

Inflammation Inflammation is a biological response of the living body to injury or other harmful insults including microbial pathogens, allergens, and physical or chemical agents. It serves to protect the body and is the precursor to wound healing. Classical signs and symptoms of inflammation include redness, swelling, heat, pain, and loss of tissue function. Thus, although inflammatory reactions are well regulated to maintain homeostasis of the body and to promote wound repair, they may result in bodily discomfort. In some instances, however, excessive inflammation may result in tissue damage. Classically, inflammation has been considered to begin with a reaction of vascular tissue that renders vessels permeable to blood cells at the site of injury, resulting in the extravasation of such cells. Recent advances in immunology and molecular cell biology have revealed the mechanisms of inflammation at the level of cellular

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interactions and molecular networks. Allergens and infection by pathogens are the major pathological triggers for inflammation at the ocular surface.

The Conjunctiva and Cornea The ocular surface is composed of the cornea, conjunctiva, lacrimal glands, and associated lid structures. Both the conjunctiva and the cornea are derived from the embryonic epidermis, and they are separated from each other by tear fluid. External insults to the ocular surface evoke different types of inflammatory reactions in the conjunctiva and the cornea that are related to the anatomic differences and physiological roles of these two structures as well as to their connection via tear fluid (Figure 1). The conjunctiva is a semitransparent membrane that covers the surface of the eye from the back surface of the eyelids to the edge of the transparent cornea. It serves as a barrier at the surface of the eyeball and helps to protect against the invasion of biological, chemical, or physical agents without interrupting the free movement of the eyeball. The surface of the conjunctiva is covered by multiple layers of epithelial cells. The conjunctival epithelium is a relatively inefficient barrier, however, with the result that pathogens, allergens, and biologically active substances readily penetrate into the stroma of the conjunctiva. The conjunctival stroma consists of conjunctival fibroblasts, loosely packed collagen fibers, a vascular system, and abundant immune cells. The triggering of an inflammatory reaction by pathogens or allergens results in enlargement of the blood vessels of the conjunctiva and consequent increased blood flow (hyperemia). The associated

increase in the permeability of the conjunctival vascular system leads to leakage of liquid components and to the development of conjunctival edema. It also allows the infiltration of blood cells into the conjunctival stroma and the consequent activation of conjunctival fibroblasts. Like the conjunctiva, the cornea faces the external environment. However, unlike the conjunctiva, the cornea is transparent, and its surface must be maintained smooth for the proper transmission of light into the eye. The anatomic structure of the cornea is relatively simple compared with that of the conjunctiva. Its surface is also covered by multiple layers of epithelial cells, but the corneal epithelium provides a much tighter barrier than does the conjunctival epithelium. In the absence of any loss or dysfunction of its component cells, the corneal epithelium prevents the entry of pathogens and allergens into the corneal stroma. The cornea does not contain a vascular system. Although a small number of immune cells such as Langerhans cells, stromal dendritic cells, and macrophages are present in the cornea, its major cellular components are epithelial cells, stromal keratocytes (corneal fibroblasts), and endothelial cells. Both the conjunctiva and the cornea are innervated by the ophthalmic branch of the trigeminal nerve, but the cornea is the most sensitive tissue at the ocular surface, and indeed maybe in the entire body, as a result of the high density of sensory nerve endings in the corneal epithelium.

Tear Fluid: A Reservoir of Inflammatory Cells and Modulators Tear fluid functions as a lubricant between the tarsal conjunctiva and the surface of the cornea and serves to

Tear fluid

Conjunctiva

Simple structure Tight epithelial barrier Avascular tissue Few immunologic cells Faces the environment Wet tissue

Biological defense system

Extensive vascular system Loose epithelial barrier Abundant immune cells Faces the environment Wet tissue

Diseases

Neutrophils Eosinophils Lymphocytes Cytokines Chemokines MMPs, etc.

Cornea Anatomic characteristics

Infections (bacterial, viral, fungal, protosoal) Physical or chemical injuries Clinical problems Loss of transparency Irregular surface-scarring

Anatomic characteristics

Lubricant Pathway for inflammatory cells and bioactive substances

Diseases Allergic conjunctivitis Infections (bacterial, viral) Chemical injuries Clinical problems Swelling, itching Fibrosis due to scarring

Figure 1 Cornea–tear fluid–conjunctiva axis in ocular surface inflammation. MMP, matrix metalloproteinase.


maintain the ocular surface wet. It is also important for ensuring the generation of a clear image on the retina. Moreover, it contributes to the biological defense system of the ocular surface, containing immunoglobulin, lactoferrin, lysozyme, and other protective proteins. With regard to inflammation at the ocular surface, tear fluid provides a pathway for the movement of inflammatory cells – such as neutrophils, eosinophils, and lymphocytes – between the conjunctiva and the cornea. It also serves as a reservoir of various inflammatory cytokines, chemokines, and growth factors as well as of nutrients and oxygen. Collagen-metabolizing enzymes such as matrix metalloproteinases (MMPs) are present in the tear fluid of individuals with certain ocular inflammatory conditions.

Allergic Reactions in the Conjunctiva The conjunctiva is a common site for allergic reactions (Figure 2). Clinical characteristics of conjunctival allergic disease include hyperemia, edema, the formation of papillary discharge, the development of corneal epithelial disorders, and, in some patients, corneal ulcer. Hyperemia and edema result from dilation and an increase in the

Allergy

permeability of the vascular system in the conjunctiva. Conjunctival fibroblasts are responsible for the formation of papillae. Mechanical injury caused by papillae as well as the effects of inflammatory cytokines, such as interleukin (IL)-4, IL-13, and tumor necrosis factor-alpha (TNF-a), are responsible for discharge and damage to the corneal epithelium. Disruption of corneal epithelial barrier-function results in the spread of inflammation to the cornea and the development of various types of corneal epithelial disorders. Corneal fibroblasts contribute to the pathology of corneal ulceration. The primary cells that mediate allergic reactions at the ocular surface include mast cells, vascular endothelial cells, eosinophils, T helper 2 (Th2) cells, and conjunctival fibroblasts, with corneal epithelial cells and corneal fibroblasts also contributing in some cases. Certain allergens that enter tear fluid from the environment are solubilized by the fluid and penetrate through the loose barrier provided by the conjunctival epithelium into the conjunctival stroma. In the stroma, the allergens trigger the secretion of histamine and inflammatory cytokines, such as IL-4, IL-13, TNF-a from mast cells, and IL-3 and IL-5 from Th2 cells. Histamine acts on the vascular endothelium to increase vessel

Histamine

Vascular endothelium

Mast cells Conjunctival stroma

Th2 cells Allergens IL-4 IL-13 TNF-α

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Permeability

Hyperemia edema

IL-3 IL-5

Eosinophils

Conjunctival fibroblasts

Eotaxin

TARC

Collagen synthesis cell proliferation Papillae

Conjunctival epithelium (loose barrier) Tear fluid

Allergens

Mechanical injury

IL-4 IL-13 TNF-α

Corneal epithelium (tight barrier)

Eosinophils

TARC

Discharge

Epithelial disorders

Disruption of barrier function

IL-4 IL-13 TNF-α Corneal stroma

TARC

Eotaxin Eosinophils Ulcer

Corneal VCAM-1 fibroblasts

MMPs

Figure 2 Clinical characteristics of allergic reactions in the conjunctiva and the cornea. IL, interleukin; MMP, matrix metalloproteinase; Th2, T helper 2 cell; TARC, thymus and activation-regulated chemokine; TNF-a, tumor necrosis factor alpha; VCAM, vascular cell-adhesion molecule.

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permeability and induce vessel enlargement, resulting in conjunctival hyperemia and edema. IL-4, IL-13, and TNF-a – released by mast cells – activate conjunctival fibroblasts and trigger their secretion of the chemokines eotaxin and thymus and activation-regulated chemokine (TARC). Eotaxin attracts eosinophils to the interstitial space, and the extravasated eosinophils are then activated by IL-3 and IL-5 released by Th2 cells. TARC attracts Th2 cells into the interstitial space, and these cells then serve as an additional source of IL-4, IL-13, and TNF-a. Exposure of conjunctival fibroblasts to IL-4, IL-13, and TNF-a also stimulates the synthesis of collagen and cell proliferation – effects that give rise to the formation of papillae. The protrusive shape of the papillae results in mechanical injury of both conjunctival and corneal epithelia; such injury together with the effects of IL-4, IL-13, and TNF-a that enter tear fluid from the conjunctiva lead to disruption of the barrier function of the corneal epithelium and to discharge. Eosinophils that enter tear fluid from the conjunctiva are then able to penetrate into the corneal stroma. IL-4, IL-13, and TNF-a also enter the corneal stroma from tear fluid and activate corneal fibroblasts to express TARC, eotaxin, and vascular cell-adhesion molecule-1 (VCAM-1) – a cell-adhesion molecule for eosinophils. The activated corneal fibroblasts also produce MMPs, which degrade collagen of the extracellular matrix in the corneal stroma, resulting in corneal ulceration. TARC released from corneal fibroblasts passes through tear fluid into the conjunctival stroma, where it further promotes the secretion of IL-4, IL-13, and TNF-a by Th2 cells in a vicious cycle. This scenario thus reveals that, although immune cells such as mast cells, Th2 cells, and eosinophils play a prominent role in allergic disorders at the ocular surface, resident fibroblasts in both the conjunctiva and cornea also contribute to the inflammatory process.

Infection of the Conjunctiva or Cornea The clinical characteristics of infection at the ocular surface include swelling, hyperemia at the conjunctiva, discharge, and epithelial defects and ulceration in the cornea. As with allergic reactions, the reactions of the conjunctiva and cornea to infection differ (Figure 3). The vascular system of the conjunctiva ensures a robust immune response to infection in this tissue, with conjunctivitis being a relatively mild clinical condition. However, the cornea is avascular and possesses few immune cells, with the result that corneal infection is more serious and may become sight threatening. If a pathogen survives the biological defense system in tear fluid, it readily penetrates the conjunctival epithelium and triggers the dilation and permeabilization of conjunctival blood vessels, resulting in swelling and

hyperemia. Neutrophils and Th1 cells enter the conjunctival stroma from the bloodstream and serve as the second line of defense against pathogens. Both neutrophils and Th1 cells secrete IL-1, with Th1 cells also secreting interferon-g (IFN-g). These cells and cytokines may be sufficient to inactivate the pathogen and limit the inflammatory response to the conjunctiva. However, pathogens also act on conjunctival epithelial cells to trigger the secretion of IL-8, IL-6, and TNF-a. These cytokines together with IL-1 and IFN-g can enter tear fluid and, in the presence of damage to the corneal epithelium, may penetrate into the corneal stroma and activate corneal fibroblasts. The tight barrier provided by the corneal epithelium normally prevents the entry of pathogens into the cornea. However, corneal epithelial injury can result in pathogen penetration into the corneal stroma. Pathogen-associated various factors such as lipopolysaccharide (LPS) of Gram-negative bacteria and peptidoglycan (PGN) of Gram-positive bacteria are recognized by toll-like receptors (TLRs) on the surface of corneal fibroblasts and trigger the production of IL-8 and the expression of intercellular adhesion molecule-1 (ICAM-1) by these cells. IL-8, IL-6, TNF-a, IFN-g, and IL-1 that enter the corneal stroma via tear fluid also induce IL-8 production by corneal fibroblasts. IL-8 then attracts neutrophils exuded (extravasated) from conjunctival blood vessels into the corneal stroma, and these cells interact with corneal fibroblasts via ICAM-1. IL-1 released from neutrophils further stimulates corneal fibroblasts. Corneal infection is associated with the production of two types of collagen-degrading enzymes: collagenase released from the pathogen and MMPs released from corneal fibroblasts. These enzymes destroy stromal collagen, eventually resulting in the development of corneal ulcer. Collagen destruction by MMPs released from activated corneal fibroblasts may continue even if the pathogen has been killed by antimicrobial treatment. Neutrophils were originally thought to destroy stromal collagen, but these cells were subsequently found to promote the production of MMPs by corneal fibroblasts rather than to degrade the collagen themselves. As with ocular allergy, corneal fibroblasts thus play a key role in the progression of the inflammatory response to corneal infection.

Tear Fluid as a Diagnostic Indicator of Inflammation The measurement of inflammatory cytokines or chemokines and the cellular components of tear fluid provides clinically important information on inflammation at the ocular surface. The presence of eosinophils in tear fluid thus confirms a diagnosis of allergic inflammation,


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Infection Vascular endothelium

Swelling

Permeability

Neutrophils

Conjunctival Stroma Pathogen

Hyperemia

Th1 cells IFN-γ IL-1

Conjunctival epithelium (loose barrier) Tear fluid

Injury

Epithelial cells

Pathogen

IL-8 IL-6 TNF-α

Corneal epithelium (tight barrier)

IFN-γ IL-1

Neutrophils

Epithelial defects

Disruption of barrier function

IL-8 IL-6 TNF-α

Discharge

IL-8

IFN-γ IL-1

Neutrophils

Pathogen Corneal Stroma

LPS PGN

Corneal fibroblasts

TLR

Ulcer

ICAM-1 IL-1

MMPs

Collagenases

Figure 3 Clinical characteristics of infection in the cornea and conjunctiva. ICAM, intracellular adhesion molecule; IFN-g, interferon-gamma; IL, interleukin; LPS, lipopolysaccharide; MMP, matrix metalloproteinase; PGN, peptidoglycan; Th2, T helper 2 cell; TARC, thymus and activation-regulated chemokine; TLR, toll-like receptor; TNF-a, tumor necrosis factor alpha.

whereas the presence of neutrophils is indicative of infectious inflammation. In addition to being of diagnostic value, the condition of the tear fluid can affect the progression of ocular surface inflammation. In individuals with dry eye, for example, the decrease in tear secretion and small volume of tear fluid may result in concentration of inflammatory cells and proteins. The condition of tear fluid should thus be taken into account in the treatment of patients with ocular surface inflammation.

Connection of the Conjunctiva and Cornea via Tear Fluid The surfaces of both the conjunctiva and the cornea are covered by epithelial cells. However, the biological responses of these two tissues to allergens or to pathogens differ markedly. The conjunctiva has a prominent vascular system and contains abundant immune cells, whereas the cornea is transparent and avascular and contains few immune cells. These anatomic differences between the

conjunctiva and cornea are reflected in the types of inflammatory condition that affect them. The conjunctiva is the principal target tissue for allergic reactions at the ocular surface, whereas the cornea is the main target for microbial infection or injury. The vascular system of the conjunctiva serves as a key source of immune cells in each of these conditions. The cornea is also affected by inflammatory reactions that occur in the conjunctiva, with the tear fluid that covers the surface of both the conjunctiva and the cornea serving as a conduit for the exchange of immune cells, cytokines, chemokines, and growth factors. The concept of inflammation was first described more than 2000 years ago as redness and swelling with heat and pain by Celsus. In the nineteenth century, the concept of loss of tissue function associated with inflammation was recognized. Recent advances in cell and molecular biology have revealed the cytokine and chemokine network that underlies inflammation. However, the availability of effective anti-inflammatory drugs other than steroids remains limited. Nonsteroidal antiallergic drugs have been developed and are effective for the treatment of allergic conjunctivitis. Nonsteroidal anti-inflammatory drugs (NSAIDs) are also effective in ameliorating inflammatory reactions.

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However, anti-inflammatory agents that halt tissue destruction are needed. Further characterization of the bidirectional regulation of conjunctival and corneal resident cells via cytokines and chemokines, as well as immune cells, released into tear fluid may provide a basis for the development of new drugs effective for the treatment of inflammation at the ocular surface. See also: Adaptive Immune System and the Eye: Mucosal Immunity; Adaptive Immune System and the Eye: T Cell-Mediated Immunity; Antigen-Presenting Cells in the Eye and Ocular Surface; Conjunctiva Immune Surveillance; Conjunctival Goblet Cells; Defense Mechanisms of Tears and Ocular Surface; Dry Eye: An Immune-Based Inflammation; Immunopathogenesis of Pseudomonas Keratitis; Molecular and Cellular Mechanisms in Allergic Conjunctivitis; Ocular Mucins; Overview of Electrolyte and Fluid Transport Across the Conjunctiva.

Further Reading Hazlett, L. D. (2005). Role of innate and adaptive immunity in the pathogenesis of keratitis. Ocular Immunology and Inflammation 13: 133–138. Kolaczkowska, E., Chadzinska, M., and Plytyez, B. (2008). Basic concepts of inflammation – from pioneer studies until now. In: Romano, G. T. (ed.) Inflammation Research Perspectives, pp. 113–168. New York: Nova Science Publishers. Kumagai, N., Fukuda, K., Fujitsu, Y., Yamamoto, K., and Nishida, T. (2006). Role of structural cells of the cornea and conjunctiva in the pathogenesis of vernal keratoconjunctivitis. Progress in Retinal and Eye Research 25: 165–187. Kumar, V., Abbas, A. K., Fausto, N., and Mitchell, R. N. (2007). Robbins Basic Pathology, 8th edn., pp. 31–58. Philadelphia, PA: Saunders-Elsevier. Ley, K. (2001). History of inflammation research. In: Ley, K. (ed.) Physiology of Inflammation, pp. 1–10. New York: Oxford University Press. Pearlman, E., Johnson, A., Adhikary, G., et al. (2008). Toll-like receptors at the ocular surface. Ocular Surface 6: 108–116. Tuli, S. S., Schultz, G. S., and Downer, D. M. (2007). Science and strategy for preventing and managing corneal ulceration. Ocular Surface 5: 23–39.

Concept of Angiogenic Privilege B Regenfuss and C Cursiefen, Friedrich-Alexander University Erlangen-Nuernberg, Erlangen, Germany ã 2010 Elsevier Ltd. All rights reserved.

Glossary Angioblast – Mesenchymal tissue that differentiates into blood cells and vascular endothelium. Angiogenesis – Formation of new blood vessels by outgrowth from preexisting vessels. Intussusception – Formation of new blood vessels by splitting of existing vasculature. Keratoplasty – Corneal transplantation. Vasculogenesis – De novo blood-vessel formation from endothelial progenitor cells.

Introduction: Angiogenesis and Lymphangiogenesis (Hem)angiogenesis describes the process of new bloodvessel formation by outgrowth from preexisting vessels. Accordingly, lymphangiogenesis means the formation of lymphatic vessels from preexisting ones. Both processes are precisely regulated and play an essential role in physiological and pathophysiological events in the organism. In the context of the eye, pathological new blood and lymphatic vessels are associated with numerous disorders reducing visual acuity. New blood-vessel formation in the organism is achieved either by angiogenesis or by vasculogenesis. Both processes can be distinguished from each other and strongly differ in the way the vessels arise. Vasculogenesis occurs mainly during embryogenesis and implies de novo blood-vessel formation by endothelial progenitor cells. During embryonic development, angioblasts, a subset of mesodermal cells, differentiate into endothelial cells and form the early vascular plexus. After establishing the primary vascular plexus, new blood vessels can be generated through angiogenesis that means by sprouting from preexisting blood vessels or by intussusception (nonsprouting angiogenesis). Angiogenesis and vasculogenesis normally occur during embryonic development. For the vascularization of the central nervous system (CNS) and the kidneys, angiogenesis seems to be the more important process. Following birth, most blood vessels remain in a quiescent state except for the once in the hair cycle, in the female reproductive system and during wound healing. In the case of unregulated angiogenesis, however, neovascularization can occur in the adult organism and usually is associated with

diseases such as arthritis, tumors, or corneal and retinal disorders. During early development of the retina, which is embryologically derived from the diencephalon, vasculogenesis and angiogenesis take place. In 1970, Ashton first described the mechanism of vasculogenesis for blood vessel formation in the retina of the human embryo. He proposed that primitive mesenchymal cells, after invading the retina, differentiate into endothelial cells, thereby, forming a capillary network. Nowadays there is evidence that vasculogenesis and angiogenesis both are responsible for the vascular development of the human fetal retina. Hughes and colleagues suggest a mechanism where vasculogenesis pioneers the establishment of a rudimentary vascular plexus, whereas angiogenesis provides further expansion of the vascular network and cares for increasing vessel density. Considering the fact that the retina is a highly metabolic active tissue both mechanisms complement one another and contribute to meet the metabolic requirements of the developing retina. The developed retina is a highly vascularized tissue that shows a dual blood supply. The inner layer of the retina is supported by the centralis retinae artery, originating from the arteria ophthalmica. The outer layer – especially the receptors – receive blood from the arteriae chorioideae. In general, the eye is an efficiently vascularized organ and shows a significantly higher blood circulation compared to other organs with the same volume; however, there are exceptions at the anterior pole of the eye being completely devoid of blood and lymph vessels. Whereas posterior structures like the retina, as mentioned earlier, show a strongly branched blood-vessel network, the sclera is relatively low vascularized and the adjacent cornea and the vitreous even are devoid of blood and lymphatic vessels. Keeping up corneal avascularity comprises an active process and needs the balance between angiogenic and anti-angiogenic factors. In this process, the cornea maintains the transparency even under inflammatory or other pro-angiogenic conditions by different molecular mechanisms which are not completely elucidated to date. This ability is called the corneal angiogenic privilege.

Corneal Angiogenic Privilege Corneal Avascularity The corneal angiogenic privilege normally prevents the ingrowths of new vessels in the cornea even under

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inflammatory or angiogenic conditions, thus maintaining corneal transparency and preventing loss of vision. A study with corneas from stillborn patients showed that this privilege is already present during intrauterine development at least from gestational age of 17 weeks. Contrary to the conjunctiva, in which blood and lymphatic vessels are detectable, no vessels are found in the cornea. This leads to the presumption that due to an early expression of anti-angiogenic and anti-lymphangiogenic factors, the cornea is primarily devoid of blood and lymphatic vessels and not as a result of regression of already existing vessels. The occurrence of neovascularization by angiogenic activity is initiated by a disbalance of angiogenic and antiangiogenic factors caused by upregulating angiogenic molecules as well as by downregulating angiogenesisinhibiting molecules. Interestingly, homozygote TSP-1 or TSP-2-knockout mice and even TSP-1/2-knockout mice showed no spontaneous corneal angiogenesis. The deficiency of an important anti-angiogenic factor like TSP-1 or TSP-2 does not result in a breakdown of the angiogenic privilege. This leads to the conclusion that at least during embryonic development the angiogenic privilege seems to be redundantly regulated by several anti-angiogenic factors. However, secondary to severe inflammation and several other diseases the angiogenic privilege can be overcome and an initial parallel outgrowth of blood and lymphatic vessels occurs. Several factors comprising angiogenic and anti-angiogenic molecules, the cornea itself, and adjacent structures like the limbus are known to be involved in affecting the angiogenic privilege (see Figure 1). Deprivation of the angiogenic privilege can lead to corneal neovascularization and, in consequence, loss of vision.

Tear film

Angiogenic and Anti-Angiogenic Molecules Involved in Corneal Neovascularization Numerous angiogenic and anti-angiogenic molecules have been identified in the cornea over the last years. Angiogenic molecules include vascular endothelial growth factors (VEGFs), basic fibroblast growth factors (bFGFs), and matrix metalloproteinases (MMPs). Angiostatin, endostatin, thrombospondins, and pigment epithelium-derived factor (PEDF) are some of the anti-angiogenic molecules detected in the cornea. The regulation of angiogenesis is due to the interaction of pro-angiogenic molecules and angiogenesis inhibitors, where tilting the balance toward pro-angiogenic factors can lead to neovascularization. Lymphangiogenesis seems to proceed in a similar way as angiogenesis, and can be activated in the adult during inflammation, immune responses, or malignant processes. Stimuli like hypoxia, for example, in the context of wound healing, can also trigger the induction of hemangiogenesis via hypoxia-inducible transcription factor (HIF), a key transcriptional regulator for VEGF-A. In contrast, lymphangiogenic VEGF-C cannot be upregulated by hypoxia but only by proinflammatory cytokines. Vascular endothelial growth factors The VEGF growth factor family currently consists of five members, VEGF/VEGF-A, PIGF, VEGF-B, VEGF-C, and VEGF-D. The growth factors are recognized by different VEGF receptors, namely VEGFR-1, VEGFR-2, and VEGFR-3. VEGF-A originally isolated from a human histiocytic lymphoma cell line U937 is secreted

Decoy receptors

Limbal barrier

Anterior border IPAS Endogenous angiogenic inhibitors

Limbal stem cells Internal border Anterior chamber

Figure 1 Several strategies are used by the normal cornea to maintain corneal avascularity (corneal angiogenic privilege): The cornea possesses several defense lines against invading vessels: an anterior border at the epithelial basement membrane, an internal border at Descemet’s membrane, and the limbal barrier beneath the limbal epithelial stem cell niche. Several mechanisms contribute to maintain the angiogenic privilege of the cornea: (a) endogenous inhibitors of angiogenesis, (b) decoy receptors neutralizing angiogenic growth factors, (c) anti-angiogenic stem cells, and (d) anti-hypoxia-driven-angiogenesis agents. ã M. Vogler.


in five different isoforms, generated by alternative splicing: VEGF115, VEGF121, VEGF165, VEGF189, and VEGF206. Vascular endothelial growth factor

VEGF-A shows numerous activities such as inducing endothelial cell proliferation and migration, proteolytic activity, and stimulating microvascular leakage – all of them promoting angiogenesis. VEGF-A mediates its function through receptors VEGFR-1 and VEGFR-2. Additionally, VEGF-A was reported to promote angiogenesis via an indirect pathway by upregulating NRP1, a neuronal receptor that has recently been shown to act as an isoform-specific receptor for VEGF165. VEGF-A can be released during hypoxia, in inflammatory situations, and during glucose deficiency. It was shown that the expression of VEGF-A is upregulated in inflamed and vascularized human corneas. In conclusion, VEGF-A seems to play an important role in inducing wound and inflammation-related corneal neovascularization. This was confirmed by the fact that corneal neovascularization could be suppressed after implantation of VEGF-A neutralizing antibodies in the corneal stroma of rats and rabbits. Whereas early data proposed that VEGF stimulates selectively hemangiogenesis but not lymhangiogenesis, recent data also suggest an (indirect) lymphangiogenic role: endogenous VEGF can promote lymphangiogenesisvia the recruitment of bone marrow-derived macrophages, releasing lymphangiogenic growth factors such as VEGF-C and -D. This broadens the impact of VEGF-A, not only for pathological hemangiogenesis, but also for lymphangiogenesis – at least in context of inflammation-induced neovascularization. A specific role for VEGF-A in the regulation of lymphangiogenesis was also described for primary tumors. The tumors were shown to overexpress VEGF-A, thereby inducing sentinel lymph node lymphangiogenesis. In a mouse model of delayed-type hypersensitivity (DTH), lymphangiogenesis was promoted by VEGF-A that was produced in the inflamed tissue. In addition, genetic variety, that is, single-nucleotide polymorphisms in the gene coding for VEGF-A, is associated with eye diseases like neovascular age-related macular degeneration and diabetic retinopathy. VEGF-C and VEGF-D

VEGF-C and VEGF-D are the main growth factors for lymphangiogenesis and both mediate their function by binding to receptors VEGFR-2 and VEGFR-3, present on endothelial cells. VEGF-C stimulates migration of cultured endothelial cells in vitro and increases – in its fully processed form – vascular permeability, migration, and proliferation of endothelial cells. Recently, the decisive role for VEGF-C during lymphangiogenic development was demonstrated in a study where homozygote as well as heterozygote VEGF-C-lacking mice were shown

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to have severe defects in the formation of lymphatic vessels. A study undertaken to analyze VEGF-C and its role in corneal neovascularization suggests VEGF-C to induce corneal lymphangiogenesis by binding to its cognate receptor VEGFR3 on lymphatic vessels in the conjunctiva. Inflammatory cells invading the cornea were identified as the main source of VEGF-C that was strongly upregulated 3 days following the injury. Besides its role as classic lymphangiogenic growth factor, VEGFC was reported to induce angiogenesis in vivo. VEGF-D also can act as a potent angiogenic factor, controlled by the nuclear oncogene c-fos and thus playing an important role in tumor invasion and tumor cell growth. Basic fibroblast growth factor Another important pro-angiogenic molecule – the basic fibroblast growth factor (bFGF/ FGF-2) – belongs to the FGF family. bFGF and acidic FGF (aFGF) expression has been demonstrated immunohistochemically in the outer retina of rat and mouse. bFGF was analyzed in several corneal neovascularization models and was recently demonstrated to induce angiogenesis as well as lymphangiogenesis in vivo in a mouse model corneal micropocket assay. Lymphangiogenesis was mediated by bFGF in an indirect way via VEGFR3 and was suppressed after inhibition of VEGFR3 signaling with anti-VEGFR3 antibodies. Both factors, bFGF and aFGF, were detectable in retinal pigment epithelial cells from choroidal neovascular membranes from human subjects with age-related macular degeneration (AMD), whereas there was only little immunoreactivity for the growth factors in retinal pigmented epithelial (RPE) cells from healthy eyes. This suggests an important role for aFGF and bFGF in the development of choroidal neovascularization. bFGF might play an indirect role in initiation of neovascularization and interacts with the VEGF signal-transduction pathways. This is supported by the fact that bFGF was found to be colocalized with VEGF in cells of epiretinal and choroidal neovascular membranes, suggesting that more than one growth factor may contribute to pathological angiogenesis. In favor for that theory is that mice with a disruption of the bFGF-coding gene can still develop choroidal neovascularization. Recently, bFGF was thought to take a role in progression and survival of retinoblastoma, a tumor producing significant amounts of bFGF. The differential production and response to isoforms of bFGF reveal bFGF as a growth factor influencing pathogenesis and chemoresistence of retinoblastoma. Inhibitory PAS (Per/Arnt/Sim) domain protein As mentioned earlier, the upregulation of angiogenic molecules like VEGF-A and angiopoietin-4 (Ang-4)

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during hypoxia is mediated by hypoxia-inducible transcription factor-1 (HIF-1) and can induce an angiogenic response. Interestingly, hypoxic conditions in the cornea appearing, for example, during overnight closure of the eye, do not induce corneal neovascularization, indicating the presence of factors suppressing hypoxia-induced angiogenesis. However, prolonged contact lens use has been associated with corneal angiogenesis and hypoxia has been implicated in this process. IPAS, a basic helix–loop–helix (bHLH)/PAS protein, expressed in mouse corneal epithelium, was suggested as a negative regulator of HIF-mediated control of gene expression: only low levels of IPAS mRNA were detectable in primary cultures of mouse corneal cells under normoxic conditions, whereas under hypoxic conditions IPAS mRNA expression was upregulated. Following transfection of primary corneal cells with an IPAS antisense vector, VEGF mRNA expression – under normoxic and hypoxic conditions – was upregulated. Furthermore, in vivo experiments with mouse corneas containing pellets with IPAS antisense oligonucleotides showed a significantly induced neovascularization compared to the eyes treated with the IPAS sense oligonucleotide.

Limbal Barrier Function The border between sclera/conjunctiva and the transparent cornea, the limbus, is of great importance for angiogenesis: the loops and arcades of conjunctival capillaries as well as the lymphatic capillaries end in the limbal region. Corneal neovascularization, however, arises in the limbal area from preexisting pericorneal vessels (hemangiogenesis as well as lymphangiogenesis). Stem cells, required for corneal epithelial cell proliferation and differentiation, are located in the basal epithelium at the corneoscleral limbus. They were described to act as a barrier between conjunctival and corneal epithelium and as important for corneal wound healing. In normal situations, the limbal stem cells prevent conjunctival epithelial cells from migrating to the ocular surface thereby inhibiting corneal neovascularization. This limbalbarrier concept may contribute to the maintenance of the angiogenic privilege. The theory is supported by the observation of conjunctivalization of the corneal surface with subsequent vascularization in situations of loss or malfunction of the stem cells. Angiogenic Privilege and Immune Privilege

Cornea and Corneal Epithelium Consistent with the assumption of a redundantly organized angiogenic privilege, numerous endogenous anti-angiogenic factors in the cornea are described to be implicated in the regulation of angiogenesis. Years ago, the corneal epithelium itself was found to have angiogenic activity. In 1978, Eliason reported data from an in vivo system suggesting corneal epithelium as a source for an unknown vasostimulating substance. In vitro experiments from Eliason and Elliott also showed a stimulating effect of corneal epithelial homogenate and epithelial-conditioned medium on the proliferation of cultured rabbit vascular endothelial cells. Recent research attributes corneal epithelium an anti-angiogenic function: an intact corneal epithelium can suppress inflammation and corneal neovascularization in the graft following orthotopic transplantation in mice. Secondly, mice with de-epithelialized corneas have significantly increased recruitment of CD45þ inflammatory cells and an increased neovascular response compared to mice with an intact epithelium. One potent mechanism for an antiangiogenic function of corneal epithelium is the ectopic constitutive expression of VEGFR-3 on normal human corneal epithelial cells. VEGFR-3 can act as a decoy receptor to bind VEGF-C, thus functioning as a sink for the angiogenic molecules and inhibiting inflammation induced corneal hemangiogenesis and lymphangiogenesis. A similar task fulfils the soluble form of VEGFR-1 – expressed in the cornea – where it can neutralize VEGF-A.

Ingrowth of blood and lymphatic vessels into the cornea is incompatible with good vision. Visual acuity is impaired not only by vascularization itself, but also by secondary changes such as lipid keratopathy, corneal edema, or bleeding into the cornea, thereby reducing corneal clarity and transparency. As mentioned earlier, actively maintaining the avascularity even under inflammatory or other angiogenic conditions is ensured by the angiogenic privilege. It contributes, at least partly, to the occurrence of the prolonged graft survival of corneal allografts, called the immune privilege of the eye. The phenomenon of the immune-privileged site was first proposed by Medawar, in 1948, and has built a foundation for numerous research. Nowadays, ocular immune privilege is commonly seen as the fact that vulnerable organs or tissues are protected from pathogens without an immunogenic inflammation that would permanently damage those tissues and/or would lead to a loss of specialized functions. An immune response after transplantation in so-called low-risk eyes can only be noticed in around 10%, although under normal circumstances there is no HLA matching and only a topical, but no systemic, immunosuppression. In contrast, immune reactions in high-risk eyes with preceding corneal inflammation or neovascularization occur in over 50%. The pathologically vascularized recipient bed prior to corneal transplantation (i.e., penetrating keratoplasty), therefore, lowers the outcome of corneal transplantation and is an important risk factor for subsequent immune reactions.


Immune responses are primarily mediated by corneal lymphatic vessels which form the afferent arc of the immune response. Via the lymphatic vessels invading antigen-presenting cells (APCs; dendritic cells from the graft or host) and antigenic material from the graft can be transported via conjunctival lymph vessels to the regional lymph nodes. The draining cervical lymph nodes were shown to be critically involved in promoting alloimmunity and allograft rejection. Following surgical removal of the cervical lymph nodes and following orthotopic corneal transplantation of fully mismatched high-risk allografts, over 90% of the hosts accepted the allograft. The importance of the lymphatic vessels, being the afferent arc of the immune response, offers new therapeutic opportunities for improving graft survival. Interfering with this pathway might restore the immune privileged status of the eye and ensures prolonged graft survival in low- and high-risk eyes. Early studies have shown that induction of donor-specific anterior chamber-associated immune deviation (ACAID) – manifestation of the ocular immune-privilege induced prolonged graft survival in high-risk eyes of C57BL/6 mice. Recently, it was shown that Integrin a5-blockade could significantly block the outgrowth of lymphatic vessels in the cornea. The angiostatic drug bevacizumab, a recombinant humanized monoclonal antibody against VEGF-A, inhibits corneal hemangiogenesis and lymphangiogenesis in vitro and in vivo. Furthermore, inhibition of corneal hemangiogenesis and lymphangiogenesis by a molecular VEGF-A trap leads to improved long-term graft survival. In addition to the inhibition of inflammatory lymphangiogenesis, alternative strategies like induction of regression of established lymphatic vessels in prevascularized corneas and influencing the recruitment of APCs could be possible methods for corneal anti-lymphangiogenic treatment. Recently, it was shown that even hemangiogenesis and lymphangiogenesis occurring following transplantation increase the risk for graft rejection after high-risk corneal transplantation.

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See also: Avascularity of the Cornea; Corneal Angiogenesis.

Further Reading Azar, D. T. (2006). Corneal angiogenic privilege: Angiogenic and antiangiogenic factors in corneal avascularity, vasculogenesis, and wound healing (an American Ophthalmological Society thesis). Transactions of the American Ophthalmological Society 104: 264–302. Cebulla, C., Jockovich, M. E., Pina, Y., et al. (2008). Basic fibroblast growth factor impact on retinoblastoma progression and survival. Investigative Ophthalmology and Visual Science 49(12): 5215–5221. Chang, J. H., Gabison, E. E., Kato, T., and Azar, D. T. (2001). Corneal neovascularization. Current Opinion in Ophthalmology 12: 242–249. Churchill, A. J., Carter, J. G., Ramsden, C., et al. (2008). VEGF polymorphisms are associated with severity of diabetic retinopathy. Investigative Ophthalmology and Visual Science 49: 3611–3616. Cursiefen, C. (2007). Immune privilege and angiogenic privilege of the cornea. Chemical Immunology and Allergy 92: 50–57. Cursiefen, C., Chen, L., Dana, M. R., and Streilein, J. W. (2003a). Corneal lymphangiogenesis: Evidence, mechanisms, and implications for corneal transplant immunology. Cornea 22: 273–281. Cursiefen, C., Seitz, B., Dana, M. R., and Streilein, J. W. (2003b). Angiogenesis and lymphangiogenesis in the cornea. Pathogenesis, clinical implications and treatment options. Ophthalmologe 100: 292–299. Folkman, J. and Shing, Y. (1992). Angiogenesis. Journal of Biological Chemistry 267: 10931–10934. Hori, J. and Niederkorn, J. Y. (2007). Immunogenicity and immune privilege of corneal allografts. Chemical Immunology and Allergy 92: 290–299. Makino, Y., Cao, R., Svensson, K., et al. (2001). Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression. Nature 414: 550–554. Niederkorn, J. Y. (1999). The immune privilege of corneal allografts. Transplantation 67: 1503–1508. Niederkorn, J. Y. (2007). Immune mechanisms of corneal allograft rejection. Current Eye Research 32: 1005–1016. Shweiki, D., Itin, A., Soffer, D., and Keshet, E. (1992). Vascular endothelial growth factor induced by hypoxia may mediate hypoxiainitiated angiogenesis. Nature 359: 843–845. Streilein, J. W. (2003a). Ocular immune privilege: The eye takes a dim but practical view of immunity and inflammation. Journal of Leukocyte Biology 74: 179–185. Streilein, J. W. (2003b). Ocular immune privilege: Therapeutic opportunities from an experiment of nature. Nature Reviews Immunology 3: 879–889.

Corneal Angiogenesis M S Cortina and D T Azar, University of Illinois at Chicago, Chicago, IL, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Angiogenesis – The formation of new vessels from preexisting vascular structures. Allograft – The tissue taken from one person for transplantation into another. Autograft – The tissue transplanted from one part of the body to another part of the same individual. Chemotaxis – The movement of neutrophils toward bacteria or an area of tissue damage. Conjunctival metaplasia – An abnormal epithelial differentiation represented by a spectrum of skin-like changes of conjunctival epithelium. Corneal extracellular matrix – The tissue that provides structural support to the cells in the cornea. Corneal pannus – The fibrovascular connective tissue that proliferates in the anterior layers of the peripheral cornea in inflammatory corneal disease. Growth factor – A naturally occurring protein capable of stimulating cellular growth, proliferation, and differentiation. Hypercapnia – High levels of carbon dioxide. Hypoxia – Oxygen deficiency. Limbal stem cell deficiency – The loss of stem cells in the limbus (ring around the base of the cornea which supports health of the corneal epithelium). Matrix metalloproteinases (MMPs) – The zincdependent endopeptidases capable of degrading all kinds of extracellular matrix proteins; they can also process bioactive molecules. Penetrating keratoplasty (PK) – The procedure in which a full-thickness button of cornea is removed from the recipient and replaced with a similar-sized or larger button of tissue from a donor. Vasculogenesis – The formation of new blood vessels from bone-marrow-derived angioblasts that occurs mainly during embryogenesis.

Introduction Under homeostatic conditions, the cornea is avascular, which is critical for corneal light transmission and proper optical performance. Corneal avascularity is maintained by tightly controlled biological anti-angiogenic events that counterbalance the effects of pro-angiogenic factors in the

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cornea. Under pathological conditions, the balance may be shifted toward angiogenesis, leading to the formation of new blood vessels and lymphatic channels. New blood vessel formation or corneal neovascularization (NV) is a sight-threatening condition usually associated with inflammatory or infectious disorders. It is a major contributor to the loss of corneal transparency. The presence of corneal NV, in turn, elevates the risk of graft rejection and decreases the success of penetrating keratoplasty (PK).

Angiogenesis Angiogenesis is the main process of blood vessel formation in nonembryonic tissue. It involves the formation of new vessels from preexisting vascular structures and is the primary mechanism underlying corneal NV. New blood vessels also form during vasculogenesis, which is the formation of new blood vessels from bone-marrow-derived angioblasts and occurs mainly during embryogenesis. Angiogenesis is a complex process that starts with vasodilation of existing vessels and an increase in vascular permeability. This leads to extravasation of plasma proteins (such as fibrin), growth factors, and inflammatory mediators. The accumulation of plasma proteins in the surrounding tissue provides a supporting structure for subsequent endothelial cell (EC) migration. The combined presence of growth factors and inflammatory mediators stimulates the degradation of the extracellular matrix (ECM), making room for EC migration as well as releasing angiogenic factors anchored in the matrix. The newly released angiogenic factors then continue to activate ECs, which migrate from preexisting vessels and form sprouting tubes. The avascularity of the cornea dictates that nutrients for this tissue be obtained from adjacent tissues. The three major sources of nutrients are tear film, aqueous humor, and the pericorneal capillary plexus at the limbus. This plexus nourishes the peripheral cornea and is derived from ciliary arteries, which are branches of the ophthalmic artery. These vessels do not normally enter the cornea. However, new blood vessels may sprout from capillaries and venules of the pericorneal plexus under pathological conditions, leading to corneal NV.

Etiology and Epidemiology of Corneal NV Although the exact incidence of corneal NV is not known, it was estimated that this condition affects 1.4 million


patients in the United States annually and that 20% of corneal buttons obtained during PK show evidence of NV. The causes of corneal NV include immune, inflammatory, infectious, degenerative, and traumatic disorders. Corneal infections are the most common worldwide causes of corneal NV leading to vision loss. A classic example is trachoma, an infectious disease characterized by the formation of a superior pannus, which can extend to the central cornea and is often associated with corneal scarring, opacification, and loss of visual acuity. The incidence of trachoma in the US is low; however, this condition remains a major cause of blindness in other parts of the world (Figure 1). Corneal NV is also commonly associated with other severe bacterial and viral infections. The herpes virus family (primarily herpes simplex and herpes zoster viruses) is the primary cause of keratitis-induced NV in transplant buttons. In herpes-simplex-induced stromal keratitis, NV is essential for the pathogenesis of keratitis, and inhibition of angiogenesis can reduce the formation of corneal lesions. Although infections account for many US cases of corneal NV, the most common cause of corneal NV in the US is the use of contact lenses. In this case, hypoxia and hypercapnia are thought to be associated with the induction of NV (Table 1). The incidence of corneal NV after PK can be as high as 40% at 6–9 months after surgery. The prognosis of transplanting grafts into heavily vascularized corneas is poor. Graft failure has been reported to contribute to >30% of the histopathological diagnoses obtained from vascularized corneal buttons. Risk factors for corneal NV after PK in patients without active inflammation, previous corneal NV, or persistent epithelial defects include suture knots buried in the host stroma, active blepharitis, and a large recipient bed.

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Immune disorders also contribute to corneal NV (Figure 2). These disorders can result in significant vision loss and include ocular pemphigoid, rosacea, atopic keratoconjunctivitis, and Stevens–Johnson syndrome. The incidence of corneal NV among patients with these disorders can be considerable. Long-term follow-up of patients with atopic keratoconjunctivitis, for example, revealed that the rate of corneal NV was as high as 60% during the disease course. Limbal stem cell deficiency, which may occur following trauma and chemical burns, is another cause of corneal NV. It may be also associated with aniridia and autoimmune disorders. Limbal stem cell deficiency produces not only corneal NV, but also corneal inflammation and conjunctivalization of the corneal epithelium. The restoration of corneal avascularity after successful limbal stem cell transplantation underscores the importance of the antiangiogenic and anti-inflammatory activity of normal corneal epithelial cells.

Clinical Manifestations Corneal NV can be classified as pannus or stromal NV. In the former, fibrovascular tissue is visible between the epithelium and Bowman layer. Inflammatory pannus is associated with prominent leukocyte infiltration and disruption of Bowman’s layer. In contrast, degenerative pannus is characterized by fewer inflammatory cells, an intact Bowman’s layer, and regression of the vascular component that leaves a layer of fibrous tissue. Stromal NV, located posterior to Bowman’s layer, is more commonly seen in the anterior two-third of the stroma. In herpetic and syphilitic interstitial keratitis, deep stromal vessels (and ghost vessels in the late quiescent stages) are seen just anterior to Descemet’s membrane (Figure 3). Visual acuity is reduced by corneal NV. Reduced visual acuity may be secondary to multiple factors. For example, opacity caused by circulating blood cells may interfere with visual acuity. Acuity may also be reduced by irregular architecture of vascular walls, a feature that induces higher-order aberrations. Other effects contributing to diminished visual acuity may include alteration in the spacing of stroma collagen fibers, fluid leakage, edema and lipid deposition in the surrounding tissue, and corneal surface irregularities (Figure 4).

Mechanisms Underlying the Maintenance of Corneal Avascularity Figure 1 Salzman’s nodular degeneration with associated superficial corneal neovascularization.

Several mechanisms have been proposed to contribute to corneal avascularity. These include:

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Immune Regulation of the Cornea and Conjunctiva and Its Dysregulation in Disease Causes of corneal neovascularization

Inflammatory disorders

Infectious keratitis

Degenerative/congenital

Traumatic/iatrogenic

Ocular pemphigoid Atopic conjunctivitis Rosacea Graft rejection Lyell’s syndrome Stevens–Johnson syndrome Graft vs. host disease

Herpes simplex Herpes zoster Pseudomonas Chlamydia Syphilis Candida Fusarium Aspergillus Onchocerciasis

Pterygium Terrien marginal degeneration Aniridia Degenerative pannus

Contact lens wear Alkali burns Ulceration Iatrogenic Stem cell deficiency

Figure 2 Corneal neovascularization and infectious keratitis in a patient with underlying severe dry eye syndrome secondary to rheumatoid arthritis.

Figure 3 Superficial and deep stromal neovascularization in a patient with neurotrophic cornea.

1. Corneal dehydration resulting in tightly packed collagen lamellae. The relationship between corneal NV and corneal edema was first reported in 1949 by Cogan, who postulated that the distention and bursting of the vessels preceding formation of the capillary sprouts were due to a decrease in external pressure that reduced

Figure 4 Lipid deposition secondary to corneal neovascularization. Note relative small vessel crossing the host-graft junction responsible for significant lipid deposit.

vessel wall support. However, subsequent studies showed that corneal edema alone is not sufficient to trigger corneal NV. 2. The angiostatic nature of corneal epithelial cells. Blood vessels are known to be capable of growing into corneas in the absence of epithelium. Early research suggested that corneal epithelial cells are a source of angiogenic factors. More recent studies suggest that the corneal epithelium has an anti-angiogenic effect. The presence of soluble vascular endothelial growth factor (VEGF) receptors 1 and 3, and other naturally occurring antiangiogenic factors, in the corneal epithelium contributes to its angiostatic nature. 3. The immune privilege of the cornea. The mechanisms underlying corneal immune privilege include low expression of major histocompatibility complex (MHC) antigens on corneal cells, expression of Fas ligand by the cornea, a relative paucity of mature antigen-presenting cells, and the presence of immunomodulatory molecules in the anterior chamber. Importantly, this state can be reversed by inflammation, and such reversal may contribute to vascularization. For example, polymorphonuclear leukocytes have the potential to initiate corneal NV through


4.

5.

6. 7.

the release of chemical mediators. Accordingly, there is a clear association between corneal inflammation and NV. Nevertheless, corneal NV may occur in the absence of inflammation. Low corneal temperature, extensive innervation, and movement of the aqueous humor across the cornea. The roles of these factors in the maintenance of corneal avascularity are currently unclear. The barrier function of limbal cells. The limbus is thought to prevent corneal NV by acting as a barrier to conjunctival growth over the cornea. This barrier function may be attributable to the ability of limbal stem cells to replenish the corneal epithelium, thus preventing invasion of conjunctival epithelium and avoiding NV. This hypothesis has been used to account for corneal NV following experimental limbal damage and stem cell dysfunction. It is also one of the explanations for corneal pannus observed in aniridia. This hypothesized barrier function of limbal cells supports the use of limbal stem cell transplantation as a definitive treatment for ocular-surface disorders. However, a physical barrier may not completely explain corneal avascularity. Low levels of angiogenic factors and active production of potent anti-angiogenic factors in the cornea during homeostasis. Active production of potent anti-angiogenic factors. Although the five previously mentioned factors likely contribute to maintenance of corneal avascularity, available evidence supports this as the main mechanism responsible for maintaining corneal avascularity.

Corneal Angiogenic Privilege: The Balance between Angiogenesis and Anti-Angiogenesis Multiple local and systemic signals are responsible for regulating growth and regression of new blood vessels. These signals include cyclic adenosine monophosphate (cAMP), steroid hormones, protein kinase C (PKC) agonists, polypeptide growth factors, oxygen, free radicals, glucose, cobalt, and iron. In the cornea, the tight equilibrium between these pro- and anti-angiogenic signals may be disrupted under pathological conditions. Ultimately, this may tip the balance toward an upregulation of proangiogenic factors or a downregulation of anti-angiogenic factors, in either case leading to corneal NV (Table 2). When the cornea is injured, wound healing often occurs in the absence of NV. This is the case for most adequately treated corneal infections. Healing after corneal surgery is also usually avascular. Corneal wound healing involves four phases. In the first phase, keratocytes near the area of epithelial debridement undergo apoptosis. In the second phase, adjacent keratocytes proliferate to repopulate the wound within 24–48 h after wounding. These keratocytes

Table 2

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Factors involved in the regulation of angiogenesis

Angiogenic factors

Anti-angiogenic factors

Fibroblast growth factor (FGF) Vascular endothelial growth factor (VEGF) Placenta growth factor (PGF) Transforming growth factor-a (TGF-a) Transforming growth factor-b (TGF-b) Insulin-like growth factor (IGF) Leptin Integrins (anb3) Platelet-derived growth factors (PDGF) Matrix metalloproteinases (MMPs) Angiogenin

Endostatin Angiostatin

Hepatocyte growth factorscatter factor (HGF-SF) Tumor necrosis factor-a (TNF-a) Connective tissue growth factor (CTGF) Interleukin-8 (IL-8) Monocyte chemoattractant protein-1 (MCP-1) Platelet-activating factor (PAF)

Prolactin Matrix metalloproteinases (MMPs) Tissue inhibitor of MMPs (TIMPs) Thrombospondin Arresten Canstatin Tumstatin Pigment-epitheliumderived factor (PEDF) Tumor necrosis factor a (TNF-a) Interleukin-4 (IL-4) Interleukin-13 (IL-13) Fibulin Endoperellin Antithrombin Plasminogen activator inhibitor (PAI) Vasostatin Neostatin-7

transform into fibroblasts and migrate into the wound area. This process may take up to a week and is not accompanied by corneal NV. In the third phase, fibroblasts may transform into myofibroblasts. This occurs in laser-inflicted wounds lacking Bowman’s layer and in incisional wounds. Myofibroblasts may take up to a month to become apparent. Corneal NV is also absent in this phase of wound healing. The fourth and final phase involves stromal remodeling and is dependent on the original wound. When wound healing is accompanied by ECM turnover, angiogenesis in granulation tissue is usually observed. Some of the molecules that regulate angiogenesis are discussed below.

Angiogenic Molecules Vascular endothelial growth factor VEGF is a dimeric 46-kDa glycoprotein. This growth factor stimulates angiogenesis by increasing EC proliferation, migration, proteolytic activity, and capillary tube formation. It also significantly increases vascular permeability. The VEGF family includes VEGF-A, -B, -C, -D, placenta growth factor (PlGF), and the viral VEGF

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homolog VEGF-E. VEGF-B promotes nonangiogenic tumor progression, while VEGF-C and -D participate in angiogenesis and lymphangiogenesis. VEGF-A also participates in angiogenesis and increases vascular permeability. Five isoforms of VEGF-A (VEGF115, 121, 165, 189, and 206) can be generated by alternative splicing of the same gene. The longer isoforms (VEGF189 and 206) are matrixbound, whereas the shorter isoforms (VEGF121 and 165) are freely diffusible. These VEGF isoforms produce different actions when secreted. For example, all isoforms increase in vascular permeability, but only VEGF121 and VEGF165 have mitogenic activity. VEGF121 has greater angiogenic activity than VEGF165 or VEGF189. On the other hand, VEGF165 is more potent than VEGF121 in induction of inflammation, intercellular adhesion molecule-1 (ICAM-1) expression in ECs, and chemotaxis of monocytes. This suggests that alternate splicing of VEGF messenger RNA (mRNA) can be regulated to achieve a range of physiologic actions. The VEGF family members act through binding to high-affinity receptor tyrosine kinases. Two high-affinity receptor tyrosine kinases have been identified for VEGFA: VEGFR-1 (fms-like tyrosine kinase-1 or Flt-1) and VEGFR-2 (kinase insert domain-containing receptor or KDR). VEGFR-3 (fms-like tyrosine kinase-4 or Flt-4) serves as a high-affinity receptor for VEGF-C and -D. Both VEGFR-1 and -2 are expressed primarily in vascular ECs, while VEGFR-3 is predominantly expressed in lymphatic ECs. VEGF-B binds to VEGFR-1 and has mild mitogenic activity. In contrast, binding of VEGF-D and -C to VEGFR-3 regulates the growth and differentiation of blood vessels and lymphatic endothelium. VEGF is produced by macrophages, T cells, astrocytes, pericytes, fibroblasts, retinal pigment epithelial cells, and smooth muscle cells. In addition, VEGF is expressed in all three cellular layers of the cornea. It is highly expressed in vascular ECs of limbal vessels and in new stromal vessels. Under inflammatory conditions, VEGF expression is increased in epithelial and vascular ECs, particularly near macrophage infiltrates and fibroblasts in corneal scars. Following corneal cautery, VEGF165 and 189 mRNA is increased at 48 h and returns to baseline by day 7. Immunohistochemistry has revealed that VEGF is initially expressed in neutrophils and later expressed in macrophages, demonstrating that VEGF production by leukocytes is associated with corneal NV. In addition, VEGF concentration is significantly increased in vascularized corneas as compared to normal corneas. In limbal-deficiency-induced corneal NV, VEGF mRNA and protein are induced after injury and are both temporally and spatially correlated with inflammation and NV. VEGF is not only induced during NV, but is also required for corneal angiogenesis. The indispensable role of VEGF in angiogenesis is shown by the finding that

stromal implantation of anti-VEGF antibodies inhibits NV in a rat model. Conversely, implantation of a Hydron pellet containing VEGF into the stroma induces severe corneal NV without significant inflammation. The effects of VEGF in the cornea are not limited to NV, as this growth factor has also been shown to regulate goblet cell migration. Studies analyzing the correlation between cornea NV and conjunctivalization showed that VEGFR-1 is present in the conjunctiva-like epithelium covering the cornea as well as in goblet cells, invading leukocytes, and the corneal vasculature. Inhibition of VEGF activity inhibited not only corneal NV, but also goblet cell density, suggesting that VEGF may promote goblet cell migration. Evidence suggests that VEGF also participates in corneal lymphangiogenesis. Corneal lymphangiogenesis may contribute to graft sensitization and rejection, following high-risk keratoplasty of vascularized corneas. VEGF-C binds to VEFGR-3 and induces lymphatic growth in the cornea. Interestingly, inhibition of lymphatic growth is observed after administration of a VEGF trap that neutralizes VEGF-A, but not VEGF-C or -D. This could be explained by the chemotactic effect on macrophages that release VEGF-C in inflamed corneas observed with VEGF-A. Thus, VEGF-A amplifies signals essential for lymphatic growth. In general, corneal lymphangiogenesis seems to correlate well with the degree of corneal hemangiogenesis. Recent studies have shown that VEGF, although present in the cornea, does not promote angiogenesis under normal conditions. VEGF-A found in corneal tissue is mostly bound to an alternative spliced secreted isoform of VEGFR-1 (sflt-1), which acts as a trap for secreted VEGF-A and in this way contributes to maintenance of corneal avascularity. In addition, VEGFR-3 is expressed in endothelial as well as epithelial cells in the cornea. When VEGF-C and -D bind to endothelial VEGFR-3, they stimulate proangiogenic signaling. In contrast, VEGFR-3 expressed by corneal epithelium acts as a decoy receptor sequestering VEGF but yet rendering it available when an angiogenic response is needed to enhance the immune defense. This VEGFR-3 sink system is a potent mechanism that inhibits inflammatory-induced angiogenesis. Basic fibroblast growth factor Basic fibroblast growth factor (bFGF) is another potent angiogenic factor. It is a member of the fibroblast growth factor (FGF) family, which includes 23 heparin-binding peptides widely expressed during cell differentiation, angiogenesis, mitogenesis, and wound healing. bFGF functions are mediated by the receptors FGFR-1, -2, -3, and-4. FGF recptor-1 (FGFR-1) is expressed in normal corneal epithelium, while bFGF is upregulated following injury. It is also upregulated following co-culture of corneal epithelial cells with vascular EC and keratocytes. The affinity of bFGF for its receptor differs according to the extent of


maturation of new vessels. This may be due to varying expression of heparan sulfate proteoglycans and highlights the role of ECM proteins in the regulation of corneal angiogenesis. Matrix metalloproteinases

The matrix metalloproteinases (MMPs) constitute a multigene family of zinc-binding proteolytic enzymes that participate in ECM remodeling. Many of the growth factors that modulate angiogenesis also influence MMP expression. These growth factors include VEGF, FGF-2, and tumor necrosis factor-alpha (TNF-a). Vascular ECs respond by secreting proteolytic enzymes that degrade the ECM to facilitate migration and differentiation of ECs. The MMPs that have identified in the cornea are collagenases I and II (MMP-1 and -13), stromelysin (MMP-3), matrilysin (MMP-7), membrane-type MMP (MT-MMP-14), and gelatinases A and B (MMP-2 and -9). Both MMP-2 and MMP-9 are proteolytically activated primarily by MT1MMP during capillary formation. Several reports suggest that these MMPs participate in vascular invasion by directly degrading the matrix or releasing matrix-bound cytokines and growth factors. Accordingly, inhibition of MMP-9 activity in the cornea decreases angiogenesis. However, given their ability to degrade ECM, MMPs exhibit a dual action in angiogenesis. For example, MMP-2 activation may release anti-angiogenic fragments, allow the production of potent angiostatic factors, or facilitate angiogenesis. Lipid mediators

One of the initial events that occurs after corneal injury is the release of arachidonic acid. In the corneal epithelium, arachidonic acid is then metabolized by cyclooxygenase (COX) to generate eicosanoids (such as 12- and 15-HETE), lipoxin A4 (LXA4), and prostaglandins. 12(S)-HETE is a powerful angiogenic factor, and COX inhibitors have been shown to reduce corneal angiogenesis in animal models. Plateletactivating factor is another potent lipid mediator released from the cell membrane after corneal injury. It contributes to corneal NV by increasing expression of VEGF, MMP-9, and urokinase plasminogen activator (uPA), all of which subsequently stimulate vascular EC migration.

Anti-Angiogenic Molecules Angiostatin

Angiostatin results from the cleavage of plasminogen. Several MMPs can cleave plasminogen to generate angiostatinlike molecules. The inhibitory effect of angiostatin on vascular ECs may be due to inhibition of adenosine triphosphate (ATP) synthesis in these cells, an effect that decreases EC migration and proliferation. Angiostatin binds to integrin alpha-v beta-3 (avb3) and affects angiogenesis as well as

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developmental NV. It also induces vascular EC apoptosis mainly in areas of NV. All three layers of the cornea are able to synthesize plasminogen and angiostatin. Tears collected after overnight eye closure contain a significant amount of angiostatinrelated molecules known to have anti-angiogenic properties. This has also been shown in tears of contact lens-bearing patients, suggesting that these molecules play a role in preventing NV under hypoxic conditions. Corneal NV occurs following injection of anti-angiostatin antibodies into corneas having undergone post-excimer laser keratectomy. This supports the idea that plasminogen and angiostatin are important for the maintenance of corneal avascularity. Endostatin and neostatins Endostatin is a 20-kDa proteolytic fragment of collagen XVIII that exhibits anti-angiogenic activity. It was originally discovered as an angiogenic inhibitor purified from conditioned media of murine hemangioendothelioma cells. Endostatin inhibits bFGF-induced corneal NV as well as VEGF-induced vascular EC migration and proliferation. Collagen XVIII is localized mainly in the corneal vascular and epithelial basement membrane. Smaller fragments of collagen XVIII, known as Neostatins -7 and -14, are generated by the enzymatic activity of MMPs -7 and -14, respectively. They have potent antiangiogenic and anti-lymphangiogenic properties. Local production of endostatin and Neostatins -7 and -14 may occur during wound healing. Endostatin is Food and Drug Administration (FDA)-approved for the treatment of cancer-related NV. Pigment-epithelial-derived factor Pigment-epithelial-derived factor (PEDF) is a potent anti-angiogenic and neurotrophic factor that is found in multiple eye tissues including the cornea. In contrast to VEGF, which is induced under low oxygen conditions, PEDF expression is suppressed during hypoxia. PEDF induces EC apoptosis. It also has antipermeability and anti-inflammatory activity that counterbalances VEGF actions. Studies have shown that PEDF-blocking antibodies induce corneal NV when implanted into the stroma and that recombinant PEDF inhibits bFGF-induced corneal NV. These findings are consistent with an essential role for PEDF in maintaining the avascularity of ocular tissues. Given its effectiveness at countering VEGF activity, PEDF may be a good pharmacological inhibitor of angiogenesis. Arresten, canstatin, and tumstatin Arresten is a 26-kDa protein derived from the noncollagenous (NCl) domain of the type IV collagen a1 chain. This molecule has been shown to inhibit bFGFstimulated proliferation, migration, and tube formation of

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cultured ECs. It also inhibits NV in vivo. Canstatin is a 24-kDa fragment of the type IV collagen a-chain. It also inhibits EC proliferation and tube formation. The mechanism of action appears to involve phosphoinositide 3-kinase/ protein kinase B (PI3K/Akt) inhibition and depends on signaling events transduced through membrane-death receptors. Tumstatin, a 28-kDa fragment of the type IV collagen a3 chain, also has anti-angiogenic activity.

Therapy Identifying and adequately treating the underlying cause of corneal NV is critical. Therapies for corneal NV may range from antimicrobial therapy for infectious keratitis to systemic immunosuppression for autoimmune diseases such as ocular cicatricial pemphigoid. Some of the established and investigational medical and surgical treatments for corneal NV are discussed below. Medical Treatments Anti-inflammatory compounds, such as steroids, have a long history of use for the suppression of inflammation and associated angiogenesis. The anti-angiogenic effects of steroid treatment are likely secondary to their antiinflammatory actions and include inhibition of chemotaxis and cytokine synthesis. Steroids have also been shown to inhibit vascular EC proliferation and migration. Unfortunately, the side effects of these compounds make long-term administration difficult in some patients. Moreover, their role in corneal NV that is not associated with inflammation is limited. Advances in our understanding of the mechanisms underlying ocular NV has led to the identification of new pharmacologic targets. Given the key role of VEGF in NV of the eye, attention has been directed to developing drugs that will counteract the activity of this factor. Bevacizumab is an anti-VEGF antibody that binds to all VEGF isoforms. This molecule inhibits VEGF-receptor interactions and in this way, blocks all VEGF actions. It is currently approved by the FDA to treat metastatic colorectal cancer. It has also been tested for the treatment of wet (neovascular) age-related macular degeneration (AMD). Ranibizumab is another anti-VEGF antibody that has been approved for use in the eye to treat wet AMD. Bevacizumab treatment of corneal NV has gained popularity since the successful use of this molecule to treat choroidal NV. Subconjunctival injection as well as topical application of this molecule has also been used with promising results to treat herpes simplex virus (HSV) keratitis, recurrent pterygia, rejection of corneal grafts, and Stevens–Johnson syndrome. However, data on these treatments are limited, and adverse effects such as loss of epithelial integrity and progression of thinning have been reported in a small number of patients.

Further investigation is required to establish efficacy, adequate dosing, and safety in the different clinical scenarios that present with corneal NV. Other forms of anti-VEGF therapy are currently undergoing clinical trials. One example is VEGF TRAP, a highaffinity VEGF antagonist designed to bind and neutralize VEGF in the circulation and within tissues. It binds to all isoforms of VEGF and to placental growth factor, which is a related pro-angiogenic factor. SIRNA-027, another anti-VEGF therapy, is a short interferon RNA designed to downregulate VEGFR-1 expression. PKC412 is an orally administered tyrosine kinase inhibitor that binds to the intracellular, enzymatically active domain of the VEGF receptor and prevents phosphorylation and activation of the VEGF signaling cascade. Some of these compounds may be available for use in the near future. Surgical Treatment One surgical approach for the treatment of corneal NV is laser therapy. The use of laser photocoagulation with a 577-nm yellow dye for the treatment of established corneal NV has been investigated. The effectiveness of this technique has been tested in clinically significant corneal NV resistant to medical therapy both before and after PK. Some reduction of corneal NV can be achieved; however, the benefit of laser photocoagulation prior to high-risk keratoplasty is unclear, and this technique does not appear to be useful for treating extensive corneal NV. An alternative to laser occlusion is fine-needle diathermy. This procedure is easy to perform, requiring only a 10-0 nylon suture and a unipolar diathermy unit. It produces occlusion of 50–100% of corneal NV and has been show to moderately benefit visual acuity in a series of 17 patients. Photodynamic therapy is currently used to treat choroidal NV. In this technique, a photo-sensitizer selectively accumulates in new vessels and is subsequently activated by a laser beam. This technique is currently under investigation in animal models of corneal NV. Finally, in some cases, conjunctival, limbal, or amniotic membrane transplantation may be required to restore the ocular surface. Conjunctival autograft and allograft transplantation have been shown to decrease corneal NV. Amniotic membrane has anti-angiogenic properties as well. Limbal autograft transplantation has been successful in cases of stem cell deficiency and conjunctival metaplasia. This technique not only treats the stem cell deficiency and decreases the angiogenic stimulus from chronic ulceration, but also directly inhibits vascular ECs. No ideal treatment is currently available for corneal NV. However, significant progress in the understanding of corneal angiogenesis has opened a new field of investigation that may lead to the development of novel therapeutic agents for the treatment of this condition.

Corneal Angiogenesis See also: Concept of Angiogenic Privilege.

Further Reading Ambati, B. K., Nozaki, M., Singh, N., et al. (2006). Corneal avascularity is due to soluble VEGF receptor-1. Nature 443: 993–997. Azar, D. (2006). Corneal angiogenic privilege: Angiogenic and antiangiogenic factors in corneal avascularity, vasculogenesis, and wound healing. Transactions of the American Ophthalmological Society 104: 264–302. Chang, J. H., Gabison, E. E., Kato, T., and Azar, D. T. (2001). Corneal neovascularization. Current Opinion in Ophthalmology 12: 242–249.

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Cursiefen, C., Chen, L., Saint-Geniez, M., et al. (2006). Nonvascular VEGF receptor 3 expression by corneal epithelium maintains avascularity and vision. Proceedings of the National Academy of Sciences of the United States of America 103: 11405–11410. Dorrel, M., Uusitalo-Jarvinen, H., Aguilar, E., and Friedlander, M. (2006). Ocular neovascularization: Basic mechanisms and therapeutic advances. Survey of Ophthalmology 52: 3–19. Ma, D. H., Chen, J. K., Zhang, F., et al. (2006). Regulation of corneal angiogenesis in limbal stem cell deficiency. Progress in Retinal and Eye Research 25: 563–590. Zhang, S. X. and Ma, J. X. (2007). Ocular neovascularization: implication of endogenous angiogenic inhibitors and potential therapy. Progress in Retinal and Eye Research 26: 1–37.

Avascularity of the Cornea R J C Albuquerque and J Ambati, University of Kentucky, Lexington, KY, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Alloimmunity – A condition in which the body gains immunity, from another individual of the same species, against its own cells. Aniridia – Congenital disorder characterized by the abnormal deficient development of the iris and associated with corneal angiogenesis and poor vision. Atopic keratoconjunctivitis (AKC) – Allergic conjunctivitis where the conjunctiva is red and swollen. Untreated, AKC can progress to ulceration, scarring, cataracts, keratoconus, and corneal vascularization. Hemangiogenesis – It pertains to the specific growth of blood vessels. Limbus – The edge of the cornea where it joins the conjunctiva and the sclera. Lymphangiogenesis – It pertains to the specific growth of lymphatic vessels. Neovascularization – Formation of new blood and lymphatic vessels. Perforating keratoplasty – Corneal transplant with replacement of all layers of the cornea, but retaining the peripheral cornea.

Light, the substrate of vision, is required to transverse the full diameter of the eye globe and reach retinal photoreceptors giving rise to the intricate biophysical phenomenon of sight. The cornea, interfacing the outer world and the intra-ocular tissues, serves as an entry window through which light comes into the eye. Avascularity and optical transparency are directly related and a requirement for optimal vision. The absence of vessels in the cornea is also one of the pillars of corneal immune privilege, an important physiological phenomenon that is associated with the maintenance of corneal clarity and responsible for the high success rate of corneal transplants. The growth of blood and lymphatic vessels into the normally avascular cornea (neovascularization) is considered pathological as it impairs the passage of light resulting in severely deteriorated vision or complete corneal blindness, which together afflict over 200 million people worldwide. The absence of vascular structures in the cornea has been known for over 1000 years. But only recently, advances in molecular biology have led to improved

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understanding of the homeostatic mechanisms underlying such phenomenon. The absence of vasculature (blood and lymphatic vessels) in the cornea is remarkably intriguing given the highly vascularized nature of the neighboring tissues such as the ocular conjunctiva. The abrupt and precise delineation of the limbal vasculature (Figure 1) suggests that corneal avascularity is an active process in which endogenous pro- and anti-angiogenic mechanisms are in harmony and that these molecular modulators of angiogenesis are differently expressed in these interfaces between the cornea and its neighboring tissues preventing the blood and lymphatic vessels from invading the avascular cornea.

Corneal Histology Histologically, the cornea is comprised of five layers (Figure 2). The epithelial layer coats its outermost surface and is composed of a thin nonkeratinized squamous stratified epithelium (only a few cells thick). Unlike the stratified squamous epithelium of the epidermis, that contains indented dermal papillae, the corneal epithelium lays flat on a thick basement membrane called Bowman’s membrane. The subjacent layer of the cornea, its stroma or substantia propria, is formed by tightly packed collagen fibers that are uniquely organized in a parallel fashion affording the cornea its crystal clear disposition. The corneal stroma is devoid of blood and lymphatic vessels and is populated by fibroblasts. In addition to fibroblasts, the substantia propria is also endowed with a heterogeneous population of cells including bone-marrow-derived cells, and antigen presenting dendritic cells, most of which, under normal physiological conditions, are still immature and remain quiescent. Descement’s membrane, a thick lamina propria, separates the corneal stroma from its innermost cellular layer: the corneal endothelium, which consists of a single layer of low cuboidal cells. The corneal endothelium is critical for water homeostasis as it actively transports excess fluid from the corneal stroma into the anterior chamber. This peculiar histological organization of the cornea with a thin epithelium layer and an active endothelium allows oxygen from the room-air and nutrients from the aqueous humor to diffuse through its full thickness. Because the loss of avascularity in the cornea results in impaired light transmission and poor vision, these unique histological features evolved over time and bestowed the cornea with the ability to remain viable and clear in the absence of a direct blood supply.


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EPI BM

STR

DM END Figure 1 Photograph of the human limbus showing the abrupt termination of the conjunctival vasculature in the interface between the cornea (C) and the conjunctiva (CJ). Dotted line delineates the limbus.

Corneal Avascularity and Optical Clarity The visual impairment associated with the loss of corneal avascularity is not only related to the physical obliteration caused by the opaque vessels within the visual field. Corneal neovascularization also reduces visual acuity because the infiltrating vasculature disrupts the tightly packed collagen bundles eliciting opacities, especially in areas surrounding the newly formed vessels. Vascular leakage and edema, usually associated with inflammatory neovascularization, overwhelms the endothelium drainage capacity, creates fluid accumulation, disrupts corneal clarity, and perturbs light transmission. Additionally, the neovascular corneas may also have diminished transparency due to lipid deposits. This is observed in vascularized corneas following corneal herpetic infections. Altogether, these observations speak of the tight correlation that exists between corneal avascularity, corneal transparency, and optimal optical performance.

Endogenous Anti-Angiogenic Mechanisms The molecular homeostatic mechanisms supporting the lack of vessels in the cornea were unknown until recently. Long ago, it was postulated that corneal avascularity was a

Figure 2 Photomicrograph of the human cornea stained with H&E showing its histological layers. The epithelial layer (EPI) overlaying Bowman’s membrane (dotted line). The corneal stroma (STR) displaying its tightly packed parallel collagen fibers. Descement’s membrane (DM) interfacing the substantia propria and the corneal endothelium (END).

passive process. It was thought that the cornea was avascular simply because pro-angiogenic forces were not present. It is quite intriguing, however, that the avascular cornea is surrounded by extremely vascularized tissues, such as the ocular conjunctiva and iris. Because pro-angiogenic factors are a requirement for endothelial cell survival one may postulate that the cornea must be armed with angiostatic capabilities in order to counteract the constant angiogenic stimuli that derives from the adjacent vascular beds. This alternate hypothesis has challenged the previous beliefs, became accepted as a working hypothesis, and still stands as a paradigm of modern vascular biology. Currently, it is well known that corneal avascularity is an extremely active phenomenon, requiring an exact balance between pro- and anti-angiogenic forces, and not the mere absence of pro-angiogenic stimulation. Extrinsic and intrinsic mechanisms have been proposed to underlie the absence of blood and lymphatic vessels in the cornea. The aqueous humor, fluid that circulates through the anterior chamber of the eye, has been regarded as a major extrinsic inhibitor of corneal angiogenesis. It contains several soluble angiostatic molecules including heparan sulfate proteoglycans. Because vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), the foremost studied angiogenic factors, bind to

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these glycoproteins with relative high affinity, it has been proposed that the aqueous humor sequesters VEGF and bFGF from the cornea into the anterior chamber. However, it is important to bear in mind that not all isoforms of VEGF, like VEGF121, shown to be expressed in the cornea, bind heparan sulphate. This suggests that additional mechanisms are in place to secure corneal avascularity. In reality, it has long been suggested that corneal avascularity is a phenomenon supported by a redundant system. Additional factors contributing to corneal avascularity include the intrinsic synthesis of angiostatic molecules. It has been shown that the cornea expresses several of these anti-angiogenic factors, like angiostatin, endostatin, thrombospondins (TSPs), interleukin-1 receptor antagonist, pigmented epithelium-derived factor (PEDF), non vascular VEGF receptor-3 and soluble VEGF receptor-1. The current paradigm of intrinsic angiostatic mechanisms in the cornea derived from the clinical observation that corneal epithelial dysfunction was often associated with neovascularization. This suggested that the antiangiogenic powers of the cornea resided primarily in its epithelial layer. In fact, most of the anti-antiangiogenic factors identified in the cornea have been localized primarily to the corneal epithelium. Angiostatin, a by-product of plasminogen, was first isolated in 1994. Its angiostatic effect was initially demonstrated in a cancerous tumor growth and metastasis model. Mechanistically, angiostatin is thought to inhibit ATP synthase activity, block endothelial cell migration and proliferation and also cause vascular endothelial cell apoptosis. Angiostatin has been detected in healthy human corneal extracts and it has been shown that human corneal epithelium in culture is capable of converting exogenous plasminogen into its angiostatic by-product. Angiostatin has also been implicated in the maintenance of angiogenic privilege in the normal cornea and after wound healing. Endostatin was first described in 1997. It is a proteolytic fragment of the caroboxyterminus of collagen XVIII. It has been portrayed as a potent inhibitor of angiogenesis and tumor growth. Collagen XVIII is a component of the basement membrane ubiquitously expressed and it also has been shown to exist in ocular tissues. Immunohistochemical studies have localized collagen XVIII and endostatin to the corneal epithelium, principally in the basal epithelium. Endostatin has been shown to block VEGFinduced phosphorylation of VEGFR-2 and inhibit endothelial cell proliferation and migration. The synthesis of endostatin in the cornea is upregulated during injury and has been shown to inhibit injury-induced corneal neovascularization. Although endostatin has been shown in the uninflammed cornea, mice deficient in collagen XVIII have normal avascular corneas, suggesting that endostatin is only one of many factors contributing to the maintenance of corneal avascularity.

TSP-1 and -2 are potent anti-angiogenesis protein. TSP-1 directly inhibits the migration and survival of endothelial cells by activation transforming growth factor-beta (TGF-b). TSP-2 inhibits vascular endothelial cell proliferation independently of TGF-b activation. Both molecules have been detected in the mouse and human corneas under normal physiological conditions. Exogenous administration of TSP-1 and/or -2 have been associated with diminished suture-induced corneal angiogenesis. However, the systemic ablation of TSP-1 and -2 was not associated with spontaneous angiogenesis in the cornea, suggesting that alternate redundant angiostatic mechanisms are operative in the cornea. Interleukin (IL)-1 receptor antagonist, a key modulator of IL-1 activity, was shown to be expressed in the normal human cornea. It was localized to the corneal epithelium and some stromal fibroblasts. In a mouse model of sutureinduced corneal neovascularization IL-1 receptor antagonist was shown to have anti-angiogenic properties and its exogenous administration was also associated with diminished infiltration of inflammatory cells into the cornea. IL-1 receptor antagonist deficiency in mice had no bearing in corneal avascularity, once again implying that corneal avascularity is secured by a multifactorial and redundant system. VEGFR-3, normally expressed in lymphatic endothelial cells (LECs), corneal dendritic cells, and macrophages, was shown to be ectopically expressed in corneal epithelial cells of human and mice. These ectopic receptors have been shown to work as an inhibitor of injuryinduced corneal angiogenesis in mice. Since its discovery in 1993, soluble VEGFR-1 (sVEGFR-1) has been extensively studied as a powerful inhibitor of VEGF-induced angiogenesis. It has been implicated in several pathological states, including preeclampsia, sepsis, arthritis, and cancer. Interestingly, VEGF, a powerful driver of angiogenesis, is expressed in the normal avascular cornea. Recently, it has been shown that sVEGFR-1 is co-expressed by the corneal epithelium serving as a VEGF manacle. Because the systemic ablation of VEGFR-1 gene is not compatible with survival, corneal specific deletion of VEGFR-1 was employed in mice and led to spontaneous corneal neovascularization. sVEGFR-1 is considered to be singularly essential for maintaining corneal avascularity of the uninjured cornea. The expression of sVEGFR-1 in the cornea was also shown to be conserved among mammals, including humans. One exception is the Manatee, whose cornea is spontaneously vascularized and lacks sVEGFR-1 expression. A similar splice variant of VEGFR-2, sVEGFR-2, was recently identified and described as the first specific endogenous inhibitor of lymphangiogenesis. In the cornea, sVEGFR-2 was shown to be singularly essential to maintaining the cornea devoid of lymphatic vessels, as its genetic deletion cause spontaneous invasion of lymphatic, but not blood vessels into the cornea.


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Loss of Corneal Avascularity

Corneal Transplant and Avascularity

Corneal neovascularization occurs when the precise equilibrium between pro- and anti-angiogenic forces is disrupted. The loss of corneal avascularity is pathological. Several ocular disorders are hallmarked by corneal neovascularization. These neovascular disorders of the cornea range from benign contact lens-associated neovascularization to congenital and hard to manage ocular anomalies such as aniridia. Extended contact lens wear may induce neovascularization because the associated hypoxia triggers a steep rise in VEGF expression, overwhelming the natural antiangiogenic barriers. Other corneal disorders are coupled with pathological angiogenesis because of direct damage to the corneal epithelium. Corneal trauma, such as corneal abrasions or chemical (alkali) burn of the ocular surface, infections (herpetic keratitis), immune diseases such as atopic keratoconjuctivitis or rheumatoid arthritis, limbal cell deficiency, or congenital anomalies such as aniridia are all associated with the loss of avascularity. These clinical entities commonly require surgical management, like refractive surgery or perforating keratoplasty, both of which are negatively impacted by the preexistence of vessels in the cornea. This represents a significant clinical predicament and speaks of the critical nature of unveiling molecular targets that may be manipulated to treat the aberrant and disordered growth of vessels into the cornea.

In 1905, ophthalmologist Edward Zim performed the first corneal transplant in a human subject. Since then, corneal transplants have become the most common type of solid tissue transplantation in the world. Nearly 46 000 corneal transplants are preformed yearly in the US. In addition to being the most prevalent, corneal allograft transplantation, it is also the most successful intervention among other commonly transplanted organs. However, the long-term outcome of this intervention is greatly influenced by pre-operative risk factors, with corneal neovascularization (high-risk group) being an important negative predictor of corneal allograft survival. While graft survival is approximately 90% in the low-risk group (no pre-operative inflammation or neovascularization), these numbers are drastically reduced to roughly 35% in the high-risk group. Recent studies targeting corneal angiogenesis with VEGF-A binding molecules (VEGF-trapW) demonstrated that allograft survival is inversely related to the amount of neovascularization in the murine corneal transplantation model corroborating the aforementioned epidemiological observations. The loss of corneal avascularity is therefore a significant clinical quandary. The surgical procedures used in corneal allograft transplantation require delicate techniques to prevent adverse inflammatory reactions which may compromise outcome. The corneal graft is attached to the recipient’s ocular surface with the placement of small sutures. Paradoxically, in a vastly employed injury model of corneal angiogenesis, similar intrastromal sutures are used as a method of eliciting blood and lymphatic vessel growth. Because suture placement is a requirement for corneal transplantation as well as a pro-angiogenic stimulus, it is critical to understand the molecular underpinnings related to the growth of blood and lymphatic vessels into the cornea, so potential molecular targets could be identified and manipulated to promote corneal avascularity, optimal optical performance, and prevent corneal blindness.

Immune Privilege of the Avascular Cornea The absence of blood and lymphatic vessels in the cornea is known to play a critical role in maintaining its immune privilege, but other immune-protective mechanisms have been described. One such mechanism is referred to as anterior chamber-associated immune deviation (ACAID). ACAID is regarded as the ability of antigen-presenting cells (APCs) and antigens from anterior chamber-associated tissues (i.e., cornea) to directly enter the blood circulation through the trabecular meshwork homing to the spleen where immune tolerance is induced. Additionally, tissues from the anterior segment of the eye have been reported to express Fas-ligand which induces apoptosis in activated immune cells (Fas-receptor positive), thus protecting the cornea from damage by stimulated lymphocytes. These mechanisms are thought to collectively downregulate inflammation in the cornea, thereby preserving corneal clarity which is essential for optimal vision. Corneal avascularity is therefore one factor of several redundant active mechanisms aimed at preserving corneal transparency and optical light transmission.

Corneal Alymphaticity and Allograft Rejection Because the growth of blood and lymphatic vessels into the cornea are intimately intertwined, the individual contribution of each of these vasculatures to the fate of corneal allografts is not clearly understood. However, recent evidence suggests that the growth of lymphatic vessels into the cornea may be more tightly associated with loss of corneal immune privilege and critical for corneal allograft rejection than corneal hemangiogenesis. Substantial progress in the study of corneal lymphangiogenesis has taken place since the discovery of VEGFR-3

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and its ligands VEGF-C and –D. The identification of specific cellular makers preferentially expressed by LECs, such as LYVE-1, Prox1, and Podoplanin, has also propelled great advances to the field of lymphangiogenesis. Corneal lymphangiogenesis generally occurs after corneal injury and inflammation, which in turn is associated with increased levels of VEGF-C. The newly formed corneal lymphatic vessels give rise to an afferent route through which corneal transudate and APCs are carried from the interstitial space into the lymphatic system and later back into the blood circulation. This drainage pathway becomes extremely deleterious in the context of corneal transplantation. Under these circumstances, the alternative route bypassing the standard outflow pathway (i.e., trabecular meshwork in the anterior chamber) allows for antigens from the donor cornea to escape through the lymphatic system and into the draining lymph node where a graft rejection reaction is initiated. The significance of this alternate drainage pathway to corneal alloimmunity and graft rejection has been portrayed in studies demonstrating that removal of cervical lymph nodes significantly increases the transplant survival rates in the low-(noninflamed, nonvascularized) and high-risk (neovascularized) groups. Together, these observations suggest that improved molecular understanding of corneal lymphangiogenesis, as well as the identification of endogenous compounds with the ability to uncouple lymphangiogenesis from hemangiogenesis would shed light into our current understanding of allograft rejection and potentially unveil therapeutic targets to enhance the survival of corneal allograft.

The Avascular Cornea as an Angiogenesis Study Platform The avascular disposition and its ready accessibility have made the cornea an important platform for the study of angiogenesis allowing scientists to test the pro-and/or anti-angiogenic effects of several compounds in vivo. Numerous assays have been developed to study angiogenesis modulation utilizing the cornea. Direct intra-stromal injection of angiogenesis compounds have been performed in the mouse, rat and rabbit cornea. Models in which a transient chemical (alkali), physical (scraping), or thermal (cautherization) injury are incurred to the cornea to provoke an angiogenic response have been widely used. Prolonged injury of the cornea has been achieved with intra-stromal suture placement. The insertion of a small pellet containing pro-angiogenic molecules has also been described and termed corneal micropocket assay. The cornea stroma has even been utilized for the implantation of tumor cells. The reliability of these models and the easy visualization of corneal vessels have placed the cornea in

the forefront of discovery and in vivo testing of drugs for the treatment of disorders hallmarked by aberrant angiogenesis, particularly cancer. The ability of analyzing these molecules in vivo provides valuable insight regarding the angio-modulatory effects of such compounds.

Conclusions Avascularity of the cornea is intimately related to optical transparency and optimal vision. Hence, the loss of corneal avascularity is pathological and often results in impaired vision or corneal blindness. A precise balance between pro- and anti-angiogenic factors is essential to maintain the avascular disposition of the cornea. Even though it is well known that corneal immune privilege is a function of a constellation of factors, the absence of blood and lymphatic vessels in the cornea has proven to be one of its most important underlying mechanisms. A more precise understanding regarding the individual contribution of blood and lymphatic vessel growth to corneal alloimmunity is needed. The cornea, given its avascular nature and accessibility, is an ideal the platform for the in vivo testing of angiogenesis modulators. See also: Corneal Angiogenesis; Tear Drainage.

Further Reading Albuquerque, R. J. C., Hayashi, T., Cho, W. G., et al. (2009). Alternatively spliced vascular endothelial growth factor receptor-2 is an essential endogenous inhibitor of lymphatic vessel growth. Nature Medicine 15: 1023–1030. Ambati, B. K., Nozaki, M., Signh, N., et al. (2006). Corneal avascularity is due to soluble VEGF receptor-1. Nature 443: 993–997. Azar, D. T. (2006). Corneal angiogenic privilege: Angiogenic and antiangiogenic factors in corneal avascularity, vasculogenesis, and wound healing (an American Ophthalmological Society thesis). American Ophthalmological Society 104: 264–302. Cursiefen, C. (2007). Immune privilege and angiogenic privilege of the cornea. Chemical Immunology and Allergy 92: 50–57. Cursiefen, C., Chen, L., Saint-Geniez, M., et al. (2006). Nonvascular VEGF receptor 3 expression by corneal epithelium maintains avascularity and vision. Proceedings of the National Academy of Sciences of the United States of America 103: 11405–11410. Folkman, J. (2007). Angiogenesis: An organizing principle for drug discovery? Nature Reviews Drug Discovery 6: 273–286. Hirsch, E., Irikura, V. M., Paul, S. M., et al. (1996). Functions of interleukin 1 receptor antagonist in gene knockout and overproducing mice. Proceedings of the National Academy of Sciences of the United States of America 93: 11008–11013. Krachmer, J., Mannis, M., and Holland, E. (2004). Cornea. Amsterdam: Mosby. Lawler, J. (2000). The functions of thrombospondin-1 and -2. Current Opinion in Chemical Biology 12: 634–640. O’Reilly, M. S., Boehm, T., Shing, Y., et al. (1997). Endostatin: An endogenous inhibitor of angiogenesis and tumor growth. Cell 88: 277–285.

Avascularity of the Cornea O’Reilly, M. S., Holmgren, L., Shing, Y., et al. (1994). Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79: 315–328. Spencer, W. H. (1996). Ophthalmic Pathology: An Atlas and Textbook. Philadelphia, PA: W.B. Saunders.

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Whitcher, J. P., Srinivasan, M., and Upadhyay, M. P. (2001). Corneal blindness: A global perspective. Bulletin of the World Health Organization 79: 214–221. Yamagami, S., Dana, M. R., and Tsuru, T. (2002). Draining lymph nodes play an essential role in alloimmunity generated in response to high-risk corneal transplantation. Cornea 21: 405–409.


IV. VISUAL ACUITY RELATED TO THE CORNEA AND ITS DISORDERS


Pupil P D R Gamlin and D H McDougal, University of Alabama at Birmingham, Birmingham, AL, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Accommodation – A change in the refractive power of the crystalline lens of the eye. Intrinsically photosensitive retinal ganglion cells (ipRGCs) – The ganglion cells expressing a photopigment, melanopsin, that is intrinsically light sensitive. Miosis – Pupillary constriction. Mydriasis – Pupillary dilation. Pupillary light reflex (PLR) – The constriction of the pupil that is elicited by an increase in illumination of the retina.

Advantages of a Mobile Pupil The normal human pupil can change diameter from 8 to 1.5 mm, which corresponds to approximately a 30-fold change in area and almost a 1.5-log unit change in retinal irradiance. Although the visual system can operate over a 10-log unit range of lighting levels through the process of adaptation, it can take several minutes for optimum sensitivity to return after an abrupt increase or decrease in retinal illumination. The rapid control of retinal irradiance by the iris allows the visual system to more quickly regain optimal sensitivity by dampening fast changes in ambient lighting levels and by requiring less retinal adaptation for a given change in environmental lighting levels. However, changes in pupil size affect not only retinal illumination, but also diffraction, optical aberrations, and depth of focus of the eye. These factors differentially affect visual performance and, given changing environmental lighting conditions and visual tasks, the nervous system continuously modulates pupil diameter for optimal visual performance. The diffraction of light rays by an aperture is a major limiting factor in the resolution of an image in any optical system. The amount of disruption in image quality caused by diffraction at a circular aperture decreases as the size of the opening increases. Therefore, as pupil diameter increases, there is decreased degradation in retinal image quality caused by diffraction. In contrast to diffraction, the image-degrading effects of optical aberrations increase as aperture diameter increases. Therefore, as pupil diameter increases, the degradative effects of optical aberrations

also increase, and offset the benefits gained by reduced diffraction at larger pupil diameters. Over the normal range of pupillary diameter, diffraction impacts image quality less than optical aberration, and the optimal pupil diameter is therefore approximately between 2 and 4 mm. Along with diffraction and optical aberrations, defocus is an important determinate of retinal image quality. Although the pupil does not refract or focus light, it influences the depth of field of the eye. Depth of field is the range of distance in depth in which objects appear to be in focus. For example, when one reads a book, the power of the crystalline lens of the eyes changes in order to bring the text on the page into focus through a process called accommodation. With the eyes accommodated on the book, all objects within a range in front of and behind the book will also appear in focus. This range is called the depth of field and it is primarily dependent both on viewing distance and pupil diameter. When the viewing distance is held constant, the depth of field increases with decreases in pupil diameter, and therefore the pupil diameter can affect the focus of the retinal image. Clearly, a mobile pupil allows the nervous system to optimize retinal irradiance, diffraction, ocular aberrations, and depth of focus despite differing conditions and visual tasks. For example, across a range of daylight (photopic) luminances, pupil size corresponds to that required for the highest visual acuity, and the maximal information capacity of the retinal image. On the other hand, under low light (scotopic) conditions in which poorer retinal image quality can be tolerated due to the lower resolution of rod photoreceptors, the pupil dilates sufficiently to maximize the retinal illumination. Further evidence for the optimization of pupil diameter for differing visual tasks is evident in the pupillary near response (PNR). When the viewing distance changes from far to near, the pupils constrict to increase the field of view and reduce the retinal image defocus. This compensates for the decrease in the effective field of view that naturally occurs when viewing distance decreases (see the section titled ‘Pupillary near response’ for more details).

Overview of the Pathways Controlling Pupil Diameter A summary diagram of the afferent, central, and efferent pathways controlling pupil diameter is shown in Figure 1. This figure shows the iris musculature innervated by autonomic efferents from both the parasympathetic and sympathetic components of the autonomic nervous system

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Ciliospinal center Figure 1 Anatomical drawing showing the direct and consensual pupillary light reflex (PLR) pathways and the parasympathetic and sympathetic innervation of the iris in primates. The bilateral projection from the retina to the pretectum is also shown. The pretectal olivary nucleus receives input from the temporal retina of the ipsilateral eye and the nasal retina of the contralateral eye. The pretectal olivary nucleus projects bilaterally to the Edinger–Westphal (EW) nucleus, which contains parasympathetic, preganglionic, and pupilloconstriction neurons. The axons of these preganglionic neurons travel in the third cranial nerve to synapse upon postganglionic pupilloconstriction neurons in the ciliary ganglion. The axons of these postganglionic neurons leave the ciliary ganglion and enter the eye through the short ciliary nerves, and travel through the choroid to innervate the sphincter muscle of the iris. The sympathetic preganglionic pupillodilation neurons are found at the C8-T1 segmental levels of the spinal cord. The axons of these neurons project from the spinal cord through the dorsal roots and enter the sympathetic trunk, and then project rostrally to the superior cervical ganglion where they synapse with the postganglionic neurons. These postganglionic neurons project from the superior cervical ganglion through the neck and carotid plexus, and into the orbit of the eye. These fibers enter the eye either by passing through the ciliary ganglion and entering the short ciliary nerves, or bypassing the ciliary ganglion and entering via the long ciliary nerves (for clarity, only one of these alternative pathways is shown). Upon entering the eye, these axons travel through the choroid and innervate the dilator muscle of the iris. From McDougal, D. H. and Gamlin, P. D. R. (2008). Pupillary control pathways. In: Basbaum, A. I., Kaneko, A, Shepherd, G. M., et al. (eds) The Senses: A Comprehensive Reference, Vol 1: Vision 1, pp. 521–536. San Diego, CA: Academic Press.

(ANS). The parasympathetic component of the ANS innervates the sphincter pupillae muscle of the iris. The preganglionic parasympathetic fibers controlling the sphincter pupillae originate from neurons in the Edinger–Westphal (EW) nucleus, the autonomic subdivision of the third cranial nerve nucleus, and travel through the third cranial nerve to the ciliary ganglion, which is located within the orbit of the eye (see Figure 1). Within the ciliary ganglion, the preganglionic pupilloconstriction neurons form cholinergic, nicotinic synapses with the postganglionic neurons.

The axons of these postganglionic neurons leave the ciliary ganglion to enter the eye through the short ciliary nerves and travel to the iris. Here, they release acetylcholine, which acts on the muscarinic receptors of the sphincter pupillae (see Figure 2). The sympathetic component of the ANS innervates the dilator pupillae muscle. The preganglionic sympathetic neurons, which control pupillary dilation, are located in the C8-T1 segments of the spinal cord, a region termed the ciliospinal center of Budge (and Waller). The

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Pupillary ruff

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Figure 2 Low-power photomicrograph of a cross section of the macaque iris. Scale ¼ 200 mm. From McDougal, D. H. and Gamlin, P. D. R. (2008). Pupillary control pathways. In: Basbaum, A. I., Kaneko, A, Shepherd, G. M., et al. (eds.) The Senses: A Comprehensive Reference, Vol 1: Vision 1, pp. 521–536. San Diego, CA: Academic Press.

axons of these preganglionic neurons project to the sympathetic chain and travel in the sympathetic trunk to the superior cervical ganglion. Within the superior cervical ganglion, the preganglionic axons form nicotinic, cholinergic synapses with postganglionic pupillodilation neurons. The axons of these postganglionic neurons project from the superior cervical ganglion to the orbit, where they enter the eye through the short and long ciliary nerves and travel to the iris (see Figure 1). Here, they release norepinephrine, which acts on the adrenoreceptors of the dilator muscle (see Figure 2).

Iris Musculature In a cross-section of the iris, the sphincter pupillae can be seen as an annular band of smooth muscle (100–170 mm thick; 0.7–1.0 mm wide) encircling the pupil (Figure 2). The sphincter, which is located in the posterior iris immediately anterior to the pigmented epithelium, interdigitates with the surrounding stroma and connects to the dilator muscle fibers. The smooth muscle cells of the sphincter are clustered in small bundles and connected by gap junctions. These gap junctions ensure synchronized contraction of the sphincter muscle. The sphincter receives muscarinic, cholinergic innervation from the short ciliary nerves – parasympathetic, postganglionic fibers arising from the ciliary ganglion. The dilator pupillae is composed of radially oriented smooth muscle fibers that are myoepithelial in origin. Individual fibers are approximately 50 mm long and 5–7 mm wide. In the pupillary zone, dilator muscle processes fuse with the sphincter pupillae, while peripherally, their processes attach to the ciliary body. Contraction of the dilator muscle pulls the pupillary margin toward the ciliary body.

Pupillary Light Reflex Description The pupillary light reflex (PLR) is the constriction of the pupil that is elicited by an increase in illumination of the retina. The direct PLR, present in virtually all vertebrates, is the constriction of the pupil in the same eye as that stimulated with light. The consensual PLR is the constriction of the pupil in the eye opposite to the eye stimulated with light. In mammals with laterally placed eyes, such as the rat and rabbit, the direct PLR is more pronounced than the consensual PLR. However, in those mammalian species with frontally placed eyes, such as humans and monkeys, the direct and consensual PLRs are essentially equal. An example of a human consensual PLR produced by two different wavelengths of light is shown in Figure 3. The PLR has traditionally been divided into two separate pathways based on the clinical manifestations of the defects in this reflex. The afferent pathway is composed of both the retinal cells that project to the pretectum as well as their recipient neurons, which project bilaterally to the EW nucleus (Figure 1). The efferent pathway is composed of the preganglionic pupilloconstriction fibers of the EW nucleus and their postganglionic recipient neurons in the ciliary ganglion, which project to the sphincter muscle of the iris (Figure 1).

Afferent Pathway The first neurons in the afferent pathway of the PLR are retinal ganglion cells. It has recently been recognized that this reflex in rodents and primates is driven predominantly by a unique subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) which project to the pretectal olivary nucleus (PON), a small nucleus in the

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Figure 3 Pupilloconstriction elicited by a 10-s light stimulus of 493-nm wavelength light at 14.0 log quanta cm–2 per second irradiance (blue trace), and 613-nm wavelength light at 14.1 log quanta cm–2 per second irradiance (red trace). Note that a 473-nm stimulus, which effectively activates the intrinsic photoresponse of intrinsically photosensitive retinal ganglion cells (ipRGCs), drives a larger pupillary response than the 613-nm stimulus (red trace), which does not effectively activate the intrinsic photoresponse of ipRGCs at this irradiance level. Note that the pupilloconstriction induced by the 473-nm light is maintained following stimulus offset. From McDougal, D. H. and Gamlin, P. D. R. (2008). Pupillary control pathways. In: Basbaum, A. I., Kaneko, A, Shepherd, G. M., et al. (eds.) The Senses: A Comprehensive Reference, Vol 1: Vision 1, pp. 521–536. San Diego, CA: Academic Press.

pretectum; the pretectum is located in the dorsal lateral aspect of the midbrain at the level of the superior colliculus (see Figure 1). Intrinsically photosensitive retinal ganglion cells

Prior to 2000, it was assumed that the PLR was driven by retinal ganglion cells which received light signals exclusively from rod and cone photoreceptors, which up to that time were the only known photoreceptive cells in the retina. However, recent studies have demonstrated that the PLR is driven predominantly by retinal ganglion cells which, unlike any other retinal ganglion cell class, are intrinsically photosensitive. The intrinsic photoresponse of these neurons, which is mediated by the photopigment melanopsin, presumably compensates for the adaptation of rod and cone photoreceptors, and serves to maintain pupilloconstriction during steady-state exposure at all photopic (daylight) illuminance levels. In addition to their intrinsic light-driven signal, it is clear that ipRGCs receive rod and cone inputs. In response to a pulse of light, intracellular recordings from these cells show a characteristic transient burst of neural activity at stimulus onset, which rapidly decays to a plateau of sustained activity that

often extends well past stimulus offset. The initial burst of neural activity is mediated by a rapidly adapting conemediated photoresponse, while the sustained activity is driven predominantly by the intrinsic response of these cells, although there is growing evidence for a rod contribution to this sustained activity under steady-state lighting conditions. IpRGCs project to the PON of rodents and primates, and they play a major role in pupillary responses. Monkeys and rodents with nonfunctional rod and cone photoreceptors but functional melanopsin-containing ipRGCs display a PLR; however, the reflex has a higher irradiance threshold than normal. Mice with ipRGCs lacking melanopsin also display PLR, but their pupils fail to constrict maximally in bright lights. Taken together, these results show that both the intrinsic photoresponse of ipRGCs and their classical photoreceptor inputs provide signals of retinal irradiance that drive the PLR. Additional studies further suggest that the influence of rod and cone photoreceptors on the pupillary light reflex is mediated exclusively through their inputs to ipRGCs. The intrinsic photoresponse of ipRGCs can also affect pupillary behavior in the absence of ongoing light stimulation. As noted above, ipRGCs encode stimulus irradiance through an elevation of firing rate that continues well beyond stimulus offset. Indeed, bright light stimuli can produce a prolonged pupillary constriction in humans that can persist up to 20 min after the light has been extinguished (see Figure 3). Experiments in primates, including humans, demonstrate that this prolonged pupillary constriction in darkness is mediated almost entirely by the intrinsic photoresponse of ipRGCs. Pretectal olivary nucleus The first relay in the afferent pathway of the PLR consists of luminance neurons within the PON, which receives direct retinal input. PON luminance neurons are characterized by tonic firing rates that increase with increases in retinal illuminance. In primates, these neurons exhibit a transient burst of activity followed by sustained tonic activity in response to increases in retinal illuminance. In addition, the tonic firing rate of these cells is proportional to retinal illuminance over at least a 3 log unit range of stimulus intensities in primates and in rats. Electrical microstimulation of the PON in rats and monkeys elicits pupilloconstriction at short latencies, and lesions of the PON in rats produce deficits in pupillomotor function. These results strongly suggest that luminance neurons within the PON mediate the PLR. In addition to retinal afferents, the PON also receives significant cortical, ventral thalamic, and midbrain inputs which may also have an influence on the PLR or other pupillary movements. Owing to its importance for the PLR, the best-described efferent projection of the PON is to the EW nucleus. However, the PON has been shown to project to a number

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of other targets, such as the hypothalamus, pons, and medulla that may also influence pupillary behavior. Efferent Pathway The efferent leg of the PLR begins with preganglionic pupilloconstriction neurons of the EW nucleus that project through the third cranial nerve to the ciliary ganglion (see Figure 1). The EW nucleus is a distinct nucleus of the midbrain, lying immediately dorsal to the oculomotor complex. It is located just ventral and lateral to the cerebral aqueduct at the level of the superior colliculus (see Figure 1). Evidence for the course of the efferent parasympathetic pupillary pathway and the importance of the EW nucleus in pupilloconstriction comes from electrical stimulation studies in the vicinity of EW nucleus that elicit pupilloconstriction in a variety of animal models. Within the ciliary ganglion, which is approximately 3 mm in size, and located 2–3 mm posterior to the globe and lateral to the optic nerve, the axons of the preganglionic neurons synapse with the postganglionic pupilloconstriction neurons. The axons of these postganglionic neurons leave the ciliary ganglion to enter the eye via the short ciliary nerves to innervate the sphincter muscle of the iris. Sympathetic Influences on the PLR It is generally agreed that the parasympathetic pathway discussed above is the primary route of pupillary constriction associated with the PLR. However, there is some evidence that increases in retinal illumination may cause a reduction in the tone of the dilator muscle of the iris through the sympathetic pathway outlined in Figure 1, and thus enhance the PLR. Studies in cats have shown a light-induced inhibition of postganglionic pupillodilation fibers at the level of the long ciliary nerves, and preganglionic, pupillodilation fibers at the level of the cervical sympathetic nerve. These studies found that the pupillodilation fibers were inhibited by light in an intensitydependent manner, that is, a more intense light brought about a greater inhibition in firing rate. However, these findings have not been replicated in primates, in which the evidence suggests that the sympathetic system does not contribute to the dynamics of the PLR and only contributes to tonic modulation of pupil diameter.

The Pupillary Near Response Description The PNR is the pupillary constriction associated with a change in viewing distance from far to near that occurs in primates including humans. When the eyes move from viewing a far object to viewing a near object, three

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oculomotor responses occur. The eyes converge to bring the image of the object onto the fovea of each retina, the refractive power of the crystalline lens is adjusted to bring the image of the object into focus on the retina, and the pupil constricts. These collective processes are classically referred to as the near response or the near triad. Efferent Pathway of the PNR The PNR is thought to be driven solely by an increased drive to the sphincter muscle of the iris through the parasympathetic efferent pathway. Therefore, the neural control pathway of the PNR shares a common efferent pathway with the PLR, although the afferent inputs responsible for the PNR are more complex. The neural signals driving these two reflexes most likely converge at the EW nucleus, since the activity of PON luminance neurons is not correlated with pupil constriction during near viewing. Further, certain clinical neurological conditions are characterized by an intact PNR despite the absence of the PLR (light-near dissociation). It is generally accepted that preganglionic neurons in the EW nucleus drive the PNR as well as the PLR. However, it has not been determined if separate subpopulations of neurons exist in EW nucleus devoted exclusively to either the PLR or the PNR, or whether the same population of neurons drives pupillary constriction in both reflexes, although the latter seems most likely. Afferent Influences on the PNR Early investigations attempted to determine whether the PNR was driven primarily by ocular convergence or accommodation, the other two components of the near triad. Some studies found that the PNR was more closely associated with accommodation than with convergence. Other studies found a greater association with convergence, and even reported that the PNR was totally absent during some blur-driven accommodative responses. These conflicting results are likely a product of an incomplete disassociation between the convergence and accommodation systems during these experiments, as these two systems have been shown to be highly interdependent. A more modern view of the afferent influences controlling the PNR has recently emerged. In this view, the PNR is not seen as resulting from either accommodation or convergence alone, but as a separate output of the neural pathways that drive both accommodation and convergence. A number of brain areas play a role in controlling the near triad. These include cortical areas, such as extrastriate cortex, parietal cortex, frontal eye fields, as well as the cerebellum and the midbrain. Of particular interest to the PNR, is the supraoculomotor area of the midbrain, which lies just dorsal and lateral to the oculomotor nucleus. The supraoculomotor area contains near response cells which

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are modulated by both vergence and accommodation. These cells project to medial rectus motoneurons, and thus contribute to vergence eye movements. It seems likely that these cells also project to EW nucleus and are responsible for carrying the signal from the accommodation and convergence controller to the preganglionic, pupilloconstrictor neurons.

Additional Cortical Influences on Pupillary Responses In addition to cortical afferents mediating the PNR, the pupil is also influenced by both visual and nonvisual cortical regions. These afferents manifest themselves as small changes in pupil diameter during presentation of visual stimuli such as colored stimuli and gratings, as well as nonvisual stimuli such as auditory tones, and even during higher-order cortical functions such as problem solving. These observations provide clear evidence that cortex exerts an influence on pupillary behavior, which therefore cannot be thought of as entirely reflexive in nature. Visually Mediated Cortical Influences on Pupillary Behavior Small pupillary constrictions have been shown to occur in both human and monkeys with the presentation of complex visual stimuli, even when the stimuli do not involve a change in viewing distance or retinal illuminance. Changes in stimulus attributes such as color, spatial frequency, or apparent motion produce such cortically mediated pupillary responses. Deficits in these pupillary responses are observed in humans with lesions to cortical areas involved in processing one or more of these stimulus characteristics. In addition, lesions of rostral inferior temporal cortex but not V4 in macaques abolish pupillary responses to chromatically modulated gratings. Task-Evoked Pupillary Responses In the early 1960s, Hess and colleagues published a series of papers which reported modulations in human pupillary diameter associated with complex cognitive processes such as subjective attitudes or mental activity. Later studies failed to replicate the findings relating pupillary dynamics to subjective attitudes, although the findings related to mental activity have been replicated and extensively studied. The small pupillary dilations associated with increased mental activity, or task-evoked pupillary responses (TEPRs), have now become a well-established tool of cognitive psychology. These pupillary responses are generally reported to vary in magnitude from 0.2 to 0.7 mm, and have been shown to correlate with cognitive

load across diverse functions, such as sensory perception, memory, language, and attention. TEPRs have been repeatedly shown to monotonically vary with the degree of mental activity required by a task as measured by other objective criterion such as reaction time and the extent of cortical activation indicated by positron emission tomography (PET) scan, and this has allowed TEPRs to be utilized successfully to empirically test theories of language processing and intelligence. Although the behavioral phenomenon of TEPRs has been extensively studied and quantified, little is known of the underlying neurophysiology that drives these responses. It has been suggested that they may be driven by noradrenergic projections from the locus ceruleus since the activity of neurons in this nucleus has been show to correlate with both pupil diameter and task-related events.

Influence of Alertness on Pupillary Behavior Since the muscles of the iris are controlled by the ANS, environmental or physiological conditions which cause changes in overall autonomic function can have a significant effect on pupillary behavior. Even though the environment or physiological conditions which produce the change in autonomic tone may not have a direct influence on the visual system, they may still manifest themselves through an affect on pupil diameter. Arousal Situations or stimuli which produce an emotional or startle response often produce a profound pupillary dilation. This effect is mediated through the hypothalamus, the brain area responsible for the integration of autonomic function. This integration allows for the coordination of the various functions of the ANS and often leads to global changes in the balance between the sympathetic and parasympathetic branches of the ANS. For example, an unexpected loud noise may produce a startle response which is characterized by increases in heart rate, respiratory rate, and pupil diameter; it is caused by a systemic increase in sympathetic tone mediated through the hypothalamus. This global increase in sympathetic tone can affect pupil diameter via activation of the pupillodilation centers of the spinal cord and inhibition of the pupilloconstriction neurons of EW nucleus. Neurons within the hypothalamus project to the sympathetic preganglionic pupillodilation neurons of the thoracic spinal cord. This direct effect of hypothalamic activation on pupil diameter can be shown through microstimulation of the posterior hypothalamus, which often causes rapid pupil dilation. Increase in sympathetic tone can also produce inhibition

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of pupilloconstriction neurons of EW nucleus via the influence of ascending neuromodulatory pathways. The hypothalamus is also the site at which autonomic function is regulated by the central nervous system through connections with the limbic system and cortical structures. The limbic system of the brain, which is responsible for emotions and short-term memory, has a direct connection to the hypothalamus and therefore can have significant effects on autonomic balance. Situations or stimuli which produce an intense emotional response are often accompanied by pupillary dilation, which is certainly mediated through limbic connections to the hypothalamus. In addition, cortical influences on the hypothalamus allow a wide variety of stimuli to effect autonomic tone and thus pupil diameter.

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affect pupillary behavior in a wide range of animal models and human studies. These differential effects are most likely due to the both interspecies variability in the projections of these neuromodulatory fibers, as well as the differential activation of multiple brain areas implicated in pupillary behavior due to the extensive projections of these neuromodulators.

Acknowledgments This work was supported by NIH grant EY09380 and the EyeSight Foundation of Alabama. See also: Acuity.

Sleep Sleep has a pronounced effect on the ANS, specifically a reduction in sympathetic outflow and an increase in parasympathetic outflow. Given this overall trend, it is not surprising that pupillary behavior during sleep is characterized by prolonged constriction of the pupil. It has been shown that sleep-induced pupillary constriction persists in animals with lesions of the preganglionic sympathetic pupillodilation fibers. This suggests that the sleepinduced pupillary changes are mediated by an activation of the preganglionic parasympathetic pupilloconstriction fibers of the EW nucleus. Ascending Neuromodulatory Systems The ascending neuromodulatory systems of the midbrain and brainstem can have a variety of effects on pupillary behavior. These nuclei are the origin of neuromodulatory fibers which release dopamine, norepinephrine, histamine, and serotonin at a number of brain areas implicated in pupillary control. These neuromodulatory systems appear to be critical in the regulation of sleep and arousal, as well as autonomic regulation and cortical plasticity. In addition to these global neuromodulatory effects, all or some of which could have a profound influence on pupillary behavior, there is some evidence for a direct inhibition of pupilloconstriction neurons in the EW nucleus by adrenergic neurons originating from the locus ceruleus in a number of animal models. However, other studies in humans and rabbits have failed to find this direct noradrenergic inhibition of EW nucleus pupilloconstriction neurons and it has been suggested that this effect might be mediated by dopaminergic neurons in these species. Drugs which agonize or antagonize these neuromodulatory neurotransmitters have been found to differentially

Further Reading Barbur, J. L. (2003). Learning from the pupil – studies of basic mechanisms and clinical applications. In: Chalupa, L. M. and Werner, J. S. (eds.) The Visual Neurosciences, pp. 641–656. Cambridge, MA: MIT Press. Beatty, J. and Lucero-Wagoner, B. (2000). The pupillary system. In: Cacioppo, J. T., Berntson, G., and Tassinary, L. G. (eds.) Handbook of Psychophysiology, 2nd edn., pp. 142–162. Cambridge: Cambridge University Press. Berson, D. M. (2003). Strange vision: Ganglion cells as circadian photoreceptors. Trends in Neuroscience 26: 314–320. Bron, A. J., Tripathi, R. C., Tripathi, B. J., and Wolff, E. (1997). Wolff’s Anatomy of the Eye and Orbit. London: Chapman and Hall Medical. Busettini, C., Davison, R. C., and Gamlin, P. D. R. (2009). Vergence eye movements. In: Squire, L. (ed.) Encyclopedia of Neuroscience, vol. 10, pp. 75–84. Oxford: Elsevier. Charman, W. N. (1995). Optics of the eye. In: Bass, M. (ed.) Handbook of Optics, pp. 24.3–24.54. New York: McGraw-Hill. Gamlin, P. D. (2000). Functions of the Edinger–Westphal nucleus. In: Burnstock, G. and Sillito, A. M. (eds.) Nervous Control of the Eye, pp. 117–154. Binghamton, NY: Harwood Academic. Gamlin, P. D. (2005). The pretectum: Connections and oculomotorrelated roles. Progress in Brain Research 151: 379–405. Gamlin, P. D., McDougal, D. H., Pokomy, J., et al. (2007). Human and macaque pupil responses driven by melanopsin-containing retinal ganglion cells. Vision Research 47(7): 946–954. Kardon, R. H. (2005). Anatomy and physiology of the autonomic nervous system. In: Miller, N. R., Walsh, F. B., Biousse, V., and Hoyt, W. F. (eds.) Walsh and Hoyt’s Clinical Neuro-Ophthalmology, vol. 3, pp. 649–714. Baltimore, MD: Lippincott Williams and Wilkins. Kawasaki, A. (2005). Disorders of pupillary function, accommodation, and lacrimation. In: Miller, N. R., Walsh, F. B., Biousse, V., and Hoyt, W. F. (eds.) Walsh and Hoyt’s Clinical Neuro-Ophthalmology, vol. 3, pp. 739–804. Baltimore, MD: Lippincott Williams and Wilkins. Loewenfeld, I. E. and Lowenstein, O. (1993). The Pupil: Anatomy, Physiology, and Clinical Applications. Ames, IA: Iowa State University Press. McDougal, D. H. and Gamlin, P. D. R. (2008). Pupillary control pathways. In: Basbaum, A. I., Kaneko, A., Shepherd, G. M., et al. (eds.) The Senses: A Comprehensive Reference, Vol 1: Vision 1, pp. 521–536. San Diego, CA: Academic Press. Oyster, C. W. (1999). The Human Eye: Structure and Function, pp. 411–446. Sunderland, MA: Sinauer Associates.

Acuity M D Crossland, UCL Institute of Ophthalmology/Moorfields Eye Hospital, London, UK ã 2010 Elsevier Ltd. All rights reserved.

Glossary Cycles per degree – The number of complete phases of a grating (e.g., the distance between the center of a white bar and the center of the next bright bar in a square-wave grating; or the distance between two adjacent areas of maximum brightness on a sine-wave grating) contained in 1 of visual angle. Minimum angle of resolution – The size of the angle subtended at the eye of the smallest feature which can be reliably identified on an optotype. Minute of arc – One-sixtieth of a degree. Optotype – A letter, symbol, or other figure presented at a controlled size to measure vision. Visual angle – The angle, which a viewed object subtends at the eye.

Detection and Resolution Acuity Visual acuity can be defined in two broad ways. Detection acuity is measured by determining the size of the smallest object which can be reliably seen (is there a circle on the first or second screen?). Detection can be elicited reliably with targets, which subtend an angle at the eye as small as 1 s of arc (1/3600 ). Even a small point of light will stimulate several photoreceptors due to the point-spread function of the eye: that is, the way in which light is diffracted through the eye’s optics (Figure 1(a)). Tests that require the identification of a target are a measurement of resolution acuity. These tests frequently involve identifying a letter or reporting an object’s orientation (what direction is this letter C facing?). Acuity for these tests depends on the separation of the target features: if they are too close, the point-spread function from each element will overlap and they will not be identified (Figure 1(b)). The smallest separation of the elements required for identification of the target (Figure 1(c)) is known as the minimum angle of resolution (MAR). For an adult observer with good vision, a typical MAR for a centrally presented, high-contrast target can be as good as 30 s of arc (1/120 ). Figure 2 shows the feature critical for the MAR for some commonly used tests of visual acuity.

494

Measurement of Visual Acuity Visual acuity tests have been used for millennia: the ancient Egyptians are reported to have used discrimination of the twin stars of Mizar and Alcor as a measurement of vision. The most familiar clinical test of visual acuity, the Snellen chart, was introduced in 1862, and is still widely used today. Detection acuity is often measured psychophysically by means of a temporal two-alternative forced-choice experiment (did the light appear in the first or the second interval?). Detection acuity is rarely measured clinically. In psychophysical experiments of the visual system, resolution acuity is commonly measured by asking observers to report the orientation of a grating with variable separation between each dark and light bar (Figure 2(b)). In clinical practice, gratings are rarely used, with the exception of forced-choice preferential looking tests in preverbal children. These tests consist of a uniform gray field with an isoluminant grating toward one side of the chart (Figure 3(a)). In a featureless room, the test is presented to the child and the clinician observes whether the child looks toward the grating. The finest grating toward which the child repeatedly looks is recorded as the visual acuity. For cooperative patients, optotypes are more often used to measure clinical resolution acuity. The Landolt C (Figure 2(c)) is the standard to which letter visual acuity tests are compared. This target consists of a ring of fixed width with a gap, of height equal to the stroke width, at the top, left, right, or bottom of the circle. The observer is asked to report the position of this gap. The smallest gap whose position can be reliably reported is equivalent to the MAR. The National Academy of Sciences standard for visual acuity measurement advocates the presentation of 10 optotypes, of equivalent difficulty to the Landolt C, at each acuity size. The horizontal spacing between each optotype should be at least one character width, and vertical spacing between lines should be 1–2 times the height of the larger optotypes. It suggests that the number of characters on each line should be equal, and that the size difference between consecutive lines is 0.1 log units: in other words, for each target size, the next line should be approximately 1.26 times smaller. The Snellen chart (Figure 3(b)) does not meet these recommendations: the number of letters per line and step size between the lines are variable, as is the horizontal and

Acuity

495

Target

(a)

(b)

(c) (a)

2-D PSF

1-D PSF

(b)

Figure 1 Schematic illustration of the point-spread function of three visual targets: (a) a point target; (b) two adjacent lines, too close to be resolved; and (c) two adjacent lines, with sufficient separation to be resolved. Middle row: two-dimensional representation of the target point-spread function; bottom row: one-dimensional representation of the point-spread function; and red line indicates the sum of energy incident on the retina. PSF, point-spread function.

θ

(a) θ

(b)

θ

(c)

θ

(d)

Figure 2 Examples of the limiting feature for four commonly used resolution tasks: (a) two-point discrimination task; (b) grating; (c) Landolt C; and (d) Sloan letter E (note that white gap size is equal in width to black bar elements).

vertical spacing on the chart. There is also a marked difference in the legibility of different letters on the Snellen chart: a W, for example, has far less separation between the elements of the letter and is more difficult

(c)

Figure 3 (a) A forced-choice preferential looking test consisting of a grating against an isoluminant background. Note the peephole in the center for the clinician to observe the child’s visual behavior; (b) the Snellen chart; and (c) The ETDRS chart. ETDRS, Early treatment of diabetic retinopathy study.

to identify than a letter L. In the 1950s, Sloan suggested the use of 10 letters with a selection of vertical, horizontal, oblique, and round strokes which are each about as legible as a Landolt C. These Sloan letters are C, D, H, K, N, O, R, S, V, and Z. Each of the Sloan letters has a stroke width of the MAR and has a total height and width of five times the MAR. The Bailey–Lovie chart, introduced before the recommendations of the National Academy of Sciences, conforms to most of these requirements, although it only has five letters per line. Further, the letters on the Bailey–Lovie chart are taller than they are wide: their height-to-width ratio is 5:4 and they are selected from the British Standards set of letters (D, E, F, H, N, P, U, V, R, and Z). The ETDRS chart (Figure 3(c)), developed for the early treatment of diabetic retinopathy study (ETDRS), is similar in design but does use the recommended 5 5 Sloan letters. A criterion of 7/10 letters being read correctly for a line to be marked as seen was suggested by the National Academy of Sciences. This threshold reduces the chance of the line being scored correctly by chance (by a blind observer) to around 1 in 9 000 000. On a chart with five letters per line, recording a visual acuity where four of the five letters are read correctly equates to a chance success rate of 1 in 46 000. There is a theoretical advantage if the observer knows there are only 10 letters which can be presented on the chart: if an observer guesses from all 26 letters rather than the ten Sloan letters, the probability of the observer getting four out of five letters correct reduces to about 1 in 100 000.

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Test–retest variability of the Snellen chart is around 0.3 logMAR, while the ETDRS chart has far better repeatability (test–retest variability 0.1–0.2 logMAR). Despite the many limitations of the Snellen chart, it is still widely used in clinical practice. While this is likely to be largely due to clinicians’ familiarity with the Snellen chart, there is also a perception that Snellen acuity measurement is quicker than that on the Bailey–Lovie or ETDRS charts. Various modified versions of the ETDRS chart exist: for example, a version with an altered letter set (A, B, E, H, N, O, P, T, X, and Y) has been developed for use by readers of most European languages, including those based on Cyrillic or Hellenic alphabets. For observers unable to report letters on a sight chart, other frequently used optotypes include the tumbling E chart (formerly and less politically correctly known as the illiterate E chart), where a letter E is shown in each of four rotations; the HOTV chart, where only these four letters are used; symbols such as the Lea or Kay pictures; and simple shapes, such as the Cardiff card.

In much of Europe, the Snellen fraction is reduced into a decimal fraction. A further confusion with the Snellen system is that in countries not using the metric system, distances are expressed in feet rather than meters, with 20/20 being exactly equivalent to 6/6 but with a test distance of 20 ft rather than 6 m. Although Snellen recommended adoption of the metric system in 1875 and, in 1980, the US National Academy of Sciences favored adoption of a standard defined in meters, given the imminent adoption of the metric system, the feet system is still widely used in the USA, and among lay people in the UK. The accepted standard for expressing visual acuity in clinical research, and increasingly in clinical practice, is to use the base 10 logarithm of the MAR (logMAR), such that 0.0 logMAR is equivalent to 6/6 or 20/20, and 1.0 logMAR is the same as 6/60 or 20/200. Table 1 gives approximately equivalent values in MAR, cycles per degree, Snellen fractions in meters and feet, decimal acuity, and logMAR for a range of visual acuities.

Reporting Visual Acuity

Optical and Neural Limits on Visual Acuity

Clinicians have traditionally used Snellen fractions to record visual acuity, where the numerator is the test distance and the denominator the target size. The target size is expressed, counterintuitively, as the distance from which the target has an MAR of 1 min of arc. Therefore, a visual acuity of 6/6 indicates that from 6 m, letters with MAR 1-min arc are correctly identified, while a visual acuity of 3/36 indicates that from 3 m, the targets identified have a MAR of 1 min of arc when viewed from 36 m. The reciprocal of the Snellen fraction gives the visual acuity in MAR: so a visual acuity of 3/36 indicates a MAR of 12 min of arc.

Visual acuity is limited by many factors: the optics and refraction of the eye; the clarity of the optical media; the spacing and function of the retinal photoreceptors; the ratio of retinal ganglion cells to photoreceptors; and the resolution of the primary visual cortex and higher areas of visual processing. Each diopter of myopia reduces visual acuity: a –1.00DS myope will typically have uncorrected visual acuity of around 0.5 logMAR (6/18; 20/60) and a two-diopter myope will have vision of around 0.8 logMAR on a distance test. Hypermetropia can often be relieved by accommodation in young people, but each diopter of hypermetropia

Table 1

Visual acuity conversion table

a

MAR (min)

Cycles/ degree

Snellen (metric)

Snellen (feet)

Decimal

Log MAR

60 20 10 6.3 4 3.2 2 1.6 1.3 1 0.83 0.67 0.5 0.33

0.5 1.5 3 4.7 7.5 9.4 15 18.8 23 30 36 44 60 91

1/60 3/60 6/60 6/36 6/24 6/18 6/12 6/9 6/7.5 6/6 6/5 6/4 6/3 6/2

20/1200 20/400 20/200 20/120 20/80 20/60 20/40 20/30 20/25 20/20 b 20/17 b 20/13 20/10 20/7

0.017 0.05 0.1 0.17 0.25 0.33 0.5 0.67 0.8 1 1.2 1.5 2 3

1.8 1.3 1 0.8 0.6 0.5 0.3 0.2 0.1 0 0.1 0.2 0.3 0.4

a

Each row contains approximately equivalent values of visual acuity. Log MAR values have been rounded to 1 decimal place. On US Snellen charts, these lines are 20/16 and 20/12 respectively.

b

Acuity

Visual Acuity across the Retina Nonfoveal vision is limited by many elements. First, the eye’s optics are not optimized for viewing off the visual axis, and peripheral vision is subject to greater aberration than central vision. Second, the size of photoreceptors increases and their density falls with increasing eccentricity. The number of photoreceptors per retinal ganglion cell also increases, from less than one photoreceptor per ganglion cell in the fovea to more than 20 photoreceptors per ganglion cell in the far periphery. The volume of visual cortex devoted to noncentral retina is also proportionally lower. It is unsurprising, therefore, that visual acuity falls quickly with increasing distance from the fovea (Figure 4). This is one reason for the severely reduced visual acuity of people with central vision loss from diseases such as age-related macular disease.

Visual Acuity over Life Over the first year of life, visual acuity assessed by a preferential looking test appears to be reasonably stable

250 Letter visual acuity (min arc)

beyond the accommodative ability of the eye will reduce visual acuity by a similar amount to an equivalent degree of myopia. Astigmatism, particularly where the meridia of astigmatism are oblique, will also reduce uncorrected vision significantly. Other aberrations of the eye beyond defocus and astigmatism further limit visual acuity. Retinal image quality can be improved by viewing monochromatic stimuli (to reduce chromatic aberration) and by using a deformable mirror to correct coma, trefoil, and other higher-order aberrations of the eye. Under these ideal conditions, Williams and colleagues have shown that subjects are able to resolve gratings of up to 55 cycles per degree, equivalent to a visual acuity of approximately –0.30 logMAR (6/3; 20/10). Assuming that an image is perfectly focused on the retina, the next limit on visual resolution is the spacing of the retinal photoreceptors. In order to detect a grating, alternate black and white bars must fall on adjacent photoreceptors. This theoretical limit of vision, known as the Nyquist limit, is equivalent to a grating with light to dark separation of 1/√D, where D is the center-to-center separation of two photoreceptors. In the fovea, D is approximately 3 mm, equivalent to a visual angle of approximately 55 cycles per degree – almost identical to the value found by Williams. This confirms that in people with good vision, all of the limits on visual acuity are precortical. Amblyopia, where vision is reduced despite the absence of any eye disease, is dealt with elsewhere in the encyclopedia.

497

200

150

100

50

0

0

10

20

30 40 Eccentricity (⬚)

50

60

Figure 4 Letter visual acuity measured in peripheral vision as a function of degrees of eccentricity. Data from Anstis, S. M. (1974). Letter: A chart demonstrating variations in acuity with retinal position. Vision Research 14(7): 589–592.

at around 6 min of arc. Between a child’s first and third birthday, visual acuity improves exponentially to reach 1 min of arc. A further small improvement in resolution ability to approximately 0.75 min of arc is achieved by age 5 years. In the absence of eye disease, this value remains relatively constant until the sixth decade. In a populationbased study of nearly 5000 older adults, Klein found a decrease in visual acuity to a mean value of approximately 2 min of arc in those aged over 75 years. Of course, this reflects the age-related nature of many diseases which affect visual acuity, such as cataract, glaucoma, diabetic retinopathy, and age-related macular degeneration. Figure 5 plots data from the studies of Mayer and Klein. Visual Standards In most countries, there is a visual-acuity requirement for car drivers. While the level and measurement technique varies between countries, the acuity limit is usually approximately 0.3 logMAR. Commercial airline pilots are required to have a binocular visual acuity of 0.0 logMAR. Best corrected binocular visual acuity of 1.0 logMAR or poorer is used as a definition of low vision or partial sight in many countries, with acuity of worse than 1.3 logMAR being described as severe sight impairment.

Hyperacuity Some visual tasks can be performed with a far greater degree of precision than would be suggested by the MAR. Alignment tasks such as Vernier discrimination (where the offset of one line with respect to another is detected, Figure 6(a)) can be performed with misalignment of less

498


6

Visual acuity (min arc)

5 4 3 2 1 0

1

10 Age (years)

100

Figure 5 Variation in visual acuity over life. From Mayer, D. L. and Dobson, V. (1982). Visual acuity development in infants and young children, as assessed by operant preferential looking. Data from Vision Research 22(9): 1141–1151 and Klein, R., Klein, B. E., Linton, K. L., and De Mets, D. L. (1991). The beaver dam eye study: Visual acuity. Ophthalmology 98(8): 1310–1315.

must be for it to be seen. If a target moves with velocity of 40 s1, the MAR is increased to about 2 min of arc, while at 80 s1, acuity is about 3 min of arc. In peripheral vision, slow image motion (less than 10 s1) slightly improves visual acuity for peripherally presented targets, perhaps because it breaks the phenomenon of Troxler fading. Target motion at the retina can be induced by target movement, by eye motion, or by head motion. Many eye diseases, particularly those of the macula, are associated with poor fixation stability of the eye. This poor eye stability increases retinal image motion, and is significantly associated with poorer visual function. Small degrees of head motion do not significantly decrease visual acuity under normal conditions, but have a marked deleterious effect for subjects viewing through telescopic spectacles. Therefore, subjects with macular disease who have poor fixation stability and who view through telescopic low-vision aids have a marked impairment in their dynamic visual acuity. See also: Amblyopia; Contrast Sensitivity; Pupil.

Further Reading

(b)

(a) Figure 6 Examples of hyperacuity tasks. Misalignment of the lower element is easily visible. (a) Vernier alignment; (b) dot alignment: the offset of the middle dot with respect to the upper and lower dot is easily discerned.

than 5 s of arc – considerably less than the center-tocenter spacing of a foveal photoreceptor. This is thought to be due to interpolation of the inputs of two or more adjacent neural elements.

Dynamic Visual Acuity Throughout this article, visual acuity has been discussed for static targets. If the target is moved, central visual acuity decreases: the faster the target moves, the larger it

Anstis, S. M. (1974). Letter: A chart demonstrating variations in acuity with retinal position. Vision Research 14(7): 589–592. Bailey, I. L. and Lovie, J. E. (1976). New design principles for visual acuity letter charts. American Journal of Optometry and Physiological Optics 53: 740–745. Bennett, A. G. and Rabbetts, R. B. (eds.) (1989). Visual acuity and contrast sensitivity. In: Clinical Visual Optics, pp. 23–72. Oxford: Butterworth-Heinemann. Brown, B. (1972). Resolution thresholds for moving targets at the fovea and in the peripheral retina. Vision Research 12(2): 293–304. Committee on vision. (1980). Recommended standard procedures for the clinical measurement and specification of visual acuity. Report of working group 39. Advances in Ophthalmology ¼ Fortschritte der Augenheilkunde ¼ Progres en Ophtalmologie 41: 103–148. Assembly of Behavioral and Social Sciences, National Research Council, National Academy of Sciences, Washington, DC Crossland, M. D., Culham, L. E., and Rubin, G. S. (2004). Fixation stability and reading speed in patients with newly developed macular disease. Ophthalmic and Physiological Optics 24: 327–333. Demer, J. L. and Amjadi, F. (1993). Dynamic visual acuity of normal subjects during vertical optotype and head motion. Investigative Ophthalmology and Visual Science 34(6): 1894–1906. Klein, R., Klein, B. E., Linton, K. L., and De Mets, D. L. (1991). The beaver dam eye study: Visual acuity. Ophthalmology 98(8): 1310–1315. Liang, J., Williams, D. R., and Miller, D. T. (1997). Supernormal vision and high-resolution retinal imaging through adaptive optics. Journal of the Optical Society of America. A, Optics, Image Science, and Vision 14: 2884–2892. Mayer, D. L. and Dobson, V. (1982). Visual acuity development in infants and young children, as assessed by operant preferential looking. Vision Research 22(9): 1141–1151. Plainis, S., Tzatzala, P., Orphanos, Y., and Tsilimbaris, M. K. (2007). A modified ETDRS visual acuity chart for European-wide use. Optometry and Vision Science 84(7): 647–653.

Acuity Rosser, D. A., Cousens, S. N., Murdoch, I. E., Fitzke, F. W., and Laidlaw, D. A. (2003). How sensitive to clinical change are ETDRS logMAR visual acuity measurements? Investigative Ophthalmology and Visual Science 44: 3278–3281. Thibos, L. N., Cheney, F. E., and Walsh, D. J. (1987). Retinal limits to the detection and resolution of gratings. Journal of the Optical

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Society of America. A, Optics, Image Science, and Vision 4: 1524–1529. Westheimer, G. (1987). Visual acuity. In: Moses, R. A. and Hart, W. M. (eds.) Adler’s Physiology of the Eye: Clinical Application, pp. 415–428. St Louis, MO: Mosby.

Contrast Sensitivity P Bex, Schepens Eye Research Institute, Boston, MA, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Channels – The groups of visual sensors that are selective for a narrow range of image spatial or temporal structure. Contrast constancy – At high contrasts, apparent contrast is relatively independent of the parameters that strongly influence contrast-detection threshold. Contrast-detection threshold – The statistical contrast boundary below which contrast is too low for an image to be detected reliably and above which contrast is high enough for frequent image detection. Often defined as the contrast that produces 75% correct target identifications in forced-choice paradigms. Contrast sensitivity – The reciprocal of contrastdetection threshold that also represents the transition between visible and invisible images. Critical flicker frequency – The highest flicker rate of a full contrast image that can be detected reliably. Forced-choice paradigms – Robust behavioral method used to measure detection or discrimination thresholds. Observers are forced to select between two or more intervals, of which only one contains a target. Fourier analysis – Analytical method that calculates the simple sine-wave components whose linear sum forms a given complex image. Resolution limit – The highest spatial frequency of a full contrast image that can be detected reliably. Spatial frequency – The number of image cycles that fall within a given spatial distance, typically 1 of visual angle. Temporal frequency – The number of image cycles that fall within 1 s. Wavelets/gabors – A local filter that is the point-wise product of a two-dimensional (2D) spatial sine wave and a 2D Gaussian envelope.

Most people are familiar with image brightness and contrast from their controls on computer and television displays. The brightness control adjusts the mean luminance of the display uniformly, in order that the intensity of every point in the image increases when brightness is increased or decreases when brightness is reduced. The contrast control adjusts the difference between the lightest and darkest areas of the image. Increasing contrast

500

makes areas that are below mean luminance darker and areas that are above mean luminance lighter, without changing the mean value. Decreasing contrast draws all values toward the mean, thus making the whole image fainter, similar to viewing the image through fog. Figure 1 illustrates the effect of changing the contrast of a sine-wave striped pattern (the reasons for using a sine-wave pattern are described below). The top panel shows images of gratings whose contrast increases from 12.5% on the left to 100% on the right. The mean luminance of each image is the same. The traces in the bottom row plot luminance versus position for a horizontal slice through each image.

Contrast-Detection Threshold A powerful measure of visual sensitivity can be obtained by finding the minimum contrast that is necessary for an image to be detected. This minimum contrast is referred to as contrast-detection threshold (Cthresh) and it is important because it defines the transition at which an image moves from invisible to visible. One method to estimate Cthresh might be to allow a subject to adjust the contrast until an image is just visible. However, this method is highly subjective and large differences in individual criteria for just visible make this measure unreliable.

Psychophysical Assessment of Vision To overcome these problems, most researchers employ forced-choice procedures that require an observer to identify which of two or more intervals (the more the better) contain the target. An example of a four-alternative forcedchoice (4AFC) detection task is shown in Figure 2(a). In this case, a computer presents a target in one of four positions at random around a central fixation point. The observer’s task is to fixate the central dot and to indicate the location of the target, usually by pressing a computer button. Targets that are below Cthresh (sub-threshold) are rarely detected, whereas targets that are above Cthresh (supra-threshold) are usually detected. Contrast-detection thresholds are therefore probabilistic and are defined as the contrast at which they are correctly detected midway between chance and perfect performance. It is difficult to cheat on forced-choice methods or to change criteria – the target is either seen, in which case its position is correctly identified, or it is not seen, in which

Contrast Sensitivity

12.5%

25%

(a)

50%

(b)

(c)

501

100%

(d)

Figure 1 Image contrast. The top row shows the appearance of two-dimensional (2D) sine-grating patterns that are routinely used in vision research. The contrast of the sine grating increases from left (12.5%) to right (100%) as shown by the caption. The bottom row plots a horizontal section through each image and shows that contrast changes the luminance range separately from mean luminance.

Proportion correct

1 0.75 0.5 0.25 0 0.001

0.01

0.1

1

75% correct at approximately 2.5% contrast. The slope (s) can be used to infer how easily nearby contrasts can be discriminated from one another – a shallow slope means that a large contrast difference is required to achieve a given change in performance, whereas a steep slope means that a small change in the stimulus produces a large change in performance.

Contrast

(a)

(b)

Figure 2 Contrast detection. (a) Example of a four-alternative forced-choice (4 AFC) task. The observer is required to fixate the central dot and to indicate whether the target appeared top left, top right, bottom left, or bottom right. The target contrast is adjusted by computer to a level that produces 75% correct detection. (b) A typical psychometric function. Circles show the proportion of trials the target was detected (ordinate) as a function of the target contrast (abscissa). Error bars show 1 standard deviation. The curve shows the best-fitting cumulative normal function, from which the interpolated 75% correct point is taken as contrast-detection threshold.

case the subject is forced to guess. Notice that when guessing, the subject is still correct sometimes (25% if there are four alternatives, 33% if there are three, or 50% if there are two, etc.), as shown in the frequency of seeing curve in Figure 2(b), where, at low contrasts, performance is 25% correct. The data have been fit with a curve known as a psychometric function, in this case a cumulative Gaussian: Y ¼ g þ ð1 g Þ erf ðz=sqrtð2ÞÞ=2

where z ¼ (X – m)/s; g is the guess rate (0.25 in a 4AFC experiment). The mid-point (m) of the psychometric function is often taken as Cthresh – for a 2AFC task, this is 75% correct. In the example shown, the observer achieved

Spatial Frequency Channels Based on behavioral observations in humans and single unit recordings in mammalian visual systems, researchers discovered around half a century ago that the visual system analyses images at a series of relatively narrow spatial scales and orientations known as channels. Thus, fine and coarse image details are encoded separately and Fourier analysis can be used to study the image structure that is encoded by different visual processing channels. Fourier analysis computes the sum of basic sine waves whose linear sum produces the image. To illustrate the representations of an image that are available at different spatial scales, Figure 3(a) shows a typical image, together with its coarse (Figure 3(b)) and fine (Figure 3(c)) spatial structure. Visually responsive neurons in primary visual cortex, the first cortical projection from the retina through the lateral geniculate nucleus of the thalamus, respond to images only within a limited area of the visual field, known as the classical receptive field, and are selective for a limited range of spatial frequencies and orientations. These receptive fields are now routinely modeled as Gabor or wavelet functions, defined as:

502


(a)

(b)

(c)

Figure 3 Spatial frequency in real images. (a) An image of Albert Einstein’s face is encoded at a range of spatial scales, from (b) coarse – low spatial frequency to (c) fine – high spatial frequency.

02 x þ g2 y 02 x0 þ ’ G ðx; y; l; ’; s; gÞ ¼ exp sin 2p 2s2 l

where x 0 ¼ x cos y þ y sin y and y 0 ¼ x sin y þ y cos y, l represents the wavelength, y the orientation, and c the phase of the sine-wave component. For the Gaussian window, s is the standard deviation and g is the spatial aspect ratio. Examples of Gabors are illustrated in Figure 4. On the top row, spatial frequency increases from left to right and all Gabors are of the same orientation 0 and contrast. On the bottom row, spatial frequency is fixed, but orientation is 45 , 90 , or 135 (from left to right). The visual system encodes image structure with a bank of such wavelet filters that represent the retinal image through patchwise local analysis. Figure 5 provides compelling demonstrations that our visual system employs a set of spatial frequency and orientation-selective channels. These demonstrations show that after prolonged viewing of a particular pattern (termed adaptation) the appearance of other patterns can be altered (termed an aftereffect). In these demonstrations, adapting to a pattern of one spatial frequency or orientation produces a loss in sensitivity in the channel that responds most to that pattern, but little change in channels tuned to other spatial frequencies or orientations. This localized loss in sensitivity produces a relative shift in the responses of our visual channels that cause us to experience changes in the appearance of the image. These observations have led to the widespread use of sine-wave grating patterns in basic and clinical vision research. In order to derive a measure of vision that reflects the sensitivity across our set of visual channels and to reflect the fact that functional vision requires us to detect and interact with objects of various sizes, contrastdetection thresholds are measured for gratings of a range of bar widths, expressed as spatial frequency or the number of grating cycles per unit distance. Figure 4 illustrates Gabors of differing spatial frequency; however, the size of one grating cycle on the retina depends on the distance

Figure 4 Gabor (wavelets) of differing spatial frequency and orientation. Top row: spatial frequency increases from left to right, orientation is fixed at 0 . Bottom row: Orientation increases from left to right: 45 , 90 , and 135 , spatial frequency is fixed.

from which it is viewed. Therefore, image sizes are usually calculated in terms of visual angle, which specifies the retinal image size. Figure 6 shows how visual angle is calculated and its relationship to image size and viewing distance. A convenient rule is that 1 cm viewed from 57 cm subtends a visual angle of 1 and roughly corresponds to a finger nail viewed at arm’s length.

Contrast Sensitivity Function Many researchers have shown that for sine-grating patterns, Cthresh strongly depends on spatial frequency. This fundamental observation is demonstrated in the classic image shown in Figure 7. Spatial frequency increases from left to right and contrast increases from top to bottom, so that contrast is constant across any horizontal line. Contrast-detection thresholds can be visualized on this figure as the imaginary curve along


Figure 5 Demonstration of spatial frequency- and orientationselective aftereffects. First note that when you fixate the centre gray dot, the gratings in the middle row are of the same spatial frequency and orientation. Next, look back and forth between the black dots in the top row for around 10 s. Now, when you look at the center gray dot, the grating on the left appears to be of higher spatial frequency than the grating on the right. Next, look back and forth between the white dots in the bottom row for around 10 s. Now, when you look at the center gray dot, the grating on the left appears tilted counterclockwise, while the grating on the right appears tilted clockwise. These aftereffects are robust even though you know that the gratings in the middle row are the same. These demonstrations provide compelling evidence that visual processing involves channels that are narrowly tuned for spatial frequency and orientation.

which the grating changes from invisible (toward the top of the figure) to visible (toward the bottom of the figure). Most people report that the function peaks somewhere near the middle of the figure. Notice that the peak shifts as you move the figure closer or further away. This demonstrates the importance of visual angle rather than physical image size. Note that the highest spatial frequency that can be detected at maximum contrast is given by the rightmost point on a contrast sensitivity function (CSF). This is referred to as the resolution limit and is a quick and convenient method of assessing visual sensitivity than measuring the entire CSF. When measured with forced-choice procedures, (Figure 2) contrast-detection thresholds are lowest for

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gratings around 2–5 cycles per degree of visual angle (c deg–1). By convention, the inverse of Cthresh (1/Cthresh) is usually reported and is termed contrast sensitivity. The rationale for the use of contrast sensitivity over contrastdetection threshold is most likely because the shape of the CSF is the same as that of the underlying modulation transfer function of the system. The circles in Figure 8 show the author’s contrast sensitivity as a function of spatial frequency measured with a forced-choice procedure. Error bars show 95% confidence intervals. The data have been fit (green curve) with the outputs of a set of spatial frequency channels shown by the colored curves. The channels are log spaced in spatial frequency (with peaks at 0.5, 1, 2, 4, 8, 16, or 32 c deg–1) and have the same bandwidth (1.4 octaves). The summed outputs of the set of filters provide a good fit to the data and this channel-based system is now a widely accepted model of early visual processing. The spatial frequency aftereffect shown in Figure 5 is easily explained with this channel-based model. Adapting to one spatial frequency reduces the responses of the channel that is most sensitive to that spatial frequency, but has little effect on the responses of other channels. When a different spatial frequency is subsequently viewed, the overall activity across the channels is shifted away from the adapted channel. This shift in the population response produces a shift in apparent spatial frequency away from the adapting frequency. An analogous model explains the shifts in orientation in the lower row of Figure 5, except that orientation-selective channels are adapted rather than spatial-frequency-selective channels. The CSF is highly dependent on the mean luminance of the display on which it is measured. This can easily be experienced by viewing Figure 7 with a pair of dark sunglasses (possibly two pairs), which moves the curve down (reducing sensitivity) and shifts the peak to lower spatial frequencies. The data in Figure 8 were collected on a standard computer monitor that has a mean luminance of 50 cd m–2 (candelas per square meter). Photopic, mesopic, and scotopic vision and changes in visual performance show that sensitivity to high spatial frequencies increases with mean luminance. This property is important because CSFs are routinely measured on relatively dim displays (e.g., 50–100 cd m–2) in the laboratory and in the clinic; however, the luminance of the real world is typically much greater. For example, the luminance of a cloudy sky is around 35 000 cd m–2, suggesting that standard experimental conditions may underestimate sensitivity to fine spatial structure.

Temporal Contrast Sensitivity In addition to a dependence on spatial frequency, contrast sensitivity also depends strongly on temporal frequency. Figure 1 illustrates spatial variation in luminance, but

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a = 0.5° 2094 miles

240 000 miles

Figure 6 Visual angle and viewing distance. The angular size of an object is calculated as 2*tan((0.5*h)/d), where h is the height of the object and d is the distance from which it is viewed. The example, which is not to scale, shows the angular size subtended by the moon is 0.5 . For comparison, the nail of the average index finger viewed at arm’s length subtends 1 .

200

Sensitivity

150

100

50

0 0.5

5

50

Spatial frequency (c deg–1) Figure 8 Spatial contrast sensitivity. Circles show contrast sensitivity (the reciprocal of contrast-detection threshold) for sine gratings of a range of spatial frequencies. Sensitivity peaks at around 2 c deg–1 under the conditions employed here and decreases at lower or higher spatial frequencies. The black curve is the summed sensitivity of the set of log-scaled channels shown by the colored curves and provides a good fit to the data. Figure 7 Illustration of the contrast sensitivity function (CSF). Spatial frequency increases from left to right, contrast increases from top to bottom. The contrast along any horizontal line is fixed. Different spatial frequencies become visible at different contrasts and define an imaginary curve that separates seen from unseen structure. Notice that if you move the image closer to your eye, the peak moves to the right and if you move it further away, the peak moves to the left. This demonstrates that contrast sensitivity depends on retinal not physical image size. If you wear one or two pairs of dark sunglasses, the curve shifts down and the peaks moves left, which demonstrates the dependence of the CSF on mean luminance.

imagine instead that the x-axis represents time, rather than space. Now the figure illustrates flicker. Flicker frequency can be varied in the same way as spatial frequency is varied in Figures 3 and 7. The circles in Figure 9 show how the author’s contrast sensitivity varies as a function of temporal frequency for a 2 c deg1 grating pattern. Sensitivity peaks around 5 Hz, at the mean luminance

used here (50 cd m2) and decreases at lower or higher temporal frequencies. These data are well fit (black curve) by a model with only two temporal channels, compared with the multiple channels that support spatial contrast sensitivity. One channel (red curve) is low-pass or sustained and is most sensitive to structure that is stationary or slowly changing over time. The second channel (blue curve) is band-pass or transient and is most sensitive to structure that changes at around 5 Hz. The spatial resolution limit falls steadily with distance from the fovea, an effect that can be experienced by viewing Figure 7 while fixating away from the center of the image. As you fixate further away, the threshold curve moves further down the figure and its peak shifts further to the left. Unlike spatial resolution, temporal resolution (the highest flicker rate that can be detected at any contrast, often called critical flicker fusion frequency)


a phenomenon termed contrast constancy.Contrast constancy can be experienced in Figure 7 – while the transition between visible and invisible gratings has a curved shape, toward the bottom of the figure, the gratings appear to have similar contrast regardless of spatial frequency. This has important implications for image enhancement, which should therefore target only image components that are below their Cthresh.

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250 200 150 100 50 0 0.5

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See also: Acuity. 5 Temporal frequency (Hz)

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Figure 9 Temporal contrast sensitivity. Circles show contrast sensitivity (the reciprocal of contrast-detection threshold) for sine gratings of a range of temporal frequencies. The black curve is the summed sensitivity of the two log-scaled channels shown by the red and blue curves. The red curve has peak sensitivity at low temporal frequencies – that is, static images – and is termed a sustained channel. The blue curve has peak sensitivity around 6 Hz and is termed a transient channel.

increases moderately with distance from the fovea. This explains why older, 60-Hz computer displays can sometimes be seen to flicker when seen in the peripheral visual field, but not when viewed directly. Just as spatial contrast sensitivity depends on luminance, so does temporal contrast sensitivity. A 35-mm film is generally recorded at 24 frames per second, a refresh rate that could be easily detected at moderate light levels, as can be seen from Figure 9. For this reason, movie theaters are generally dark because sensitivity to high flicker rates is poor under those conditions. In addition, the visible 24-Hz image update rate is masked by flashing the illuminant at 48 Hz, so each frame is flashed twice. At supra-threshold contrasts, apparent contrast is relatively independent of spatial or temporal frequency,

Further Reading Bracewell, R. (1999). The Fourier Transform and Its Applications, 3rd edn. London: McGraw-Hill. Campbell, F. W. and Robson, J. G. (1968). Application of Fourier analysis to the visibility of gratings. Journal of Physiology 197: 551–566. Field, D. J. and Tolhurst, D. J. (1986). The structure and symmetry of simple-cell receptive-field profiles in the cat’s visual cortex. Proceedings of the Royal Society of London. Series B. Biological Sciences 228(1253): 379–400. Georgeson, M. A. (1990). Over the limit: Encoding contrast above threshold in human vision. In: Kulikowski, J. J. (ed.) Limits of Vision, pp. 106–119. London: Erlbaum. Hubel, D. H. and Wiesel, T. N. (1959). Receptive fields of single neurones in the cat’s striate cortex. Journal of Physiology 148: 574–591. Kelly, D. H. (1961). Visual responses to time-dependent stimuli. 1. Amplitude sensitivity measurements. Journal of the Optical Society of America. A, Optics, Image Science, and Vision 51: 422–429. Kulikowski, J. J. and Tolhurst, D. J. (1973). Psychophysical evidence for sustained and transient detectors in human vision. Journal of Physiology 232(1): 149–162. Landis, C. (1954). Determinants of the critical flicker-fusion threshold. Physiological Reviews 34(2): 259–286. O’Shea, R. P. (1991). Thumb’s rule tested: Visual angle of thumb’s width is about 2 deg. Perception 20(3): 415–418. Rovamo, J., Virsu, V., Laurinen, P., and Hyvarinen, L. (1982). Resolution of gratings oriented along and across meridians in peripheral vision. Investigative Ophthalmology and Visual Science 23: 666–670.

Astigmatism M J Cox, University of Bradford, Bradford, UK ã 2010 Elsevier Ltd. All rights reserved.

Glossary Against-the-rule – Ocular astigmatism in which the meridian with greater optical power in the eye is horizontal. Astigmat – An individual with ocular astigmatism. Axis meridian – The meridian of a cylindrical surface that is flat and consequently has no optical power. Emmetropization – The active process of reduction in refractive error toward an ideally focused system that occurs in the human eye during the first 2 years of development. High-order wave front aberrations – Wave front aberrations that are expressed using cubic, or higher, powers of the light ray’s distance from the pupil center to predict the amount of aberration. Meridional amblyopia – A lack of contrast sensitivity to high and medium spatial frequency contours oriented along a particular meridian without any refractive error or ocular pathological process affecting the visual function. Paraxial optics – Image forming through an optical system where only rays traveling close to the optical axis of the system and/or at small angles to this axis are considered. Penetrating keratoplasty – A surgical procedure to remove corneal material and replace it with material from a donor cornea. Stokes lens – A lens constructed from two symmetrically counter-rotating cylindrical lenses of equal absolute power but opposite sign. These combine to make a continuously variable power crossed-cylinder lens. Wave front aberrations – Deviations of a wave front of light propagating through an optical system from a perfect spherical wave front. With-the-rule – Ocular astigmatism in which the meridian with greater optical power in the eye is vertical.

The Definition and Etymology of Astigmatism The earliest forms of correction for visual loss caused by refractive errors in the eye used spherical spectacle lenses.

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These are rotationally symmetrical about the optical axis and, considering paraxial optics, produce a point image from a point object on the axis of the lens. This type of image forming is known as stigmatic, from the Greek stigma, meaning a branding mark. Astigmatism describes an optical system where any nonpoint image is formed from a point object. In practice, astigmatism commonly refers to the simplest extension of stigmatic image formation, namely where the optical system forms two perpendicular line images from an axial point object, each at a different distance along the optical axis. In an astigmatic optical system the power varies as a function of the meridian, with a maximum and minimum power in meridians that are perpendicular. This variation in power approximates very well to a sinusoidal function, as seen in the formula Fy ¼ FSph þ

FCyl FCyl þ cos½2ðy aÞ 2 2

½1

where Fy is the power in the meridian at y , measuring angles anticlockwise from the horizontal, FSph is the power in the meridian of minimum power, FCyl is the difference in power between the meridians of maximum and minimum power, and a is the angle of the meridian of maximum power. The double angle ½2ðy aÞ in eqn [1] demonstrates that the power varies through a complete cycle as we rotate the meridian 180 around the optical axis. Newton is said to be the first to describe the variation in optical power with meridian and the consequent formation of line foci, but did so in the rather specialized form of oblique astigmatism. It was left to Thomas Young to first describe and measure ocular astigmatism, a finding which was a by-product of his attempts to measure his own refractive error as a starting point for investigating accommodative mechanisms in the human eye. He found his own astigmatism to be around 1.75 D and even had additional evidence to suggest that its source was a tilt in his crystalline lens. Airy was the first individual to measure and correct his ocular astigmatism. Wollaston, Ostwalt, and Tscherning discovered means by which spectacle lenses could be manufactured to minimize lens-induced oblique astigmatism. The above discussion concerns regular astigmatism, where two perpendicular axes of symmetry exist within the optical system and the relationship between power and the angle of the meridian is known. In irregular astigmatism this symmetry does not exist over the aperture through which light travels to form an image. Locally, over much smaller apertures, such symmetry may be present, but for

Astigmatism

the purposes of image formation by the optical system or eye it is absent. This results in objects that do not form line images but rather two elongated spreads of light, even at best focus. Furthermore, these two elongated images are not oriented perpendicularly. All eyes contain some degree of irregular astigmatism but this is rarely visually limiting except in pathological processes where the irregular astigmatism is large. Examples of such pathology include keratoconus; lenticonus; corneal scarring, inflammation, dystrophy, and degeneration; pterygium; mechanical effects on the cornea from neighboring structures such as the lids or sclera or following the use of rigid contact lenses; lens dislocation; localized lens index changes; and polycoria and ectopic pupils. In ocularly healthy individuals with unusually large pupils and statistically higher levels of irregular astigmatism, visual function can be affected in low and medium light levels.

Ocular Astigmatism: Prevalence and Age-Related Changes Ocular astigmatism is important for two main reasons. First, it prevents optimal retinal image formation and leads to a loss of contrast in the retinal image. It can be argued that it is more debilitating than either myopia or hypermetropia as, unlike myopia, there is no object distance at which a clear retinal image can be formed and unlike hypermetropia, it is not possible to overcome the refractive defect by using one’s accommodation. Second, if the high levels of astigmatism that are naturally present during early infancy do not reduce during the process of emmetropization, then permanent meridional amblyopia can occur leading to lack of visual sensitivity to small oriented details in later years, even when the astigmatism has been refractively corrected. In addition, some suggest that visual blur during early life may help to drive the development of myopia in later years. Uncorrected ocular astigmatism may be one such cause of visual blur, and an association between ocular astigmatism and myopia development has been found. Almost all neonates have significant amounts of astigmatism caused by an unusually steep cornea in one meridian, although the angle of this meridian with higher power is not consistent across the population. Early development reduces this cornea-generated astigmatism such that by 4–6 years of age only around one-twentieth of the population has ocular astigmatism in excess of 1 D and in the great majority of young astigmats, the meridian with the greatest power is vertical (or within 15 of vertical, the so called with-the-rule astigmatism). In later childhood (5–17 years of age) the proportion of individuals with at least 1 D of astigmatism increases up to around a quarter, with a higher risk for Asians and Hispanics (around a third), and a lower risk for African-Americans (around a fifth).

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In adults, about two-thirds of the population have measureable astigmatism (> 0.25 D), but the majority of this is at low levels (1 D) in adults ranges from about 10–20%, dropping to only 0.5% for astigmatism in excess of 4 D. A general trend can also be found in the adult population concerning the angle of the meridian of greatest power. In younger adults (+3.50 D) are up to 13 times more likely to develop strabismus by 4 years of age if left uncorrected, and they are 6 times more likely to have reduced visual acuity than infants with low hyperopia or emmetropia. Children who had significant hyperopia during infancy are much more likely to develop amblyopia and strabismus by 4 years of age. The presence of anisometropic hyperopia further increases the risk of strabismus and amblyopia, especially if found beyond 3 years of age. The American Optometric Association has published guidelines stating that levels greater than 1.00 D of hyperopic anisometropia and 5.00 D of isometropic hyperopia are amblyogenic. Early detection and treatment of hyperopia may reduce the incidence and severity of consequential amblyopia and strabismus and is a major justification for universal vision evaluation of young children.

Importance of Early Detection of Significant Hyperopia Atkinson and colleagues showed that uncorrected hyperopia (>3.5 D in one meridian) might contribute to poor motor and cognitive development in younger children (9 months to 5.5 years) and/or learning problems in some older children. The precise mechanism of this relationship is unclear, but optical blur, accommodative and binocular dysfunction, and fatigue all appear to play roles. In fact, uncorrected infant hyperopia has been associated with mild delays in visuo-cognitive and visuo-motor development, but has appeared to reach the level of their emmetropic counterparts after 6 weeks of full time hyperopic spectacle wear in 3–5-year-olds. The substantial number of school-age children and young adults who have uncorrected significant hyperopia is evidence of the potential impact of this learning-related vision problem and the need for early detection screening programs.

Examination Techniques of Hyperopia Optical correction should be based on both static (normal accommodation) and cycloplegic (e.g., 1% cyclopentolate) retinoscopy, accommodative and binocular assessment, and AC/A (accommodative convergence/ accommodation) ratio. The correction should then be modified as needed to facilitate binocularity and compliance. Plus-power spherical or sphero-cylindrical lenses are prescribed to shift the focus of light from behind the eye to a point on the retina. Accommodation plays an important role in determining the prescription. Some older patients with hyperopia do not initially tolerate the full correction indicated by the manifest refraction, and many patients with latent hyperopia do not tolerate the full correction of hyperopia indicated under cycloplegia.

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However, young children with accommodative esotropia and hyperopia generally require only a short period of adaptation to tolerate full optical correction. Patients with latent hyperopia who prove intolerant to the use of full or partial hyperopic correction may benefit from initially wearing the correction only for near viewing; or alternatively, trial use of a short-acting cycloplegic agent may enhance acceptance of the optical correction. Patients with absolute hyperopia are more likely to accept nearly the full correction, because they typically experience immediate improvement in visual acuity.

Management of Hyperopia The specific elements of treatment (e.g., final spectacle prescription) should be tailored to individual patient needs. Among the factors to consider when planning treatment and management strategies are: the magnitude of the hyperopia (under dry and cycloplegic conditions), the presence of astigmatism and/or anisometropia, the patient’s age, the presence of an associated esotropia and/or amblyopia, the status of the accommodative and convergence systems, the demands placed on the visual system, and any symptoms. Among several available treatments for hyperopiarelated symptoms, optical correction of the refractive error with spectacles and contact lenses is the most commonly used modality. Newer high-index lens materials and aspheric lens designs have reduced the thickness and weight of high plus-power lenses, increasing wear ability and patient acceptance. Spectacles, especially those with lenses of polycarbonate material, provide protection against trauma to the eye and orbital area and are imperative in children. Soft or rigid contact lenses are an excellent alternative for some patients. Contact lenses not only provide better cosmesis and compliance, but they reduce aniseikonia in persons with anisometropia, improving binocularity. Multifocal or monovision contact lenses may be considered for patients who require additional near correction, but resist the use of multifocal spectacles because of their appearance. Patients who wear contact lenses are at increased risk for ocular complications due to corneal hypoxia, mechanical irritation, or infection; but nevertheless, improved vision makes contact lens wear a valuable treatment option for compliant patients. Vision therapy and modification of the patient’s habits and environment can be important in achieving definitive long-term remediation of symptoms. Such modifications include, but are not limited to, improved lighting, longer near working distances, using better-quality-printed material, taking frequent breaks when reading or working on a computer for sustained periods of time. These modifications all work to reduce the accommodative

demand placed on the patient and can allow, irrespective of spectacle wear, the patient to perform their daily near activities with less symptoms. Several refractive surgery techniques to correct hyperopia are under development. The major procedures currently being studied as possible therapies for hyperopia are – Holmium: YAG laser thermal keratoplasty, automated lamellar keratoplasty, spiral hexagonal keratotomy, excimer laser, and clear lens extraction with intraocular lens implantation. The American Academy of Ophthalmology has recently reviewed 36 research articles studying the efficacy and safety of refractive surgery for hyperopia and found that the surgery provides an effective and safe correction for lower ranges of hyperopia (

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