Series Editors

Volume Editors

Guenter K. Krieglstein, MD

Thomas Reinhard, MD

Professor and Chairman

Professor of Ophthalmology

Department of Ophthalmology

University Eye Hospital

University of Cologne

Killianstrasse 5

Joseph-Stelzmann-Strasse 9

79106 Freiburg

50931 Cologne

Germany

D. F. P. Larkin, MD, MRCPF, FRCS

Robert N. Weinreb, MD

Cornea & External Diseases Service

Professor and Director

Moorfields Eye Hospital

Hamilton Glaucoma Center

London, EC1V 2PD, United Kingdom

Department of Ophthalmology - 0946

University of California at San Diego

9500 Gilman Drive

La Jolla, CA 92093-0946

USA

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Foreword

Essentials in Ophthalmology is a new review se-

worthy advances in the subspecialty than on

ries covering all of ophthalmology categorized

systematic completeness. Each article is struc-

in eight subspecialties. It will be published quar-

tured in a standardized format and length, with

terly; thus each subspecialty will be reviewed

citations for additional reading and an appro-

biannually.

priate number of illustrations to enhance im-

Given the multiplicity of medical publi-

portant points. Since every subspecialty volume

cations already available, why is a new series

is issued in a recurring sequence during the

needed? Consider that the half-life of medical

2-year cycle, the reader has the opportunity to

knowledge is estimated to be around 5 years.

focus on the progress in a particular subspecial-

Moreover, it can be as long as 8 years between

ty or to be updated on the whole field. The clin-

the description of a medical innovation in a

ical relevance of all material presented will be

peer-reviewed scientific journal and publica-

well established, so application to clinical prac-

tion in a medical textbook.A series that narrows

tice can be made with confidence.

this time span between journal and textbook

This new series will earn space on the book-

would provide a more rapid and efficient trans-

shelves of those ophthalmologists who seek to

fer of medical knowledge into clinical practice,

maintain the timeliness and relevance of their

and enhance care of our patients.

clinical practice.

For the series, each subspecialty volume com-

prises 10-20 chapters selected by two distin-

guished editors and written by internationally

G. K. Krieglstein

renowned specialists. The selection of these

R.N.Weinreb

contributions is based more on recent and note-

Series Editors

Preface

This volume of the series Essentials in Ophthal-

outline modern matching techniques (major -

mology aims to present recent developments

triplet, minor matching) as a prophylactic im-

regarding the cornea, with a discussion of diag-

munological measure for patients with normal-

nostic measures and particular emphasis being

risk as well as those with high-risk keratoplasty.

placed on treatment.

Modern strategies of systemic immunosuppres-

The therapeutic repertoire for surface disor-

sion following high-risk penetrating kerato-

ders has increased considerably within the past

plasty are presented by Reis and coworkers.

decade. The chapter by Geerling and Hartwig

An overview is given by Seitz of how best to

reviews the application of autologous serum eye

trephine the cornea in penetrating keratoplasty

drops for this indication. Dua and coworkers in

in order to minimize postoperative astigma-

their chapters first help us to understand the

tism. Watson and Daya provide an expert opin-

limitations of amniotic membrane transplanta-

ion on a serious postoperative complication

tion and then give us an overview regarding the

following LASIK, i.e. infection.

various possibilities of limbal stem cell trans-

Adenoviral corneal opacities are still a thera-

plantation. Güell and coworkers provide an

peutic challenge. Hillenkamp and coworkers

illustration of the potential of limbal stem cell

provide an update on the different treatment

transplantation following ex-vivo expansion.

regimes. Guthoff and coworkers present con-

Anterior lamellar keratoplasty is presented

focal microscopy of the cornea as a valuable

by Melles as a promising technique for patients

tool for the in-vivo description of corneal struc-

with low grade keratoconus or opaque corneas

tures on a cellular basis. Finally, a overview of

with healthy endothelium. Endothelial immune

ocular allergic disease is given by Manzouri and

reactions may be avoided using this procedure.

coworkers.

In penetrating keratoplasty, immune reactions

All the topics covered by the book have a direct

and astigmatism in patients still represent the

clinical relevance, and we hope they will make a

major postoperative problems. Slegers and

significant contribution to the development of

coworkers illustrate immunopathological phe-

optimal diagnostic and therapeutic procedures

nomena, clinical features and risk factors of

for patients with disease of the cornea.

graft rejection. Antiangiogenic procedures are

discussed by Cursiefen and Kruse which might

contribute to minimizing the immunological

T. Reinhard

problem in the future. Böhringer and coworkers

D. F. P. Larkin

Contents

Chapter 1

2.3

Intra and Inter Donor Variations

Autologous Serum Eyedrops

of the Membrane

for Ocular Surface Disorders

2.4

Processing and Preservation

Gerd Geerling,Dirk Hartwig

of the Membrane

2.5

Clinical Studies and Outcomes

1.1

Introduction

(Definitions of Success and Grading

1.1.1

The Rationale for Using Serum

of Disease Severity)

in Ocular Surface Disorders

2.6

Efficacy of Membrane in Relation

1.1.2

Legal Aspects

to Other Established Techniques

1.2

Production and Application

and Options

1.2.1

Important Parameters

References

of the Production Process

1.2.2

Current Standard Operating

Chapter 3

Procedures Used at the University

Transplantation of Limbal Stem Cells

of Lübeck

Harminder S. Dua

1.2.3

Quality Control

1.3

Clinical Results

3.1

Introduction

1.3.1

Persistent Epithelial Defects

3.2

Stem Cells

1.3.2

Dry Eye

3.2.1

Definition

1.3.3

Other Indications

3.2.2

Characteristics of Stem Cells

1.4

Complications

3.2.3

The Stem Cell ‘Niche’

1.5

Alternative Blood Products

3.3

Limbal Stem Cells

for the Treatment

3.3.1

The Clinical Evidence

of Ocular Surface Disease

3.3.2

The Scientific Evidence

1.5.1

Umbilical Chord Serum

3.4

Limbal Stem Cell Deficiency

1.5.2

Albumin

3.4.1

Causes of Limbal Stem Cell

1.5.3

Plasma and Platelets

Deficiency

References

3.4.2

Effects of Limbal

Stem Cell Deficiency

3.4.3

Diagnosis of Stem Cell Deficiency .

Chapter 2

3.5

Limbal Transplant Surgery

Controversies and Limitations

3.5.1

Principles

of Amniotic Membrane

3.5.2

Preoperative Considerations

in Ophthalmic Surgery

3.6

Surgical Techniques

Harminder S. Dua, V. Senthil Maharajan,

3.6.1

Sequential Sector Conjunctival

Andy Hopkinson

Epitheliectomy (SSCE)

2.1

Introduction

3.6.2

Auto-limbal Transplantation

2.2

Proposed Mechanisms of Action

3.6.3

Allo-limbal Transplantation

of the Amniotic Membrane

3.6.4

Adjunctive Surgery

2.2.1

Amnion Structure

3.6.5

Postoperative Treatment

2.2.2

Amnion Composition

References

Contents

Chapter 4

6.6

Immunopathological Mechanisms

Limbal Stem Cell Culture

6.6.1

Immune Privilege

José L. Güell, Marta Torrabadella,

and Its Breakdown

Marta Calatayud, Oscar Gris,

6.6.2

Afferent Arm

Felicidad Manero, Javier Gaytan

of the Allogeneic Response

6.6.3

Efferent Arm

4.1

Introduction

of the Allogeneic Response

4.2

Epithelial Phenotype

6.7

Treatment of Rejection

4.3

Preparation of Human Amniotic

6.8

Prevention of Rejection

Membrane

6.8.1

Immunosuppression

4.4

Culture of Explanted Tissue

6.8.2

HLA Matching

4.5

Tissue Procurement

6.9

Future Prospects

4.6

Preliminary Clinical Experience . . .

References

4.6.1

Principles for Taking the Biopsy . . .

4.6.2

Advantages of Limbal

Chapter 7

Stem Cell Culture

New Aspects of Angiogenesis in the Cornea

4.7

Case Report

Claus Cursiefen, Friedrich E. Kruse

4.8

Future Standard Staging Approach

for Ocular Surface Reconstruction .

7.1

Introduction

References

7.2

“Angiogenic Privilege of the Cornea”

or “How Does the Normal Cornea

Maintain Its Avascularity?”

Chapter 5

7.3

Corneal (Hem)angiogenesis

Deep Anterior Lamellar Keratoplasty

7.3.1

General Mechanisms of Corneal

Gerrit R.J. Melles

(Hem)angiogenesis

7.3.2

Common Causes

5.1

Introduction

of Corneal (Hem)angiogenesis

5.2

Main Drawbacks

7.3.3

Clinical Consequences

of Conventional DALK

of Corneal Hemangiogenesis

5.3

Different Concepts

7.3.4

Corneal Hemangiogenesis

5.4

Important Preoperative

After Keratoplasty

Considerations

7.3.5

Corneal Angiogenesis

5.5

Psychological Preparation

Due to Contact Lens Wear

of the Patient

7.3.6

Angiogenesis as a Cause

5.6

Choice of DALK

of Disease Progression,

Surgical Technique

not a Sequel (Herpetic Keratitis) . .

5.7

Clinical Results

7.3.7

Surgery in Vascularized Corneas . .

References

7.4

Corneal Lymphangiogenesis

7.4.1

Mechanisms of Corneal

Lymphangiogenesis

Chapter 6

7.4.2

Importance of Lymphangiogenesis

Corneal Transplant Rejection

for Induction of Alloimmunity

T.P.A.M. Slegers, M.K. Daly, D.F.P. Larkin

After Keratoplasty

6.1

Introduction

7.4.3

Non-immunological Effects

6.2

Incidence

of Corneal Lymphangiogenesis . . .

6.3

Factors Predisposing

7.5

Antiangiogenic Therapy

to Corneal Graft Rejection

at the Cornea

6.4

Clinical Features

7.5.1

Established and Novel

6.5

Histopathology

Antiangiogenic Therapies

Contents

7.5.2

Novel Antihemangiogenic

9.4

Immunosuppressive Agents

112

and Antilymphangiogenic Therapies

9.4.1

History

112

to Improve Graft Survival After

9.4.2

Corticosteroids

114

Keratoplasty

9.4.3

Cyclosporine A (CSA, Sandimmun,

References

Sandimmun Optoral,

Sandimmun Neoral)

114

9.4.4

Tacrolimus (FK506, Prograf)

114

Chapter 8

9.4.5

Mycophenolate Mofetil

Histocompatibility Matching

(MMF, CellCept, Myfortic)

115

in Penetrating Keratoplasty

9.4.6

Rapamycin (Sirolimus, Rapamune)

115

Daniel Böhringer, Rainer Sundmacher,

9.4.7

RAD (Everolimus, Certican)

116

Thomas Reinhard

9.4.8

FTY 720

116

9.4.9

Biologic Agents

117

8.1

Introduction

101

9.5

Guidelines for Practitioners

117

8.1.1

Immune Reactions Constantly

9.5.1

Preoperative Evaluation

117

Threaten Graft Survival

101

9.5.2

How To Use Cyclosporine

8.1.2

Major Transplantation Antigens

in High-Risk Corneal

(HLA)

102

Transplantation

118

8.1.3

Minor Transplantation Antigens . .

105

9.5.3

How To Use MMF in High-Risk

8.2

Time on the Waiting List Associated

Corneal Transplantation

118

with Histocompatibility Matching .

106

9.5.4

How To Use Rapamycin

8.2.1

Waiting Time Variance Has Been

in High-Risk Corneal

a Barrier to Histocompatibility

Transplantation

118

Matching

106

9.5.5

How To Use Tacrolimus

8.2.2

Algorithm for Predicting the Time

in High-Risk Corneal

on the Waiting List

106

Transplantation

118

8.3

Recommended Clinical Practice . . .

107

9.5.6

Combination Therapies

119

References

108

9.6

Conclusion

119

References

119

Chapter 9

Current Systemic Immunosuppressive

Chapter 10

Strategies in Penetrating Keratoplasty

Trephination in Penetrating Keratoplasty

Alexander Reis, Thomas Reinhard

Berthold Seitz, Achim Langenbucher,

Gottfried O.H. Naumann

9.1

Introduction

109

9.2

Immunology

109

10.1

Introduction

123

9.2.1

Acute Rejection

110

10.2

Astigmatism and Keratoplasty

124

9.2.2

Major Histocompatibility Complex

110

10.2.1

Definition of Post-keratoplasty

9.2.3

Chronic Rejection

110

Astigmatism

124

9.3

Normal-Risk Versus High-Risk

10.2.2

Reasons for Astigmatism

Transplantation

111

After Keratoplasty

125

9.3.1

Normal-Risk Transplantation

111

10.2.3

Prevention/Prophylaxis

9.3.2

High-Risk Transplantation

111

of Astigmatism After Keratoplasty

129

9.3.3

Rationale for Systemic

10.3

Trephination Techniques

130

Immunosuppression

111

10.3.1

Principal Considerations

131

9.3.4

Why Is Immunomodulation

10.3.2

Conventional Mechanical Trephines 139

with Topical Steroids Not Sufficient

10.3.3

Nonmechanical Laser Trephination

144

To Prevent Immunologic Graft

10.4

Concluding Remarks

148

Rejection in High-Risk Patients? . .

111

References

149

XII

Contents

Chapter 11

Chapter 13

Infective Complications Following LASIK

In Vivo Micromorphology of the Cornea:

Adam Watson, Sheraz Daya

Confocal Microscopy Principles

and Clinical Applications

11.1

Introduction

153

Rudolf F. Guthoff, Joachim Stave

11.2

Frequency and Presentation

153

11.3

Characteristics

154

13.1

Introduction

173

11.4

Differential Diagnosis

154

13.2

Principle of In Vivo Confocal

11.4.1

Diffuse Lamellar Keratitis

Microscopy Based

(DLK,“Sands of the Sahara”)

154

on the Laser-Scanning Technique .

174

11.4.2

Steroid-Induced Intraocular

13.2.1

Slit-Scanning Techniques

175

Pressure Elevation

13.2.2

Laser-Scanning Microscopy

with Flap Oedema (Pseudo-DLK) .

155

and Pachymetry

176

11.5

Management

155

13.2.3

Fundamentals of Image Formation

11.5.1

Flap Lift

155

in In Vivo Confocal Microscopy . . .

179

11.5.2

Specimen Taking

155

13.3

General Anatomical

11.5.3

Treatment

157

Considerations

180

11.5.4

No Improvement

157

13.4

In Vivo Confocal Laser-Scanning

11.6

Special Considerations

158

Microscopy

181

11.6.2

Fungal Keratitis

158

13.4.1

Confocal Laser-Scanning Imaging

11.6.3

Viral Keratitis

159

of Normal Structures

182

11.7

Visual Outcome

159

13.5

Clinical Findings

190

11.8

Management of Sequelae

159

13.5.1

Dry Eye

190

11.9

Prevention

160

13.5.2

Meesmann’s Dystrophy

190

References

160

13.5.3

Epithelium in Contact

Lens Wearers

192

13.5.4

Epidemic Keratoconjunctivitis

195

Chapter 12

Treatment of Adenoviral Keratoconjunctivitis

13.5.5

Acanthamoeba Keratitis

196

13.5.6

Corneal Ulcer

199

Jost Hillenkamp, Rainer Sundmacher,

13.5.7

Refractive Corneal Surgery

200

Thomas Reinhard

13.6

Future Developments

201

12.1

Introduction

163

13.6.1

Three-Dimensional Confocal

12.1.1

Etiology and Clinical Course

Laser-Scanning Microscopy

201

of Ocular Adenoviral Infection

163

13.6.2

Functional Imaging

203

12.2

Socioeconomic Aspect

166

References

206

12.3

Treatment

166

12.3.1

Treatment of the Acute Phase

166

12.3.2

Treatment of the Chronic Phase . . . 168

12.3.3

Prophylaxis

169

12.4

Conclusion and Outlook

170

12.5

Current Clinical Practice

and Recommendations

170

References

170

Contents

XIII

Chapter 14

14.3.2

Perennial Allergic Conjunctivitis . .

214

Allergic Eye Disease: Pathophysiology,

14.3.3

Vernal Keratoconjunctivitis

214

Clinical Manifestations and Treatment

14.3.4

Atopic Keratoconjunctivitis

215

Bita Manzouri, Thomas Flynn,

14.3.5

Giant Papillary Conjunctivitis

217

Santa Jeremy Ono

14.4

Treatment of Allergic Eye Disease .

217

14.4.1

Antihistamines

218

14.1

Introduction

209

14.4.2

Mast Cell Stabilizing Agents

218

14.2

Pathophysiology

210

14.4.3

Dual-Acting Agents

219

14.2.1

Type I Hypersensitivity

210

14.4.4

Non-steroidal Anti-inflammatory

14.2.2

Ocular Inflammatory Reaction:

Drugs (NSAIDs)

219

Late Phase

211

14.4.5

Topical Corticosteroids

219

14.2.3

Non-specific Conjunctival

14.4.6

Calcineurin Inhibitors

220

Hyperreactivity

211

14.4.7

Future Drug Developments

220

14.2.4

T-Cell-Mediated Hypersensitivity

14.5

Conclusion

221

in Allergic Eye Disease

212

References

222

14.3

Clinical Syndromes

of Allergic Eye Disease

212

14.3.1

Seasonal Allergic Conjunctivitis . . .

214

Subject Index

225

Contributors

Daniel Böhringer, Dr.

Gerd Geerling,Prof.Dr.

University Eye Hospital

Department of Ophthalmology

Killianstrasse 5, 79106 Freiburg, Germany

University of Würzburg

Josef-Schneider-Strasse 11

Marta Calatayud, MD

97080 Würzburg, Germany

Diagonal 419 6º1a, 08008 Barcelona, Spain

Oscar Gris, MD

Claus Cursiefen, Dr.

Instituto de Microcirugia, Ocular de Barcelona

Department of Ophthalmology

Munner 10, 08022 Barcelona, Spain

Friedrich-Alexander University

Erlangen-Nürnberg

José L. Güell, MD

Schwabachanlage 6, 91054 Erlangen, Germany

Associate Professor of Ophthalmology

Instituto de Microcirurgia

M.K. Daly, MD

Ocular de Barcelona

Cornea & External Diseases Service

C. Munner, 10, 08022 Barcelona, Spain

Moorfields Eye Hospital

London, EC1V 2PD, UK

Rudolf F. Guthoff, Prof. Dr.

University Eye Hospital Rostock

Sheraz Daya, MD

Doberaner Strasse 140

Eye Bank, Queen Victoria Hospital NHS Trust

18057 Rostock, Germany

East Grinstead, West Sussex, RH19 3DZ, UK

Dirk Hartwig, Dr.

Harminder S. Dua, MD, PhD

Institute of Immunology

Chair and Professor of Ophthalmology

and Transfusion Medicine

University of Nottingham, Eye ENT Centre

University of Lübeck, Ratzeburger Allee 160

Queens Medical Centre

23538 Lübeck, Germany

Derby Road, Nottingham, NG7 2UH, UK

Jost Hillenkamp, Dr.

Thomas Flynn, MRCOphth.

Eye Hospital of the University of Regensburg

Department of Ocular Immunology

Franz-Josef Strauss Allee 11, 93042 Regensburg

Institute of Ophthalmology

Germany

University College London, Bath Street

London, EC1V 9EL, UK

Andy Hopkinson, PhD

Division of Ophthalmology

Javier Gaytan, MD

and Visual Sciences, Eye ENT Centre

Instituto de Microcirugia, Ocular de Barcelona

Queens Medical Centre, University Hospital

Munner 10, 08022 Barcelona, Spain

Derby Road, Nottingham, NG7 2UH, UK

XVI

Contributors

Friedrich E. Kruse, Dr.

Santa Jeremy Ono, PhD

Department of Ophthalmology

Department of Ocular Immunology

University of Erlangen-Nürnberg

Institute of Ophthalmology

Schwabachanlage 6, 91054 Erlangen, Germany

University College London

11-43 Bath Street, London, EC1V 9EL, UK

Achim Langenbucher, Priv.-Doz. Dr.

Department of Ophthalmology

Thomas Reinhard, Prof. Dr.

University of Erlangen-Nürnberg

University Eye Hospital

Schwabachanlage 6, 91054 Erlangen, Germany

Killianstrasse 5, 79106 Freiburg, Germany

D.F.P. Larkin, MD, MRCPF, FRCS

Alexander Reis, Priv.-Doz. Dr.

Cornea & External Diseases Service

Reis Medical Institution Est.

Moorfields Eye Hospital

Landstrasse 310, 9495 Triesen

London, EC1V 2PD, UK

Principality of Liechtenstein

V. Senthil Maharajan, MS, FRCS

Berthold Seitz, Prof. Dr.

Division of Ophthalmology

Department of Ophthalmology

and Visual Sciences Eye ENT Centre

University of Erlangen-Nürnberg

Queens Medical Centre, University Hospital

Schwabachanlage 6, 91054 Erlangen, Germany

Derby Road, Nottingham, NG7 2UH, UK

T.P.A.M. Slegers, MD

Felicidad Manero, MD

Department of Ophthalmology

Instituto de Microcirugia

University Medical Center Groningen

Ocular de Barcelona

9700 RB Groningen, The Netherlands

Munner 10, 08022 Barcelona, Spain

Joachim Stave, Prof. Dr.

Bita Manzouri, MRCOphth.

University Eye Hospital Rostock

Department of Ocular Immunology

Doberaner Strasse 140, 18057 Rostock

Institute of Ophthalmology

Germany

University College London

Bath Street, London, EC1V 9EL, UK

Rainer Sundmacher, Prof. Dr.

University Eye Hospital, Moorenstrasse 5

Gerrit R.J. Melles, MD

40225 Düsseldorf, Germany

Netherlands Institute

for Innovative Ocular Surgery

Marta Torrabadella, MD

Laan Op Zuid 390

Paseig Vall d’Hebron 119

3071 AA Rotterdam, The Netherlands

08034 Barcelona, Spain

Gottfried O.H. Naumann, Prof. Dr.

Adam Watson, FRANZCO

Department of Ophthalmology

Corneoplastic Unit

University of Erlangen-Nürnberg

Queen Victoria Hospital NHS Trust

Schwabachanlage 6, 91054 Erlangen, Germany

East Grinstead, West Sussex, RH19 3DZ, UK

Autologous Serum Eyedrops for Ocular Surface Disorders

Gerd Geerling,Dirk Hartwig

Core Messages

∑ The viability and function of the corneal

ments that have the necessary laboratory

and conjunctival epithelium is supported

facilities and are able to admit the patient

by the antimicrobial, nourishing, mechani-

for the duration of the treatment

cal and optical properties of tears, since

∑ The production of serum eyedrops should

these contain factors which promote prolif-

be well documented and measures for ap-

eration, migration and differentiation

propriate quality control (i.e. serological and

of epithelial cells

microbiological tests) should be established

∑ A lack of these epitheliotrophic factors,

∑ The results of a considerable number of

e.g. in aqueous tear deficiency, can compro-

clinical cohort studies have reported benefi-

mise the ocular surface and result in serious

cial effects of its use for persistent epithelial

disorders such as persistent epithelial

defects, severe dry eyes and other indica-

defects

tions

∑ Pharmaceutical lubricants usually replace

∑ The use of serum eyedrops implies the risk

the aqueous component of tears alone

of transmission of infectious diseases, from

and may have little efficacy in improving

the donor to other individuals involved

surface disorders

in the production and application of the

∑ Serum has biomechanical and biochemical

product

properties similar to normal tears

∑ In addition contamination of the dropper

∑ In vitro cell culture experiments have

bottle and subsequent microbial keratitis

shown that the morphology, migration and

can occur

differentiation of ocular surface epithelia

∑ Such complications can be largely avoided

are better supported by serum than by

by testing the patient for HIV, syphilis and

pharmaceutical lubricants

hepatitis B and C prior to the blood dona-

∑ A number of protocols for the production

tion and testing every serum eyedrop batch

of serum eyedrops have been published,

for bacterial contamination. In addition,

which vary considerably and can influence

the serum therapy can be combined with

the biochemical properties of this autolo-

topical antibiotics

gous blood product

∑ Due to these risks and the lack of ran-

∑ Under EU legislation production of serum

domised controlled trials, the use of serum

eyedrops requires a license to produce

eyedrops should still be considered experi-

blood products by the appropriate national

mental and informed consent should be

body, which means that an extensive evalu-

obtained from every patient treated

ation and documentation process must be

∑ Alternative blood products with epithe-

successfully completed

liotrophic potential such as plasma, platelet

∑ Alternatively the use of serum eyedrops is

concentrates or serum albumin should be

allowed on an intention to treat basis if pro-

evaluated since they are readily available as

duction and application are all performed

quality controlled blood derivatives from

by the physician, i.e. in ophthalmic depart-

blood banks on a routine basis

Chapter 1

Autologous Serum Eyedrops for Ocular Surface Disorders

protein for retinol, supports goblet cell differen-

1.1

tiation, but also reduces the surface tension

Introduction

of tears. As part of inflammatory processes,

additional proteins, such as lactoferrin, serum-

In recent years the use of eyedrops produced

IgA and complement factors, are released into

from autologous serum has gained wide accept-

the tears and support opsonisation and phago-

ance for the treatment of ocular surface disor-

cytosis of microbes by macrophages and

ders intractable to conventional medical thera-

lymphocytes. Tears thus not only have a lubri-

py. Such conditions include persistent epithelial

cating and mechanical clearance function,

defects or severe dry eyes. Autologous serum

but also epitheliotrophic and antimicrobial

was first evaluated in 1984 by Fox et al. [9] in

properties.

search of an unpreserved lubricant, which was

If the carrier, i.e. the aqueous phase, or

not available from pharmaceutical providers at

the epitheliotrophic factors of the tear film

that time. However, it was Tsubota who repopu-

are diminished, the integrity of the surface

larised their use when he described the epithe-

epithelia can become disrupted and epithelial

liotrophic potential of serum for the ocular sur-

defects evolve, which may persist and progress.

face due to its content of growth factors and

Surgical attempts to rehabilitate the ocular sur-

vitamins [46].

face in severe dry eyes also fail frequently [2, 22]

unless sufficient substitute lubrication is pro-

vided.

1.1.1

Summary for the Clinician

The Rationale for Using Serum

in Ocular Surface Disorders

∑ The tear film has lubricant and nutrient

properties

The tear film supplies the ocular surface with

∑ In severe dry eye it is not only the increased

many nutrient and wound healing modulating

biomechanical stress, but also the lack of

factors such as fibronectin, vitamins or growth

epitheliotrophic factors that promotes

factors, which support and modulate prolifera-

damage of the ocular surface

tion, migration and differentiation of the con-

junctival and corneal epithelium. These epithe-

The ideal tear substitute would possess lubri-

liotrophic factors are predominantly released

cant and nutrient properties. However - with

into the aqueous component of the tear film by

few exceptions - currently available products

the main and accessory lacrimal glands as well

are optimised for their biomechanical proper-

as conjunctival vessels, while glucose, elec-

ties only [49, 51]. Vitamin A, EGF and fibro-

trolytes and amino acids are provided by the

nectin have been used in vitro and in vivo to

aqueous humour.

encourage epithelial wound healing, but due to

For example fibronectin, a disulphide glyco-

stability concerns and limited clinical success,

protein that influences cell adhesion and migra-

these single compounds have not become part

tion of the healing epithelium, predominantly

of clinical routine management

[15,

25,

29].

originates from plasma when conjunctival

Autologous blood offers a number of character-

blood vessels become more permeable during

istic advantages:

an inflammatory reaction and the lacrimal

1. It contains a large number of substances also

gland itself secretes vitamins, neuropeptides

present in tears, such as vitamin A, epithe-

and growth factors such as substance P and

liotrophic and neurotrophic growth factors,

epidermal growth factor (EGF) [36]. However,

immunoglobulins and fibronectin. Some of

proteins of the aqueous tear film not only act as

these factors are found in serum in higher

essential nutrients for the ocular surface epithe-

concentrations than in natural tears

lia, but also determine the biomechanical prop-

(Table 1.1) [20, 27, 29, 45].

erties of the tear lipid layer. Tear-lipocalin is an

2. Serum can be prepared as an autologous

example for this, which by acting as a transport

product and thus lacks antigenicity.

1.1

Introduction

while human serum albumin fractions can be

Table 1.1. Biochemical and biophysical properties

of undiluted serum and normal, unstimulated human

purchased from pharmaceutical companies.

tears (EGF, epidermal growth factor; FGF, fibroblast

While all of these blood derived products are

growth factor; IGF, insulin like growth factor; NGF,

potentially available as growth factor contain-

neurotrophic growth factor; PDGF, platelet derived

ing solutions for topical application to the ocu-

growth factor; SP, substance P; TGF, transforming

growth factor) [11, 15, 20, 48-52]

lar surface, serum has been predominantly used

in clinical studies.

Tears

Serum

The growth and migration promoting effect

of serum on cell cultures in general and on

7.4

corneal epithelial cells is well documented [13,

Osmolality

298±10

296

46]. In dose- and time-response experiments in

EGF (ng/ml)

0.2-3.0

0.5

vitro, we found that serum maintained the mor-

phology and supported proliferation of primary

TGF-b

2-10

6-33

human corneal epithelial cells better than un-

NGF (pg/ml)

468.3±317.4

54.0

preserved or preserved pharmaceutical tear

SP (pg/ml)

157.0±73.9

70.9±34.8

substitutes [13]. It is also known that serum

IGF-1 (ng/ml)

0.031±0.015

105

induces mucin-1 expression in immortalised,

PGDF

0-1.33

15.5

conjunctival epithelial cells as a sign of higher

differentiation [45] and that it increases tran-

Vitamin A (mg/ml)

0.02

scription of RNA for NGF as well as TGF-b re-

Albumin (mg/ml)

0.023±0.016

35-53

ceptors in human keratocytes [7]. The epithe-

Fibronectin (mg/ml)

205

liotrophic properties are - again according to in

Lactoferrin (ng/ml)

1,650±150

266

vitro tests - not reduced in patients suffering

Lysozyme (mg/ml)

2.07±0.24

0.001

from systemic autoimmune disease requiring

SIgA (mg/ml)

1,190±904

2,500

systemic immunosuppression [17].

Summary for the Clinician

∑ Plasma, platelet suspensions and albumin

are available as blood derived products

3. Eyedrops from serum can be produced with-

from blood banks

out preservatives and hence toxicity due to

∑ Serum is not a standard blood product,

additives is not an issue.

but can easily be produced on special

request and currently is by far the most

A wide range of quality controlled products can

often used product in clinical studies

be derived from full blood. These include not

∑ Serum contains many epitheliotrophic

only serum, but also, e.g. plasma, platelet rich

factors also present in tears and can be

suspensions or various protein fractions such as

prepared as an autologous unpreserved

albumin. Serum is the fluid component of full

tear substitute that offers both lubricant

blood that remains after clotting. It should not

and nutrient properties

be confused with plasma, which is obtained

when clotting is prevented by mixing a full

blood donation with an anticoagulant and re-

1.1.2

moving all corpuscular elements by centrifuga-

Legal Aspects

tion (see Sect. 1.5.3). Plasma thus does not con-

tain the significant amount of platelet derived

Autologous serum eyedrops are a blood prod-

growth factors such as EGF, PDGF and TGF-b,

uct. The distribution of pharmaceuticals is reg-

which are released into serum upon activation

ulated by governmental laws in most countries.

of the platelets during clotting. Platelet rich sus-

Although in the European Union the manufac-

pensions (platelet concentrates) are available as

turing and distribution of pharmaceuticals is

a standard blood product from blood banks,

regulated by the individual country, several

Chapter 1

Autologous Serum Eyedrops for Ocular Surface Disorders

directives have been issued (1965/65, 1975/139,

∑ Every patient should give their informed

1975/318) which have been implemented in the

consent before the production of autolo-

laws of each member state of the EU. Today

gous serum eyedrops is initiated

every pharmaceutical product requires a mar-

∑ Quality control measures must be imple-

keting authorisation to be issued by a compe-

mented for the production as well as the

tent authority of each state. Authorisation de-

application

pends on the proof of efficacy in clinical trials,

∑ Serum should only be applied under the

implementation of quality controls, reports of

supervision of the prescribing doctor

adverse effects, proof of expert knowledge and

other regulatory aspects. These criteria can

probably only be fulfilled by professional phar-

1.2

maceutical manufacturers.

Production and Application

An exemption from the need to obtain mar-

keting authorisation is granted if a physician

1.2.1

manufactures a specific medical product by

Important Parameters

himself or under his supervision with responsi-

of the Production Process

bility to treat his own patient on a named basis.

This product has to be prepared according to

Although the complex composition of full

the doctor’s specifications and autologous

blood will certainly vary between individuals, it

serum eyedrops can therefore be produced only

is also known that a number of production pa-

by the physician himself or by his staff. Howev-

rameters can significantly influence the bio-

er, it remains the physician’s responsibility that

chemical composition of blood derived prod-

manufacturing and application are performed

ucts. These critical steps in the production of

correctly. Since even stricter regulations may

serum eyedrops should therefore be standard-

especially exist for blood products in individual

ised.

states, every physician producing autologous

These include:

serum eyedrops needs to inform himself about

∑ Clotting phase: duration and temperature

specific national regulations.

∑ Centrifugation: centrifugal force

In the United States, producers of drugs and

and duration

medical devices have to be registered with the

∑ Dilution: dilution factor and diluent

Food and Drug Administration (FDA). Similar

∑ Storage: container, temperature, duration

to EU regulations, registration is not necessary

“for practitioners licensed by law to prescribe or

In the absence of any controlled clinical trial

administer drugs or devices and who manufac-

evaluating the impact of such differences in the

ture, prepare, propagate, compound, or process

production, in vitro models have been helpful in

drugs or devices solely for use in the course of

assessing a large number of protocol variations.

their professional practice”. Again, special regu-

The published clinical studies often fail to men-

lations on testing and approval of drugs by the

tion important parameters such as for how long

FDA or for using blood products need to be

the blood was allowed to clot before centrifuga-

evaluated by the practitioner.

tion. If the full blood sample is centrifuged

before the clotting process is completed, the

Summary for the Clinician

release of platelet derived factors is reduced. We

∑ Serum eyedrops are a blood product that

have established that the concentration of EGF,

can be produced on a named patient basis

HGF, and TGF-b are higher after a 2-h clotting

according to the doctor’s specifications

time when compared with paired full blood

∑ It remains the physician’s responsibility

samples that were allowed to clot only for 15 min

that manufacturing and application are

and this was associated with a trend towards

performed correctly

better corneal epithelial cell proliferation [24].

Thus we allow the full blood donation to clot for

at least 2 h at room temperature. However, a

1.2

Production and Application

centrations have been shown to induce apopto-

sis [5]. Also a higher g-force can result in a low-

er concentration of TGF-b₁. As TGF-b is able to

slow down epithelial wound healing, Tsubota

suggested diluting the serum 1:5 with saline,

which, however, reduces the concentration of

other growth factors, such as EGF, that are

proven to support proliferation of corneal ep-

ithelial cells. Combinations of 1,500 rpm - in an

average size centrifuge equal to about 300 g - to

4,000 g (ca. 5,000 rpm) for 5-20 min have been

used. A 15-min centrifugation at 3,000 g results

in good separation of serum and blood clot,

without inducing haemolysis [41].

It is obvious that the dilution of the obtained

serum sample to the final concentration in the

eyedrops determines the concentration of ep-

itheliotrophic factors. In clinical studies, 20 %,

33 %, 50 % or 100 % have been used. Since the

protocols for the production of serum eyedrops

also varied in other parameters, there is no clear

clinical evidence to favour any specific concen-

tration. However, in vitro experiments show

that cell proliferation is best supported at a 20 %

Fig. 1.1.

Demonstration of the influence of

the g-

concentration of serum. It was also shown that

force on the volume of serum obtained from 50 ml of

the type of diluent has an impact and that BSS

full blood centrifuged 2 h after donation: A at 3,000 g

rather than saline should be used [24].

for 15 min; B at 500 g for 5 min

In some indications, e.g. dry eye, autologous

serum eyedrops are applied for many months.

They are also usually produced without preser-

48-h period of storage at 4 °C, to allow trans-

vatives or stabilising additives to minimise the

portation of full blood donations from periph-

risk of drug induced toxicity. Since the produc-

eral ophthalmic departments to a centralised

tion is labour intensive, a large number of

production unit, seems an equally acceptable

aliquot samples is prepared from a single blood

procedure [53].

donation to keep the number of donation and

The volume retrieved from a given blood do-

processing efforts limited. To preserve the activ-

nation as well as its biochemical composition

ity of the biological substances thought to be

are also influenced by the centrifugation, which

beneficial for the ocular surface, the drops can

is determined by the centrifugal g-force and the

be refrigerated or stored frozen. The concentra-

time used to spin a sample. The g-force itself de-

tion of growth factors, vitamin A and fibro-

pends upon the revolutions of the rotor per

nectin in pure and diluted serum was found to

minute (rpm) as well as on the diameter of the

remain stable for at least 3 months if stored at

rotor. Thus g-force and not rpm is the parame-

-20 °C and for 1 month if stored at 4 °C. How-

ter that should be stated in a protocol. The

ever, it is known that many protein concentra-

g-force in the studies published so far - if men-

tions in tears are reduced if stored for several

tioned at all - probably varies by at least 1 log.

weeks at 4 °C. While in developed countries ac-

A higher g-force not only helps to yield a larger

cess to freezers is rarely a problem, it seems

volume of serum from a full blood sample

preferable to store unused daily dosage vials of

(Fig. 1.1), but also reduces membranous platelet

serum frozen [45, 38]. From this stock one con-

remnants in the supernatant, which in high con-

tainer is then removed every morning and kept

Chapter 1

Autologous Serum Eyedrops for Ocular Surface Disorders

refrigerated at 4 °C until it is discarded at the

HIV serology (HbsAg; antibodies to HCV, HIV-

end of the day. Alternatively dilution of the

I/-II, HIV-NAT, syphilis; HCV-NAT) before

serum with chloramphenicol 0.5 %, which has

blood is donated for the production of eye-

few toxic side effects, has also been advocated to

drops. A positive serology excludes the patient

allow the use of dropper bottles for up to 1 week.

from the donation of autologous blood for

serum eyedrop production. Prior to venisec-

Summary for the Clinician

tion, the patient must be informed in writing

∑ The list of protocol variations for the pro-

about the planned therapy, its experimental na-

duction of autologous serum eyedrops is

ture, the risks involved (e.g. bacterial contami-

long

nation) and alternative methods of treatment.

∑ The biochemical composition of serum

The patient’s consent should be obtained and

depends on the time and conditions of

kept with the notes.

clotting, the centrifugation, dilution and

Venipuncture is performed at the antecubital

storage

fossa under aseptic conditions. Depending on

∑ No controlled clinical trial has determined

the expected duration of treatment, 100-200 ml

so far which of the protocols published

of whole blood is collected into sterile contain-

offers the best epitheliotrophic support

ers. For larger volumes a sterile blood pack

∑ In the absence of such trials, the production

without anticoagulant can be used to collect up

protocol has been optimised in vitro

to 470 ml. A 100-ml donation of whole blood

∑ The stock of serum eyedrops can be stored

will yield 30-35ml of serum, which diluted to

frozen for several months to preserve

20 % is sufficient for at least 3 months of serum

the biologically active components of the

eyedrops 8 times daily. Larger volumes are rec-

product

ommended in patients who require long-term

treatment, in order to minimise labour intensive

production. The containers are left standing

1.2.2

upright for 2 h at room temperature to ensure

Current Standard Operating Procedures

complete clotting before they are centrifuged at

Used at the University of Lübeck

3,000 g for 15 min. The supernatant serum is

removed under sterile conditions in a laminar

Following the principles of Good Manufactur-

air flow hood with sterile

50-ml disposable

ing Practice and based on extensive evaluation

syringes. The volume retrieved is determined

in vitro, the following standard operating pro-

and diluted 1:5 with sterile BSS. Gentle shaking

cedures are currently used at the University of

ensures homogenisation before portions of 2 ml

Lübeck (Fig. 1.2) [24, 19].

are aliquoted through a 0.2-mm filter into sterile

Patients are assessed for their suitability to

dropper bottles. The effect of filtration has not

donate according to the guidelines of the Bun-

been evaluated, but Fox recommends filtration

desärztekammer and Paul-Ehrlich Institute for

to remove fibrin strands, suspected to reduce

blood donation and use of blood products. This

the effect of serum eyedrops. The bottles are

requires them to be in reasonably good health,

sealed and labelled with the name, date of birth

with no significant cardiovascular or cere-

of the patient, the date of production and the in-

brovascular disease, and free of bacterial infec-

struction “Autologous blood serum for topical

tion. Anaemia (Hb<11 g/dl) is a relative con-

use in the eye. To be stored frozen and used

traindication. If only a small amount of blood

within 3 months after date of production. To be

(50-100 ml) is taken, mild anaemia or circulato-

discarded 24 hours after opening.” Two milli-

ry disorders need not be considered contraindi-

litres of the solution is - as required by the

cations. To minimise the danger of bacterial

European Pharmacopoeia Addendum 2000 -

contamination, no blood should be taken from

sent for microbiological evaluation.

patients suffering from suspected septicaemia.

The product is available approximately 6 h

To exclude transmission of infection, patients

after venesection, but is only dispatched once

must be tested for hepatitis-B/-C, syphilis and

negative serology and microbiology of donor

1.2

Production and Application

Fig. 1.2. Standard manufacturing protocol for the preparation, storage and use of serum eyedrops. Parame-

ters which influence the biochemical character of the product are also shown

and product are confirmed. Usually the drops

and thus should be acceptable for the rare occa-

are applied 8 times daily. A new bottle is opened

sion where the ocular surface disease is so se-

everyday. It is recommended to be stored at

vere that the use of topical autologous serum is

+4 °C and to be discarded after 16 h of use with

justified [14].

regular household waste. The remaining bottles

Summary for the Clinician

are stored frozen (ideally at -20 °C) for up to

3 months. If the domestic freezer has no ther-

∑ Every physician producing or prescribing

mometer, it is recommended to place one inside

autologous serum eyedrops should inform

and control the temperature when taking a new

himself about specific national regulations

vial out every day. If the temperature cannot be

∑ Any production protocol should follow the

adjusted to about -20 °C, the dispensing doctor

principles of Good Manufacturing Practice

may consider recommendation of shorter stor-

∑ The dropper bottles must be carefully

age episodes.

labelled with the patient’s details and

The costs of production - i.e. labour and

instructions for storage and use

consumables alone - for a day’s dosage of serum

∑ If produced without preservatives the drops

eyedrops following this protocol are well below

should be kept frozen at -20 °C until the day

5 j. This is the approximate equivalent of one

of use

bottle of preserved pharmaceutical lubricant

Chapter 1

Autologous Serum Eyedrops for Ocular Surface Disorders

cluding the dates of application and any un-

1.2.3

wanted effect) should be recorded. From this it

Quality Control

becomes obvious that strict guidelines for good

manufacturing, quality control and documenta-

In order to guarantee quality, control measures

tion must be established and maintained prior

should be initiated and if strictly interpreted

and throughout the therapeutic use of auto-

serum eyedrops should be produced only by

logous serum eyedrops. All steps of the produc-

personnel supervised by the doctor directly in

tion should be documented on a form which -

charge of the patient treated. Bacterial contam-

together with the patient’s consent - is kept with

ination of the product during the production as

the patient’s notes.

well as from the application of serum eyedrops

Summary for the Clinician

is a potential risk. Sterile manufacturing condi-

tions, beginning with thorough skin disinfec-

∑ A written protocol of the standard operat-

tion, are of the utmost importance. It is prefer-

ing procedures should be established

able that further processing is performed in a

∑ All production steps must be documented

closed system. To minimise the risk of infection

on a SOP form

to third parties

(e.g. production or nursing

∑ Serum eyedrops should only be produced

staff), it is strongly recommended that the

and released once the negative serology of

donor is tested for HIV, HBV, HCV and syphilis

the donor for hepatitis, syphilis and HIV

before donation of larger amounts of blood are

and the negative microbiology of the prod-

collected for the production process itself. For

uct have been confirmed

quality control purposes bacterial contamina-

tion resulting from the production process

needs to be ruled out by a microbiological

1.3

examination of the product prior to initiation of

Clinical Results

the clinical application and a control system

must be implemented to ensure that the product

Serum eyedrops have predominantly been used

is only used when the microbiological and sero-

for persistent epithelial defects and severe dry

logical tests are clear.

eye, but also as a supportive measure in ocular

Prior to venipuncture and application, the

surface reconstruction and for a number of

identity of the patient must be confirmed. The

other indications.

packaging and dropper bottles must be clearly

labelled with:

∑ The patient’s name and date of birth

1.3.1

∑ The name and address of the manufacturer

Persistent Epithelial Defects

∑ The date of manufacture and the date of ex-

piry

A persistent epithelial defect (PED) is defined as

∑ The instructions on how to store and use the

a defect of the corneal epithelium that - in the

drops

absence of microbial keratitis - fails to heal

∑ A comment that the material is an autolo-

within the expected time course (e.g. 2 weeks)

gous blood product, which is solely for appli-

despite topical lubricants [26, 50]. A PED can

cation with the named patient

occur as a result of many different pathologies,

including rheumatoid arthritis, neurotrophic

To minimise the variability of the product and

keratopathy or dry eye [8]. “Success” of treat-

to maximise the safety of its use, a written

ment is best defined as percentage of defects

version of the standard operating procedures

healed in a given time or as total time to com-

(SOP) should be established. Conscientious

plete epithelial wound closure.

documentation is indispensable for good med-

ical and manufacturing practice. Each step rele-

vant to manufacture as well as application (in-

1.3

Clinical Results

Table 1.2. Clinical studies using serum eyedrops to treat persistent epithelial defects. The production param-

eters and results are given. Success is defined as percentage of eyes/patients with complete epithelialisation.

Note that the scale used to measure these changes as well as the baseline level varied between the studies (NA,

not applicable; NR, not reported; rpm, revelations per minute)

Author

Con-

Diluent

Centri-

Dura-

Clott-

Frequency

Eyes

Success

centra-

fugation

tion

ing

of appli-

objec-

tion

(g force)

time

cation

(patients)

tive

Alvarado

20 %

0.9 % NaCl

5000 RPM

10 min.

17 (14)

83 %

De Souza

100 %

Hourly

70 (63)

81 %

Garcia

20 %

0.9 % NaCl

5000 RPM

10 min.

10×

11 (11)

55 %

Matsumoto

20 %

0.9 % NaCl

3000 RPM

10 min.

5-10×

14 (11)

100 %

Poon

50-100 %

0.5 %

4000 RPM

10 min.

2 h

8×

15 (13)

60 %

chloram-

(2200 G)

phenicol

Tsubota

20 %

0.9 % NaCl

1500 RPM

5 min.

6-10×

16 (15)

63 %

Young

20 %

0.9 % NaCl

1500 RPM

5 min.

6-14×

10 (10)

75 %

1.3.1.1

epithelial defects with undiluted serum hourly

Currently Available Published Data

in addition to routine medication, 45 of which

had occurred early after penetrating kerato-

In five prospective case series,

20 % serum

plasty

[8] and had persisted for a mean of

diluted in 0.9 % saline has been used 5-14 times

15±17 days. Eighty-one percent of these defects

daily for this indication

(Table 1.2). In

1999

with a relative short history healed within

Tsubota was the first to report a series of 16 eyes

14±12 days.

(15 patients) in whom the PEDs had persisted

Healing generally starts within 2 weeks after

despite medical treatment with lubricants

initiation of the serum therapy [33]. So far no

or bandage contact lenses for a mean of

study has been able to show a correlation be-

7.2±9.4 months. Ten out of these

16 defects

tween size or localisation of the defect with suc-

healed completely within 4 weeks after initia-

cess or failure, but the older and deeper stromal

tion of therapy [11, 46]. Garcia-Jimenez reported

defects tended to heal less successfully. Also,

complete epithelial wound healing in 6 of 11 eyes

when serum eyedrops are changed back to

with persistent epithelial defects with healing

pharmaceutical lubricants the epithelial defects

beginning within 3-4weeks of treatment with

may recur, as happened in 6 out of the 9 eyes in

serum eyedrops [1, 52]. In a group of 9 patients

Poon’s and 9 out of 70 eyes in De Souza’s group.

with predominantly diabetic or postherpetic

These figures are difficult to compare since

neurotrophic keratitis, all 12 epithelial defects

none of the studies was placebo controlled and

healed within 15.8±7.9 days and this was associ-

the study population seems to differ significant-

ated in 9 eyes with an improvement of corneal

ly in terms of underlying pathogenesis and

sensitivity [27] (Fig. 1.3).

duration of the PED.

A different concentration of serum was used

Summary for the Clinician

in two other studies. Poon et al. substituted

unpreserved pharmaceutical lubricants with

∑ Pathogenic factors that can be avoided or

50-100 % serum eyedrops and observed closure

treated, such as toxicity due to preserved

of a PED with a mean duration of 7.5±5.8

eyedrops, steroids or active herpetic kerati-

(1-24) weeks in 9 of 15 eyes after 3.6±2.5 weeks

tis, should be ruled out before a PED is

(3 days-8 weeks) [33]. De Souza et al. treated 70

treated with serum eyedrops

Chapter 1

Autologous Serum Eyedrops for Ocular Surface Disorders

Fig. 1.3. Epithelial defect persisting unaltered for 2 weeks in the left eye of a female patient with severe aque-

ous tear deficiency due to secondary Sjögren’s syndrome before (A, B) and 1 week after (C, D) treatment with

20 % autologous serum eyedrops 8 times daily. The defect started to heal within 2 days

∑ No signs of microbial keratitis should be

present if serum application is considered

1.3.2

for an epithelial defect, since the effect of

Dry Eye

serum in this situation is unknown and

may support bacterial growth

Dry eye is a group of disorders, of diverse

∑ Twenty percent serum eyedrops are applied

pathogenesis, that share as common manifesta-

approximately every 2 h until the defect is

tions signs and symptoms due to the interaction

healed

of both an abnormal tear film and an abnormal

∑ PEDs begin to heal generally within 2 weeks

ocular surface.It is subdivided into aqueous de-

after initiation of serum eyedrops

ficient and evaporative, i.e. lipid or mucin defi-

∑ Older and deeper stromal defects tend to

cient dry eyes. Although it is believed to be one

heal less successfully

of the most common ocular problems in the

∑ If 20 % serum fails, a higher concentration

Western world with an incidence of symptoms

of serum may help to achieve epithelialisa-

of dry eye in up to 14.6 %, ocular surface changes

tion

are observed clinically in only 0.5 %. Severe

∑ When serum eyedrops are changed back to

aqueous tear deficiency, however, can lead to

pharmaceutical lubricants the epithelial

blindness and serum eyedrops have been used

defect may recur

in this situation.

1.3

Clinical Results

In the dry eye “success of treatment” is more

Two studies have reported the use of higher

difficult to define than in PEDs, and this can be

concentrations of serum. Poon et al. found an

done either as subjective or objective improve-

improvement of subjective and objective crite-

ment compared to baseline.“Subjective” success

ria of severe dry eyes (Schirmer test <5 mm) in

is determined as reduction of a score of symp-

only three out of eight eyes receiving

50 %

toms in a questionnaire of variable length.

serum but all of three eyes receiving 100 %

“Objective” success is either determined by re-

serum [33]. Noble et al. compared the efficacy of

duction of fluorescein or rose bengal positive

3 months of autologous serum 50 % diluted in

staining of the ocular surface or improvement

0.9 % saline in a prospective clinical crossover

of histologic parameters in impression cytol-

trial against the previously used commercial lu-

ogy, although this is usually scored more or less

bricant and reported that 10 out 16 patients had

according to the subjective impression of an

improved symptoms [31], and that there were

examiner.

impression cytological findings in 6, no change

in 10 and improvement in 9 of 25 treated eyes.

1.3.2.1

The efficacy seems to be dose dependent

Currently Available Published Data

since 94 % of patients receiving eight applica-

tions daily reported reduced symptoms com-

Following the initial report of Fox in 1984, it

pared to only 58 % of those receiving four drops

took 15 years until Tsubota in 1999 and subse-

[39]. Overall the efficacy of serum eyedrops in

quently a number of studies reported on the use

dry eyes varied between 30 % and 100 % for

of serum eyedrops for dry eyes (Table 1.3). Fox

symptomatic relief, between 39 % and 61 % for

treated 30 eyes of 15 patients with 50 % serum in

reduction of fluorescein and between 33 % and

0.9 % NaCl and found that signs and symptoms

68 % for rose bengal positive staining. However,

improved in all of the patients, until this med-

the variation in study population, production

ication was replaced by 0.5 % serum or pure

and treatment protocol are again significant. In

diluent. Tsubota focussed on dry eyes due to

some studies, serum was used as an additive

Sjögren’s syndrome. When treated with 20 %

rather than a substitute for lubricants and in

serum 6-10 times daily for 4 weeks symptoms

others therapeutic contact lenses or punctal oc-

improved by only 34 %; however, fluorescein

clusion were applied in addition to the serum

and rose bengal staining decreased by 55 % and

eyedrop therapy. Comparison of the published

68 % of baseline, while tear break-up time re-

data is therefore difficult and it has to be con-

mained unchanged [45]. Two groups of authors

cluded that no definite evidence supporting the

described similar findings in patients with dry

use of serum eyedrops in dry eyes is available so

eye due to graft-versus-host disease [32, 34] with

far.

symptoms improving within days, but punctate

Summary for the Clinician

epithelial staining improving only after months.

Ogawa also reported that - although the aque-

∑ Serum eyedrops should be reserved for the

ous deficiency in his patients was less severe

most severe cases of dry eye

(£10 mm, Schirmer test) - in 50 % symptoms

∑ Punctal occlusion should be performed

recurred while the patient continued to apply

first

serum eyedrops and 43 % required additional

∑ Symptoms usually improve within days,

punctal occlusion. In a placebo-controlled pro-

but punctate epithelial staining may

spective study of severe dry eyes with a mean

decrease only after months of treatment

Schirmer test score of less than 1 mm, 20 %

∑ The use of serum in dry eyes is not evidence

serum 6 times daily was not found to be signif-

based. Using an optimised protocol for

icantly more effective in improving symptoms

the production of serum eyedrops, a

and signs than 0.9 % saline, which had been

randomised controlled trial should be

used as diluent [40], although a trend towards

performed

reduced fluorescein and rose bengal staining

was observed after 2 months of treatment.

Table 1.3. Clinical studies using serum eyedrops to treat severe dry eyes. Success was defined either as number/percentage of all eyes/patients with improved/

reduced mean baseline of objective [fluorescein (Fl) or rose bengal (RB) positive epitheliopathy or impression cytology (IPC)] or subjective (symptoms) scores.

Note that the scale used to measure these changes as well as the baseline level varied between the studies (NR, not reported; NS, not significant; rpm, revelations per

minute; replacement, frequency equivalent to previously applied pharmaceutical tear substitute)

Author

Concen-

Diluent

Centri-

Duration

Clotting

Frequency

Eyes

Success objective

Success subjective

tration

fugation

time

of application

(patients)

Fox

33 %

0.9 % NaCl

500 g

10 min

2-hourly

30 (15)

RB 41 %

51 %; 100 %

Noble

50 %

0.9 % NaCl

48-72 h

Replacement

32 (16)

IPC 36 %

63 %

Ogawa

20 %

0.9 % NaCl

1,500 rpm

5 min

¥10

28 (14)

Fl: 61 %; RB: 40 %

30 %

Poon

50-100 %

0.5 % chlor-

4,000 rpm

10 min

¥8

11 (9)

Fl: 55 %; RB: 45 %

55 %

amphenicol

(2,200 g)

Rocha

33 %

0.9 % NaCl

500 g

10 min

Hourly

4 (2)

100 %

Takamura

20 %

0.9 % NaCl

3,000 rpm

10 min

¥4-8

NR (26)

“Improved”

77 %

Tananuvat

20 %

0.9 % NaCl

4,200 rpm

15 min

¥6

12 (12)

Fl: 39 %; RB: 33 %; IPC 44 %

36 % (NS)

Tsubota

20 %

NaCl

1,500 rpm

5 min

¥6-10

24 (12)

Fl: 55 %; RB: 68 %

34 %

1.3

Clinical Results

traumatic RES with a mean of 2.2 recurrences/

1.3.3

month in a prospective cohort study with

Other Indications

unspecified, unpreserved lubricants and 20 %

serum eyedrops TDS for 3 months in a taper-

Other indications which reportedly have been

ed fashion. During a mean follow-up of

treated with autologous serum include recur-

9.4±3.7 months the recurrence rate decreased to

rent erosion syndrome, superior limbic kerato-

0.028/month. No information is given whether

conjunctivitis and as adjunctive therapy in sur-

previous treatment modalities were suspended

gical ocular surface reconstruction (Tables 1.4,

for the time of the serum application. Unfortu-

1.5).

nately the authors also do not state the duration

of the history of RES, which may have been

1.3.3.1

rather short. Given the self-healing nature of the

Adjunctive Use

post-traumatic variant of the condition, these

in Ocular Surface Reconstruction

data have to be taken with caution [4].

Absolute aqueous deficiency prevents a suc-

1.3.3.3

cessful surgical ocular surface reconstruction.

Superior Limbic Keratoconjunctivitis

Tsubota used 20 % autologous serum as adjunc-

tive treatment in a prospective cohort study on

This is a rare, chronic, inflammatory disease

14 eyes of 11 patients, in which due to Stevens-

thought to result from a localised reduction of

Johnson syndrome or ocular cicatricial pem-

goblet cells and tear film deficiency at the 12:00

phigoid, the Schirmer test result with nasal

limbus with subsequently reduced wettability

stimulation was 0 mm. Surface reconstruction

and positive rose bengal staining of the corneal

included a limbal stem cell graft, amniotic

and conjunctival epithelium. Goto used 20 %

membrane and/or penetrating keratoplasty.

serum eyedrops as additional therapy 10 times

Within the short follow-up of 20 weeks, a stable

daily for bilateral superior limbic keratocon-

corneal epithelium was observed in 12 of the 14

junctivitis (SLK) in a prospective cohort study

eyes [44] and these findings were confirmed by

on 22 eyes. Within 4 weeks symptoms were im-

Lagnado et al. [23]. Poon used 50 % of serum in

proved in 9 of 11 and epitheliopathy in all pa-

two eyes undergoing keratoplasty for PEDs.

tients. Also, tear break-up time increased signif-

Again a stable epithelium resulted. However,

icantly and conjunctival squamous metaplasia

epitheliopathy recurred when the serum treat-

was reduced. When the serum application was

ment was discontinued in these patients. In an-

discontinued, discomfort recurred [16].

other study, Tsubota also observed that in four

Summary for the Clinician

children (mean age 9 years) with severe OSD

and absolute dry eye due to Stevens-Johnson

∑ Autologous serum can be used in other

syndrome surface reconstruction failed despite

ocular surface disorders such as superior

the use of autologous serum eyedrops [47].

limbic keratoconjunctivitis or if ocular

surface disease - e.g. due to dry eye or PED

1.3.3.2

- becomes so severe that a surgical inter-

Recurrent Erosion Syndrome

vention is required

∑ However, the surface disease is likely to

Insufficient adhesion of the basal epithelial lay-

recur if the serum treatment is stopped

ers to the underlying basement membrane is

observed following trauma or in corneal base-

ment membrane dystrophy. This can lead to

recurrent erosion syndrome (RES), which in-

cludes repeated episodes of irritation, pain,

epiphora and conjunctival hyperaemia. Del

Castillo treated 11 patients with unilateral post-

Table 1.4. Clinical studies using serum eyedrops for ocular surface reconstruction. Success was defined as number of all eyes with a stable postoperative ocular

surface score (h, hours; NR, not reported; rpm, revelations per minute)

Author

Concen-

Diluent

Centri-

Duration

Clotting

Frequency

Eyes

Success objective

tration

fugation

time

of application

(patients)

Lagnado

20 %

0.9 %

4,500 rpm

15 min

10-12 h

1-2 hourly

14 (14)

100 %

Poon

50-100 %

0.5 % chloramphenicol

4,000 rpm (2,200 g)

10 min

¥8

2 (2)

100 %

Tsubota

20 %

0.9 % NaCl

1,500 rpm

5 min

1/4 hourly

14 (11)

86 %

Table 1.5. Clinical studies using serum eyedrops to treat other indications. Success is defined either as number/percentage of eyes/patients with improved/reduced

mean baseline of objective [fluorescein (Fl) or rose bengal (RB) positive epitheliopathy or impression cytology (IPC)] or subjective (symptoms) score (NR, not

reported; RoR, rate of recurrence)

Author

Indication

Concen-

Diluent

Centri-

Duration

Clotting

Frequency

Eyes

Success

tration

fugation

time

of application

(patients)

objective

subjective

Del Castillo

Superior limbic

20 %

0.9 % NaCl

1,500 rpm

5 min

¥3

11 (11)

RoR: 99 %

keratoconjunctivitis

Goto

Recurrent corneal

20 %

0.9 % NaCl

1,500 rpm

5 min

¥10

22 (11)

Fl: 88 %;

21 %;

erosion syndrome

BR: 91 %;

82 %

IPC 100 %

1.4

Complications

was either an epithelial defect or ocular surface

1.4

reconstruction surgery, all patients had received

Complications

topical antibiotics at the same time. This is like-

ly to have reduced the rate of contamination of

Potential unwanted effects of the use of autolo-

the serum containers and prevent cases of

gous serum as eyedrops include worsening of

microbial keratitis.

the initial problem, infection, immunological

The incidence of dropper bottle contamina-

reactions and contact lens deposits. Most authors

tion and the risk of microbial keratitis is likely

mention no complications at all, but in five

to increase when the drops are used in a domes-

patients with dry eyes, discomfort or epithelio-

tic setting by the patients themselves and if no

pathy increased and eyelid eczema was reported

topical antibiotic is applied, e.g. in patients with

in two cases [34, 40].

other indications than epithelial defects. Even if

0.5 % chloramphenicol was added as a preserva-

1.4.1.1

tive to the dropper bottles, microbial keratitis

Risk of Infection

evolved in 3 out of 13 eyes with PEDs treated

with 50 % or 100 % serum [33]. Since laboratory

An infection in the context of topical serum ap-

evidence suggests that dilution with an antibiot-

plication can occur either locally or systemical-

ic may reduce the epitheliotrophic capacity of

ly. The risk for systemic transmission of an in-

serum eyedrops and since ocular surface dis-

fectious disease arises only if serum of a donor

ease often requires long-term treatment, hospi-

affected by a systemic infection, such as HIV,

talisation of patients for this purpose is not a

hepatitis or syphilis, is applied to a person oth-

suitable option and storage of the serum prod-

er than the donor. This can occur during the

uct in day dosage vials seems preferable.

production or application of serum eyedrops,

and a systemic as well as a topical route of entry

1.4.1.2

of the infectious agent is possible, since trans-

Immunological Complications

mission of HIV by a single serum droplet into

one eye has been reported at least in one case

In the first report by Fox in 1984 the authors

[6].Although the risk is small, the potentially fa-

mentioned [9] that some users of serum - not

tal consequences make the tight quality control

Fox himself - had encountered scleral vasculitis

as described under Sect. 1.2.3 mandatory.

and melting in patients with rheumatoid arthri-

Although it is known that serum has antimi-

tis, although the indication for the use of serum

crobial properties, this has not been quantified

in these patients remains unknown. McDonnell

for diluted, cryopreserved serum so far. Sauer et

reported one case of an immune complex depo-

al. observed that contamination of dropper bot-

sition after hourly application of 100 % serum.

tles with Staphylococcus epidermidis occurred

Poon observed the onset of one peripheral

in 3 out of 40 bottles on the 7th day of use of

corneal infiltrate and ulceration within

24h

undiluted serum stored refrigerated and ap-

after initiation of serum drops [28, 33] and

plied from week dosage containers by trained

hypothesised that this could have been a result

personnel in a hospital setting [35]. Lagnado et

of circulating antibodies which must also be

al., however, observed contamination of 18 % of

present in serum eyedrops and could have re-

containers at the end of the first day of applica-

acted with corneal antigens with a subsequent

tion, and 43 % of inpatients treated with 20 %

inflammatory response.

serum diluted with sterile saline (0.9 %) had at

one stage received serum from a day usage

1.4.1.3

dropper bottle that at the end of the day was

Contact Lens Contamination

found to be contaminated. Again most of these

contaminations were Staphylococcus epider-

We have used serum eyedrops in combination

midis, but one case of S. aureus was also report-

with a soft, class IV hydrogel contact lens con-

ed [23]. Since the indication for the use of serum

taining 45 % ocufilcon D and 55 % water (Bio-

Chapter 1

Autologous Serum Eyedrops for Ocular Surface Disorders

∑ Frequent monitoring of patients is recom-

mended since in rare cases serious inflam-

matory complications can result from the

use of autologous serum drops

∑ Serum eyedrops can be combined with

other forms of wound healing supporting

therapy, without any serious adverse effect

being reported so far

∑ If a contact lens is applied, a material

with little tendency to accumulate surface

deposits is recommended

Fig. 1.4. Hydrogel bandage contact lens (45 % ocufil-

con D, 55 % water) with contaminations in the right

eye of an 88-year-old female patient after 78 days of

1.5

treatment with 20 % serum 8 times daily. The patient

Alternative Blood Products

previously had developed a recurrent corneal epithe-

for the Treatment of Ocular Surface Disease

lial defect due to neurotrophic keratopathy secondary

to herpes zoster keratitis and severe aqueous tear de-

Serum has to be prepared from an autologous

ficiency due to secondary Sjögren’s syndrome which

blood donation for each patient individually.

persisted, perforated and recurred despite repeated

multilayer amniotic membrane grafts

This is time and labour intensive and in many

countries no standard operating protocol has

yet been evaluated or approved by the licensing

medics 55) in six eyes (six patients) with persist-

authority. Other blood derived extracts, such as

ent or recurrent epithelial defects.A contact lens

albumin, plasma or platelet concentrates, are

was applied if an epithelial defect recurred or

readily available from pharmaceutical compa-

progressed while the eye was being treated with

nies or blood banks. They are quality controlled

autologous serum. In five eyes the contact lens

and might therefore be considered for treat-

was applied immediately after transplantation

ment of ocular surface disorders.

of an amniotic membrane. Three lenses in two

eyes showed substantial numbers of large de-

posits on the anterior surface of the lens after

1.5.1

application of serum drops 8 times a day for

Umbilical Chord Serum

18-78 days (Fig. 1.4). In addition all eyes re-

ceived unpreserved ofloxacin (3 mg/ml) 4 times

Recently umbilical chord serum has been pre-

daily topically. All defects healed without signs

pared like autologous serum (5 min centrifuga-

of microbial keratitis or conjunctival hyper-

tion at 1,500 rpm), diluted to a 20 % concentra-

aemia. However, since protein deposition may

tion in 0.9 % saline and used as an alternative

induce ocular surface inflammation, it is recom-

treatment for promoting corneal epithelial

mended to use silicone hydrogel lenses, if a

wound healing. In a prospective randomised

combination with serum drops is necessary, be-

controlled clinical trial on 60 eyes, this led to a

cause this type of lens is less prone to accumu-

higher rate of healing of persistent epithelial de-

late protein on the surface [21, 42, 54].

fects than autologous serum. However, the de-

fects treated with autologous serum healed

Summary for the Clinician

faster than those treated with umbilical cord

∑ Serum eyedrops are generally well tolerated

serum. This product is not autologous but allo-

with little or no side effects

geneic and hence not only immunological prob-

∑ However, all possible measures should be

lems but also a higher risk of infection for the

taken to exclude transmission of systemic

recipient may be expected. In many countries it

disease during the production or applica-

is not supplied by a centralised blood service

tion of serum eyedrops

and thus it is more difficult to obtain. All con-

1.5

Alternative Blood Products for the Treatment of Ocular Surface Disease

cerns regarding quality control and microbio-

further use. We have termed this product

logical testing are applicable [50].

“platelet releasate” (PR). This blood product

contains large amounts of EGF and other

growth supporting mediators, but little of the

1.5.2

extracorpuscular factors such as fibronectin or

Albumin

vitamins.

Both products have so far only been tested in

One of the most abundant proteins in tears is

vitro, but from these experiments it is clear that

albumin is one of the most abundant (Table 1.1).

plasma is not suitable to support proliferation

It often acts as a carrier for other factors, such as

and migration of epithelial cells. Although

hormones, including steroids, although no

platelet releasate also does not support migra-

physiological role for its presence in tears has

tion, it has a substantially stronger proliferative

been described to date.Albumin is also used in

effect than serum and thus may be promising

medicine for treating severe albumin deficien-

for the treatment of ocular surface disorders.

cy, e.g. due to protein loss in extensive skin

However, this still has to be evaluated in a clini-

burns or liver dysfunction. Tsubota found in

cal trial [18, 19].

an animal experiment that the topical applica-

Summary for the Clinician

tion of albumin reduces enzymes involved in

apoptosis and improves epithelial cell viability

∑

Alternative blood products are routinely

and re-epithelialisation. When a 5 % solution

produced and quality controlled by blood

of recombinant albumin was applied 6 times

banks. They are currently under investiga-

daily, mean fluorescein and rose bengal, as well

tion

as break-up time and symptom scores, im-

∑

Umbilical chord serum was found to be

proved significantly from baseline, but no com-

more effective than autologous serum to

ments are made as to which solvent was used

heal epithelial defects, but it is more diffi-

and whether any non-responders were found

cult to obtain and supply is limited.Since

with this treatment. Although no complications

evidence about this allogeneic treatment

were observed - as with all blood derived prod-

modality is even more scarce, it should cur-

ucts - a minute risk of transmission of viral or

rently not be used to substitute autologous

prion mediated disease cannot be ruled out if

serum, unless the latter has failed to estab-

the albumin preparation was derived from

lish a stable ocular surface

blood [37].

∑

Albumin may be one of the components of

serum that support epithelial wound heal-

ing and can be used as a single compound

1.5.3

product without the need for an autologous

Plasma and Platelets

blood donation. It has been reported to im-

prove findings but not symptoms of ocular

Plasma is the cell free supernatant after cen-

surface disease in severe dry eyes

trifugation of full blood mixed with an antico-

∑

Platelet releasate, but not plasma, may be

agulant. Plasma therefore contains only small

suitable as an additional treatment modali-

amounts of the growth factors present in

ty for ocular surface disease, but no clinical

platelets since these are - due to the anticoagu-

data are available yet

lant - not activated by blood clotting.

∑

Similar if not enforced legal guidelines and

Platelets are a major source of growth factors

quality controls need to be applied to these

in serum. They can be obtained by means of

alternative blood products, since the risk

apheresis and stimulated with thrombin to re-

of transmission of infection is due to their

lease their content. Following centrifugation the

allogeneic nature being theoretically

clear supernatant, which is free of any cellular

increased

remnants, can be resuspended with a buffer to a

concentration of choice and stored frozen for

Chapter 1

Autologous Serum Eyedrops for Ocular Surface Disorders

13.

Geerling G, Daniels JT, Dart JK, Cree IA, Khaw PT

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Matsumoto Y, Dogru M, Goto E, Ohashi Y, Kojima

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cation in the treatment of neurotrophic keratopa-

elvie P, McCarty DJ et al. (2001) Controlled study

thy. Ophthalmology 111(6):1115-1120

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28.

McDonnell PJ, Schanzlin DJ, Rao NA (1988) Im-

tients. Cornea 20(8):802-806

munoglobulin deposition in the cornea after ap-

41.

Thomas L (1998) Labor und Diagnose, 5th edn.

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TH-Books, Frankfurt/Main, p 1494

106:1423-1425

42.

Tighe BJ, Jones L, Evans K, Franklin V (1998) Pa-

29.

Nelson JD, Gordon JF (1992) Topical fibronectin

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in contact lens deposition processes. Adv Exp

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Am J Ophthalmol 114(4):441-447

43.

Torsteinsdóttir L, Hakansson L, Hällgren R, Gud-

30.

Ng V, Cho P, Mak S, Lee A (2000) Variability of

björnsson B, Arvidson NG, Venge P (1999) Serum

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Rheumatology 38:1249-1254

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Noble BA, Loh RS, MacLennan S, Pesudovs K,

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Tsubota K, Satake Y, Ohyama M, Toda I, Takano Y,

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45.

Tsubota K, Goto E, Fujita H, Ono M, Inouc H, Saito

32.

Ogawa Y, Okamoto S, Mori T,Yamada M, Mashima

I et al. (1999) Treatment of dry eye by autologous

Y, Watanabe R et al. (2003) Autologous serum eye

serum application in Sjogren’s syndrome. Br J

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Tsubota K, Goto E, Shimmura S et al. (1999) Treat-

Bone Marrow Transplant 31(7):579-583

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Poon AC, Geerling G, Dart JK, Fraenkel GE,

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Rocha EM, Pelegrino FS, de Paiva CS,Vigorito AC,

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Tsubota K, Goto E, Shimmura S, Shimazaki J

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Ubels J,Williams K,Lopez Bernal D,Edelhauser H

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Ophthalmologe 101(7):705-709

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Scardovi C, De Felice GP, Gazzaniga A (1993) Epi-

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Vajpayee RB, Mukerji N, Tandon R, Sharma N,

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1312-1316

Controversies and Limitations of Amniotic Membrane

in Ophthalmic Surgery

Harminder S. Dua, V. Senthil Maharajan, Andy Hopkinson

Core Messages

∑ The amniotic membrane is a useful adjunct

∑ Despite the vast literature on the use of the

in the management of many ocular condi-

membrane, randomised controlled studies

tions

are virtually none

∑ Several mechanisms of action have been

∑ Lack of defined criteria of success makes

attributed to the membrane based on its

evaluation of outcomes difficult. This is

structure and biochemical composition.

rendered more difficult due to improper

Not all mechanisms are scientifically

characterisation of disease severity making

substantiated

comparisons between studies next to

∑ Many inter and intra donor variations in the

impossible

structure and function of the membrane

∑ Although the beneficial effects of the mem-

have been demonstrated. Location in rela-

brane are emphasised in several consecu-

tion to the placenta, duration of pregnancy,

tive case series, these studies often lack

parity, gravidity, onset of labour and even

proper and adequate controls and it is

age and race of the donor are all variables

important to bear in mind that existing, at

∑ Processing and preservation of the mem-

times simpler options do exist with equiva-

brane can be accomplished by different

lent or better efficacy

methods. Different methods affect the

∑ Standardisation of the membrane supplied

membrane differently and can substantially

for widespread clinical use is an important

alter the membrane. Mandatory quarantine

challenge that lies ahead. Perhaps the

of the membrane to rule out HIV contami-

generation of a ’synthetic membrane’ with

nation does not allow for use of fresh mem-

known quantities of desired ingredients,

branes. The potential risk of transmission

tailored to the intended clinical use, will be

of serious infections from one donor to a

possible in the future

number of recipients remains a concern

pears that during these ‘silent years’ it was being

2.1

used extensively in the Soviet Union and latter-

Introduction

ly in South America [13]. Its introduction to

North America in the early 1990s heralded a

The first documented ophthalmologic applica-

massive surge in the ophthalmic applications of

tion of the amniotic membrane was in the 1940’s

this membrane. The amniotic membrane is now

when it was used in the treatment of ocular

increasingly being used in ocular surface sur-

burns [9, 54, 55]. Following initial reports, its use

gery for a wide range of indications. There are

in ocular surgery, as indicated by reports in the

over 500 publications in the scientific literature

scientific literature, abated until recently. It ap-

with most of them reporting success. However,

Chapter 2

Controversies and Limitations of Amniotic Membrane in Ophthalmic Surgery

the enthusiasm to extend its clinical applica-

tural and biochemical composition of the mem-

tions and indications is not matched by the sci-

brane often without any direct evidence.

entific rigor that should be applied to any new

To understand the basis of some of the pro-

product or technique that is being used so ex-

posed mechanisms of action of the AM it is use-

tensively. There are thus several limitations of

ful to understand the structure and composi-

the amniotic membrane as applied to its oph-

tion of the membrane.

thalmic usage, which are not widely known. It is

therefore important that these limitations are

carefully considered and all applications of the

2.2.1

membrane be interpreted in the context of these

Amnion Structure

limitations. These limitations apply to the fol-

lowing areas:

The AM consists of five layers from within out-

Proposed mechanisms of action of the mem-

ward: (a) a single layer of highly metabolically

brane

active, columnar to cuboidal epithelium; (b) a

Intra and inter donor variations of the mem-

thin basement membrane; (c) a compact layer

brane

made of reticular fibres virtually devoid of cells;

Processing and preservation of the mem-

(d) a loose network of reticulum containing

brane

fibroblasts, called the fibroblast layer; and (e) a

Clinical studies and outcomes [definitions of

spongy layer of wavy bundles of reticulum

success and grading of disease severity]

bathed in mucin, which forms the interface with

Efficacy of membrane in relation to other

the chorion [4].

established techniques and options

Matrix. Amniotic basal lamina contains large

Summary for the Clinician

quantities of proteoglycans rich in heparin sul-

∑

The amniotic membrane has been used

phate. Amnion contains a large amount of colla-

in ophthalmic surgery since the mid

gen, hyaluronan and predominantly smaller

1940s

proteoglycans such as biglycan and decorin,

∑

Many ophthalmic applications are pro-

with decorin being more prominent of the two,

posed but not are supported by scientific

and is located in close connection with the col-

evidence

lagen fibrils [36]. Collagen types I, III, IV, V and

∑

The major controversies and limitations

VII [2, 25, 39, 62], laminin [2] and fibronectin

of the membrane are in relation to its pro-

[33] have been identified in amniotic basement

posed mechanisms of action, inter and intra

membrane and stromal amnion. Similarities

donor variations, its methods of processing

between the lamini-1, laminin-5, fibronectin

and preservations, the evaluation of its

and type VII collagen components of the base-

outcomes against defined criteria and in

ment membranes of conjunctiva, cornea and

comparison to existing techniques

amniotic membrane have been demonstrated

[20]. The a-subchain components of collagen

IV have been shown to be similar between am-

2.2

niotic membrane and conjunctiva but different

Proposed Mechanisms of Action

between amniotic membrane and cornea [20].

of the Amniotic Membrane

Several mechanisms of action are attributed to

2.2.2

the membrane. These include: (a) promotes ep-

Amnion Composition

ithelialisation, (b) inhibits scarring, (c) inhibits

vascularisation, (d) reduces inflammation, (e)

Some components of the membrane that are

provides a substrate for cell growth, (f) antimi-

relevant in the context of its mechanism of

crobial effects and (g) as a biological bandage

action, and that help understand its limitations,

[3, 13]. Most of these are inferred from the struc-

are mentioned below:

2.2

Proposed Mechanisms of Action of the Amniotic Membrane

Enzymes. Important amongst these are en-

components in the membrane. This is not sur-

zymes involved in prostaglandin synthesis such

prising as in any biologically active tissue such

as phospholipases, prostaglandin synthase and

as the AM, balances and counterbalances for ac-

cyclo-oxygenase [53, 57]. Prostaglandin dehy-

tion of various molecules would be expected.

drogenase, a prostaglandin-inactivating enzyme,

However, when applied surgically to the ocular

has also been demonstrated

[7]. Secretory

surface or elsewhere only the presence of the

leukocyte protease inhibitor, a potent inhibitor

desirable ones for a particular set of action(s) is

of human leukocyte elastase, has been demon-

quoted with no regard to the opposing mole-

strated in human amniotic fluid and in the am-

cules. For example, the presence of prosta-

niotic membrane. Its concentrations can be up-

glandins in the membrane would promote in-

regulated by exposing amniotic cells to IL-1a,

flammation but the presence of prostaglandin

IL-1b, and TNFa [63].

inactivating enzyme and of secretory leukocyte

protease inhibitor would suppress inflamma-

Cytokines. Interleukins-6 and -8 are the pre-

tion. The presence of anti-inflammatory cyto-

dominant cytokines associated with amnion

kines such IL-1Ra and IL-10 would suppress in-

cells [24, 47]. Expression of these cytokines was

flammation but the presence of IL-6 and IL-8

increased in the presence of IL-1b, TNFa and

would promote inflammation. In eyes that are

bacterial lipopolysaccharide. IL-10 and IL-1RA

inflamed due to injury, other proinflammatory

(receptor antagonist), both anti-inflammatory

cytokines such as IL-1a, IL-1b and TNFa could

cytokines, have been shown in amnion epithe-

also promote both pro- and anti-inflammatory

lial and mesenchymal cells [22].

cytokines and enzymes.

Similarly, the presence of various growth fac-

Growth Factors. Studies on human amniotic

tors like EGF would support epithelial growth

membrane have revealed the presence of EGF,

and TGF would support wound healing. Howev-

TGFa, KGF, HGF, bFGF, TGF-b1, and -b2 by

er, TGF would itself promote scar tissue forma-

RT-PCR for the mRNA and by ELISA for the

tion and be contradictory to the ‘anti-adhesive

protein products [29]. TGF-b3 and growth fac-

or scar suppressing’ action proposed for the

tor receptors KGFR and HGFR were also detect-

membrane in preventing corneal and conjunc-

ed by RT-PCR. A higher level of various growth

tival cicatrisation. Likewise, the presence of

factors was found in amniotic membrane with

TIMPS would disfavour vascularisation but the

epithelium than without epithelium, indicating

presence of TMPS would have the opposing ef-

an epithelial origin for these growth factors

fect. AM has been used with success in some

[29, 45]. Neurotrophic factors like NGF (nerve

cases of ocular surface burns with limbal is-

growth factor) have also been demonstrated in

chaemia. One of the main concerns in such pa-

the amniotic membrane and amniotic fluid [51,

tients is the ‘limbal ischaemia’ and procedures

60].

such as tenoplasty and conjunctival flaps where

possible have been advocated in an attempt to

Metalloproteases and Inhibitors of Metallopro-

restore limbal vascularisation. Would not appli-

teases. Tissue inhibitors of metalloproteases

cation of the membrane, with its ‘inhibitor of

(TIMPS) have been shown to be produced by

vascularisation effect’, have a contradictory

both amniotic epithelial cells and mesenchymal

effect on limbal ischaemia?

cells [22, 50]. The presence of tissue metallopro-

The mere presence of one or the other ‘factor’

teases (TMPS) has also been demonstrated in

thus cannot be presented as evidence in support

amniotic fluid and amniotic membrane, where

of a particular mode of action for the mem-

they may play a role in the mechanisms of hu-

brane. How these various factors interplay, if at

man parturition and in the regulation of host

all, in the transplanted membrane to bring

response to intrauterine infection [17, 41].

about some of its attributed effects remains to

be elucidated.

Controversies and Limitations. From the above

Of the proposed mechanisms of action of the

it is obvious that there are several contradictory

membrane the most likely manner in which it

Chapter 2

Controversies and Limitations of Amniotic Membrane in Ophthalmic Surgery

affects its beneficial effect is perhaps as a sub-

strate or basement membrane transplant. It

2.3

provides a favourable substrate by virtue of its

Intra and Inter Donor Variations

basement membrane, for new epithelial cells to

of the Membrane

migrate on, expand and adhere. Use of the

membrane as a bandage to cover inflamed or

The general functional description of the am-

exposed areas, due to injury or surgery, not only

nion is that of an epithelial lining which con-

favourably influences the healing process but

tributes to the homeostasis of amniotic fluid. It

also has a dramatic favourable effect on the

is natural therefore that its physiological role

symptom of pain and discomfort. It is our clini-

and some corresponding morphology will

cal experience that when denuded areas of the

change depending on the stage of gestation.

ocular surface, particularly the cornea, are cov-

This in fact has been amply demonstrated.

ered by amniotic membrane, the levels of pain

Many changes in the biochemical composition

and discomfort experienced by the patient are

of the membrane are known to occur nearer

significantly reduced. This too could be as a re-

term and to be induced by labour, for example

sult of the mechanical or physical presence of

increased apoptosis of amnion epithelial cells

the membrane. One study has shown that amni-

occurs just before commencement of labour

otic fluid application to the corneal surface of

and IL-6 and IL-8 are found in increased con-

rabbits following excimer laser photokeratecto-

centrations in the amniotic fluid towards the

my actually enhanced corneal sensitivity and

end of pregnancy [24].

nerve regeneration [32].

The amnion varies in histological appear-

ance from conception to maturity and several

Summary for the Clinician

different patterns are often noted even at term.

∑

The amniotic membrane is composed of a

There is an increase in prostaglandin synthase

single layer of epithelial cells, basement

in the amnion at term and during labour [53].

membrane and avascular stroma

The epithelial morphology can change to that of

∑

Several growth factors,cytokines,proteases

large flat cells and some show distinct intercel-

and their inhibitors, antimicrobials, antian-

lular channels. Also, the amniotic epithelial cells

giogenic factors and enzymes have been

are columnar towards the placenta and cuboidal

identified in these layers

away from it [43]. The apical surfaces of the am-

∑

The mechanisms of action of the membrane

niotic epithelial cells are covered by microvilli

are attributed to and inferred from its

[8, 43, 61], the density of which varies during

physical structure and its molecular con-

pregnancy. An amorphous material of un-

stituents

known substance is seen on the surface of these

∑

The fate of these substances after trans-

microvilli at term [43]. In a recent elaborate

plantation is unknown and the mere pres-

study, using TGFb as a test molecule to illustrate

ence of a factor or molecule does not imply

inter donor variations such differences were

that it is available in its active form after

clearly demonstrated (see next section below).

surgery

In a recent study, Fortunato et al. [16] have

∑

Often both pro and anti factors are present

demonstrated a racial disparity in the ability of

and a beneficial effect cannot be ascribed

the membrane to respond to infectious stimuli,

to any one of them without taking into

suggesting that race may yet be another variable

account the effect of the other

to contend with.

∑

The most uncontroversial mechanism

With increasing use of the membrane it is

of action is by its physical presence as a

our experience that the thickness and trans-

‘substrate’ transplant

parency of the membrane varies at different

sites of the membrane. Generally, the mem-

brane closer to the umbilical cord appears

thicker. This also affects the transparency or

translucency of the membrane. The variation

2.4

Processing and Preservation of the Membrane

between donors can be more pronounced. The

∑ The thickness and transparency of the

amnion can vary in thickness from 0.02 to

membrane is different at different parts of

0.5 mm [4, 8].

the membrane - thinner and clearer away

from the placenta, and in different donors

Controversies and Limitations. Many differ-

∑ The membrane undergoes considerable

ences, some yet unknown, can exist between

physiological changes near term and

amniotic membranes obtained from different

during labour

donors. Racial variations too may exist. There

∑ Racial variations between donors can exist

are many other important variables such as the

∑ Duration of pregnancy,trial of labour,

duration of gestation, trial of labour before cae-

gravidity and parity can all influence the

sarean section and perhaps parity and gravidi-

composition of the membrane

ty of the donor. Increased prostaglandin and

∑ Different pieces of the membrane from the

proinflammatory cytokines nearer term could

same donor can potentially have different

influence the effect of the membrane when ap-

effects

plied on the ocular surface. Even for the same

donor, it is the general practice to obtain nu-

merous pieces of amnion for use in multiple

2.4

operations. Clearly therefore some pieces would

Processing and Preservation

be from locations closer to the placenta and oth-

of the Membrane

ers distant to it. The thickness of these locations

can vary as can the morphology of the amniot-

Many methods of preserving and storing amni-

ic epithelium. The thickness could affect the in-

otic membrane for ocular and other uses have

tegration of the membrane with the ocular sur-

been described.Some of these are historical and

face tissues and perhaps influence the ease with

others popular and currently in vogue. Methods

which the membrane ‘comes off ’ at some point

such as lyophilisation [6, 56], air drying [35, 46],

after surgery. The transparency would naturally

glutaraldehyde and polytetrafluoroethylene

affect potential vision when applied on the

treatment [40] and irradiation [35, 46, 59] have

corneal surface. Current practice in procure-

been described but are not among the ones used

ment and supply of the membrane does not in-

commonly for ophthalmic use. Preservation by

dicate these variables that could be important to

freezing is the commonest mode of preserva-

the outcome of its usage. Age, race, parity, gra-

tion of the membrane before use. This involves

vidity, duration of gestation, whether trial of

use of two types of solutions, either DMSO in

labour was given or not and location of a partic-

phosphate buffered saline [3, 52] or Eagle’s min-

ular piece of membrane supplied need to be

imum essential medium (MEM) and glycerol

considered in evaluating outcomes and even-

[26, 27]. Recently a freeze dried preparation of

tually in bringing some semblance of standard-

the membrane has been commercially intro-

isation in the practice of amniotic membrane

duced (Ambiodry) but not much is known

transplantation. This may not be practically

about its usage. Furthermore different antibiot-

possible but only by recording these variables

ic cocktails, 0.5 % silver nitrate [21] and 0.025 %

can we begin to understand whether and

sodium hypochlorite solution [48, 49], have

what effect they may have on the outcome of

been used to render the membrane sterile. In

transplantation. One point that comes out loud

many parts of the world ‘fresh membrane’

and clear from the above is that membranes

(within days or weeks of donation) is still used.

used in transplantation are far from standard-

However, in most Western countries the use of

ised across donors and even within the same

one or the other method of preservation is

donor.

mandatory because of legislation requiring that

the membrane be adequately screened for HIV

Summary for the Clinician

contamination. To this end the donor is tested

∑ The amniotic epithelial morphology varies

at the time of delivery and 6 months later (to

from flat to cuboidal to columnar

cover the window period of infection). All

Chapter 2

Controversies and Limitations of Amniotic Membrane in Ophthalmic Surgery

processed membranes are stored in quarantine

protein level TGF-b1 is the highest expressed

until the second test is performed and reported

isoform of TGF-b, and that expression is lower

negative.

in AM than in chorion. In addition, they demon-

strated considerable variations in TGF-b1 gene

Controversies and Limitations. Several differ-

expression between membranes, with expres-

ences can therefore occur between different

sion at different locations within a single mem-

membranes depending on whether they are

brane also appearing to vary. Another impor-

used fresh or preserved and, in case of the latter,

tant observation was that maximal presence of

on the mode and duration of preservation. Most

TGF-b1 was in the acellular spongy layer, which

methods employed in the preservation of the

suggests that the spongy layer most likely acts as

membrane affect it in some manner.

a depot for chorion-derived TGF-b1. It is possi-

Kruse et al. [30] demonstrated that cryop-

ble that the spongy layer acts as a physical bar-

reservation significantly impaired the viability

rier, preventing chorionic-TGF-b1 from diffus-

and proliferative capacity of amniotic mem-

ing into the AM during gestation. They also

brane and its cells. They concluded that amniot-

showed that alterations in the method of han-

ic membrane grafts function primarily as a ma-

dling the membrane could drastically alter the

trix and not by virtue of transplanted functional

concentration of TGF-b1. Any method of pro-

cells. Kubo et al. [31] have shown that after

cessing and storage of the membrane, therefore,

2 months of freezing, at least 50 % of amniotic

that did not get rid of this layer would yield a

cells are viable and capable of proliferation. Af-

product that would enhance wound healing and

ter 18 months of cryopreservation, they were not

scarring and vice versa. This study has conclu-

able to demonstrate a significant amount of cell

sively demonstrated that, not only for this one

survival. Our own studies show that cell viability

factor but for other proteins as well, consider-

is minimal, if at all, after 6 months of preserva-

able variations exist between membranes, at

tion at -80 °C; however, at this time point the

different locations within the same membrane

membrane continues to demonstrate many

and can be profoundly affected by the method

growth factors and cytokines (Hopkinson A et

of processing and storage of the membrane

al., submitted for publication). Fujisato et al. [18]

(Fig. 2.1). Clinically, such variation between

cross-linked amniotic membrane with chemical

membranes is not considered prior to surgery,

means (glutaraldehyde) and with gamma-ray

and therefore the effect on clinical efficacy is

and electron beam. They showed that radiation

unknown. To determine the clinical significance

cross-linked membranes degraded rapidly in

of such variables, further studies are required.

vitro compared to chemically cross-linked

Koizumi et al. [28] cultivated rabbit limbal

membranes. Hao et al. [22], who demonstrated

and corneal epithelial cells on denuded human

the presence of mRNA for both antiangiogenic

amniotic tissue. They demonstrated significant-

and anti-inflammatory factors in amniotic

ly improved growth of cells on membrane de-

membrane, have suggested that amniotic mem-

nuded of epithelial cells compared with intact

brane should be applied epithelial cell surface

membrane. To the contrary, most of the mem-

down in order to deliver a high concentration of

brane supplied in the USA is with the amnioitic

these factors to the damaged ocular surface. This

epithelium in situ. It is claimed that ocular sur-

would be applicable more to fresh rather than to

face epithelial cells grow better on this surface.

preserved membranes if one were to accept (de-

This controversy remains unresolved. Clinically,

spite lack of any evidence in support) that the

it is our experience that both membranes, with

amniotic epithelial cells continue to produce the

and without the amniotic epithelium in situ,

desired ‘factors’ in biologically active forms after

seem to support growth of ocular surface cells.

transplantation onto the ocular surface.

The weight of the evidence available sup-

Hopkinson A et al. (submitted for publica-

ports the notion that viability of the tissue com-

tion) studied extensively the growth factor TGF-

ponents of the amniotic membrane is not essen-

b1 to determine intra- and intermembrane vari-

tial for its biological effectiveness. However, the

ations. They showed that at both the gene and

extent of the effectiveness could vary and result

2.5

Clinical Studies and Outcomes (Definitions of Success and Grading of Disease Severity)

Fig. 2.1 A, B. Two-dimensional gel electrophoresis

(reference markers) between membranes are indicat-

of solubilised protein from transplant ready amniotic

ed (arrows). Variation between membranes, repre-

membrane. Sixty micrograms of protein from two

sented by spots detected in some membranes but not

different donor membranes (A, B) was separated on

in others, is also indicated (A 1-4). Examples shown

18-cm pH 3-11 IPG strips (Amersham Biosciences)

are of comparable zoomed areas of the whole gel, and

and then on a 8-19 % gradient polyacrylamide gel fol-

representative experiments of a total of 24 performed

lowed by silver staining of protein spots. Comparable

are shown

spots of similar intensity representing similarities

in inconsistent results. Until the effect of these

∑ The effect of these different methods on the

different methods of preservation and storage

structural and biochemical composition of

on the membrane has been evaluated and stan-

the membrane is not fully understood. Such

dardised, success or failure of the membrane

effects could have a direct consequence on

should be qualified by the method of preserva-

the proposed mechanisms of action

tion employed.

∑ Studies have shown that preservation and

Despite the prolonged quarantine, the poten-

processing can have profound effects on the

tial risk of transmission of serious infections

membrane constituents

from one donor to a number of recipients

∑ Fresh, and to some extent even preserved,

remains a concern. This concern is heightened

membranes that have been tested twice

in countries where fresh membrane is used

carry a potential risk of spread of serious

though mitigated to some extent by the fact that

infections

only a limited number of surgeries (recipients)

are performed with a given donor.

2.5

Summary for the Clinician

Clinical Studies and Outcomes

∑ Many different methods of preservation

(Definitions of Success and Grading

and storage of the membrane exist

of Disease Severity)

∑ Wet freezing in phosphate buffered saline

or in minimum essential medium are cur-

Most published reports on use of the amniotic

rently the two most popular methods

membrane in ophthalmology have been consec-

∑ A freeze-dried membrane has recently been

utive case series or retrospective studies. Ran-

introduced in the market and initial usage

domised controlled studies are practically

seems to suggest clinical efficacy

non-existent bar one or two [5]. This must be

Chapter 2

Controversies and Limitations of Amniotic Membrane in Ophthalmic Surgery

considered a serious limitation of the use of the

amniotic membrane. Another significant short-

coming has been the lack of clear definitions of

success or failure and the evaluation of the out-

comes against such definitions. This limitation

is to some extent matched by a similar lack of

consistency in gradation and classification of

severity of the disease that make up the major

indications for use of the membrane, for exam-

ple ocular surface burns. These two factors

combine to make evaluation of the effects of the

membrane difficult and in some instances ren-

dering it even impossible to compare outcomes

between different groups [12, 15, 23, 37].

We have used a pre-determined protocol to

define outcomes and propose this as one model

that may be considered in evaluating the effica-

cy of the membrane (Maharajan et al., submit-

ted for publication). When the membrane was

used with the intention of it becoming incorpo-

rated into the recipients tissue it was termed a

graft and when the intention was for it to come

away or be removed at a certain point following

surgery, it was termed a patch. In our group of

Fig. 2.2.

A Ocular surface reconstruction with al-

patients it was used primarily as a graft or a

lolimbal transplantation and use of two membranes.

patch with four objectives: (a) to establish ep-

The inner 9-mm disc acts as a graft and the outer

ithelial cover in an area where none existed, (b)

larger membrane as a patch. The outer membrane

prevents conjunctival epithelial cells mixing with the

to prevent corneal perforation in eyes at risk

transplanted limbus-derived cells growing on the in-

due to stromal melting, (c) to limit scarring

ner membrane (author’s technique: H.S. Dua). B The

where the clinical likelihood was high or where

outer membrane has cut through sutures and retract-

scarring (symblepharon/adhesions) previously

ed, exposing the inner membrane. This was consid-

existed, and (d) to limit inflammation and neo-

ered as a partial success

vascularisation. The outcome was evaluated

against both the intended purpose of the mem-

brane, patch or graft, and whether the objective

success: when the membrane served the pur-

was achieved or not. Three outcomes were thus

pose that was intended, i.e., acted as a patch or

defined, success, partial success and failure.

graft and the objective was achieved; (2) partial

success: (a) when the membrane did not serve

Criteria for Success or Failure (Maharajan et al.,

the purpose that was intended, i.e. acted as a

submitted for publication). The purpose of

patch when intended as a graft or vice versa but

AMT was to act as a patch, graft or both. AMT

the objective was achieved, for example re-ep-

was carried out to fulfil one or more of the fol-

ithelialisation of a persistent epithelial defect

lowing objectives: (a) to establish epithelial cov-

(PED) occurred, (b) when the membrane (as a

er in an area where none existed, (b) to prevent

patch) did not persist long enough but the ob-

corneal perforation in eyes at risk due to stro-

jective was nevertheless achieved, for example

mal melting, (c) to limit scarring where the clin-

epithelialisation continued till the defect was

ical likelihood was high or where scarring (sym-

closed, (c) when multiple objectives were set

blepharon/adhesions) previously existed, and

and not all were realised; (3) failure: when the

(d) to limit inflammation and neovascularisa-

objective was not achieved even though the pur-

tion. Three outcome measures were applied: (1)

pose may have been achieved, for example if the

2.5

Clinical Studies and Outcomes (Definitions of Success and Grading of Disease Severity)

membrane was intended as a patch and acted as

such for the expected duration but the PED did

not heal.

When the above criteria were applied to 74

procedures involving use of the amniotic mem-

brane, failure of the procedure was observed in

44% of patients where the membrane was used

in the presence of stem cell deficiency, in 33 % of

procedures where the membrane was used in

the absence of stem cell deficiency and in 44 %

of patients where the membrane was used for

conjunctival reconstruction. This clearly illus-

trates that a significant proportion of failures

can occur and these should be appropriately

recognised and documented in order to refine

and temper the vastly expanding indications for

use of the membrane (Figs. 2.2, 2.3).

Controversies and Limitations. Lack of ran-

domised controlled trials, lack of clearly defined

criteria of success and failure against intended

use of the membrane and inadequate gradation

or classification of disease severity all con-

tribute significantly to highlight a serious limi-

tation surrounding the clinical use of the mem-

brane, ascertaining its efficacy for different

indications and comparing and evaluating out-

comes of use.

Summary for the Clinician

∑ The membrane can be used to serve either

as a patch, when the membrane is removed

after some time or is expected to fall off,

or as a graft (including carrier of ex vivo

expanded cells) when the membrane is

incorporated into the host tissue

∑ Important objectives of use of the mem-

Fig. 2.3.

A Corneal scarring and vascularisation de-

brane are to provide epithelial cover, arrest

spite allolimbal transplantation and use of amniotic

membrane in a case of stem cell deficiency. This

melting, limit scarring, limit inflammation

patient was considered a failure. B Failed amniotic

and neovascularisation

membrane transplant in a patient of acute ocular sur-

∑ Outcomes should be clearly defined,

face burn. C Amniotic membrane and corneal stro-

for example as success, partial success

mal melting in a patient of bullous keratopathy treat-

or failure

ed with amniotic membrane transplantation. The

∑ Proper gradation or classification of

picture was taken on day 7 after surgery

clinical conditions is necessary for proper

evaluation of the membrane

∑ Randomised controlled studies are required

to scientifically evaluate the efficacy of the

membrane

Chapter 2

Controversies and Limitations of Amniotic Membrane in Ophthalmic Surgery

ment of abnormal cells from the surface of the

2.6

cornea in cases of partial stem cell deficiency

Efficacy of Membrane in Relation

[1, 38, 58]. In a technique now established as

to Other Established Techniques

sequential sector conjunctival epitheliectomy

and Options

(SSCE), Dua has shown [10, 11, 14] that the same

effect can be achieved with simple debridement

Another interesting point of note is that many

without the use of the membrane. The studies

reports of the success of the use of the mem-

that advocated the use of the membrane for this

brane for one indication or the other do not

indication did not include any controls where

compare it with valid controls or even against

simple debridement was undertaken without

standard existing techniques for the same indi-

amniotic membrane transplant. The SSCE tech-

cation. This leads to the erroneous message that

nique can be taken as akin to controls for these

the membrane should be used for a particular

studies and illustrates that proposed indications

indication without flagging that an existing pro-

of the membrane are not always substantiated

cedure or alternative to the membrane may be

scientifically.

equally effective or in some cases better. The

Similarly, the use of the membrane for the in-

following illustrations emphasise this point:

dication of painful bullous keratopathy [42]

When the advocated use of the membrane to

needs to be evaluated against the alternative

repair leaking trabeculectomy blebs was stud-

of anterior stromal puncture and temporary

ied against the standard conjunctival advance-

placement of a bandage contact lens. The latter

ment, the latter was found to be more reliable

is a simple outpatient procedure with compara-

[5]. In some studies where the membrane has

tively little expense. In an ongoing stratified (ac-

been used to repair failing blebs, antimetabo-

cording to pain score) randomized controlled

lites too have been used as adjuncts. In the ab-

study we have performed the two different pro-

sence of any controls it is impossible to assess

cedures in 25 patients and thus far no significant

the contribution of the membrane to any suc-

difference between amniotic membrane trans-

cess, which may equally have been due to the

plantation and anterior stromal puncture is

antimetabolite [13, 19].

emerging.

Its use in pterygium surgery too is not fully

clarified. It may be a useful alternative but autol-

Controversies and Limitations. Many proposed

ogous conjunctival grafts seem to have a better

indications of the membrane are not founded

success rate than amniotic membrane grafts.

on hard evidence. In some instances use of the

Prabhasawat et al. [44] found that in pterygium

membrane may be an option but not necessari-

surgery the recurrence rate was higher with

ly a better option. In some cases it has distinct

amniotic membrane compared to autologous

disadvantages over existing techniques. Longer

conjunctival grafts. Ma et al. [34] found no dif-

follow-up studies are needed to ascertain dura-

ference between the amniotic membrane, mito-

tion of its efficacy.

mycin C or autologous conjunctival grafts in the

Summary for the Clinician

management of pterygium, but recommend use

of the membrane.

∑ Alternative options exist for many of

An important case in point is the reported

the clinical indications for use of the

success of the membrane in treating patients

membrane

with partial stem cell deficiency. The membrane

∑ In most instances the membrane has not

can offer help in instances where a fibrovascular

been compared directly with these options

pannus has to be excised, but where abnormal

to evaluate its superiority or otherwise

conjunctiva derived epithelium has encroached

∑ Clear examples of the failure of the

onto the corneal surface the membrane is often

membrane exist where other options

not required though reportedly used and advo-

have succeeded

cated. Tseng have shown good results with use

of the membrane following superficial debride-

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Dua HS (1998) The conjunctiva in corneal epithe-

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many clinical situations and in this context is

11.

Dua HS (2001) Sequential sector conjunctival ep-

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13.

Dua HS, Gomes JAP, King AJ, Maharajan VS

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(2004) The amniotic membrane in ophthalmolo-

the amniotic membrane on a sound scientific

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footing. Perhaps the generation of a ‘synthetic

14.

Dua HS, Gomes JAP, Singh A (1994) Corneal ep-

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membrane’ with known quantities of desired

408

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15.

Dua HS, King AJ, Joseph A (2001) A new classifi-

will be possible in the future.

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85:1379-1383

16.

Fortunato SJ, Lombardi SJ, Menon R

(2004)

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Transplantation of Limbal Stem Cells

Harminder S. Dua

Core Messages

∑ Most self renewing tissues are served by a

∑ Total unilateral cases can be treated with

population of stem cells

auto-limbal transplantation

∑ Potency and plasticity are two important

∑ Bilateral cases often require allo-limbal

characteristics of stem cells. They may also

transplantation from living related or

have the potential to transdifferentiate

cadaver donors

∑ Stem cells usually reside in a defined ’niche’.

∑ Auto-limbal and living related donor trans-

The corneal epithelial stem cells are be-

plantation should be avoided in the pres-

lieved to be located in the limbal palisades

ence of active inflammation. Auto-limbal

∑ Clinical and laboratory evidence strongly

transplantation should be avoided in unilat-

supports the notion that corneal epithelial

eral manifestation of a systemic disease

stem cells are located at the limbus but no

∑ Amniotic membrane transplant can be

marker yet exists that can positively identi-

combined with any of the limbal transplant

fy a limbal stem cell

procedures

∑ Limbal stem cell deficiency can be congeni-

∑ Allografts usually require long-term

tal or acquired. Ocular surface burns, im-

systemic (and/or topical) immunosuppres-

mune mediated ocular surface diseases

sion

and chronic inflammation are important

∑ All associated pathology such as lid malpo-

causes of limbal stem cell deficiency

sitions, trichiasis, secondary glaucoma and

∑ The effects of limbal stem cell deficiency

cataracts should ideally be managed prior

can range from mild, such as loss of limbal

to considering limbal transplantation,

anatomy or conjunctivalisation of the pe-

as far as clinically possible

ripheral cornea, to severe, such as corneal

∑ Buccal mucosa grafts help restore some

invasion by a thick fibrovascular pannus or

moisture to a dry ocular surface. Living

persistent epithelial defects with stromal

tissue transplants usually do not survive

melts

in a dry environment

∑ The diagnosis of limbal stem cell deficiency

∑ Long-term outcomes of auto-limbal trans-

is essentially clinical, but impression cytol-

plants are far better than those of allo-

ogy may help. Presence of goblet cells on

limbal transplants

the cornea is diagnostic

∑ Ex vivo expansion of limbus derived epithe-

∑ Limbal stem cell deficiency can be unilater-

lial cells as a sheet on different substrates

al or bilateral, partial or total

can also be used in ocular surface recon-

∑ Mild cases of partial deficiency can be

struction with good results but is also sub-

treated by sequential sector conjunctival

ject to immune rejection

epitheliectomy

Chapter 3

Transplantation of Limbal Stem Cells

self renewal and an increased potential for er-

3.1

ror-free division [65-67]. They have the ability

Introduction

to proliferate indefinitely [32] and generally live

for the duration of the organ(ism) in which they

In this chapter the general characteristics of

reside. A constant pool is maintained by differ-

stem cells (SC) and their niche are first de-

ent strategies of cell division. The most accept-

scribed. The evidence supporting the existence

ed strategy is that of asymmetric cell division

of SC at the corneoscleral limbus, both clinical

whereby one daughter cell stays back in the

and scientific, is then explored providing the

stem cell niche and the other follows the path

scientific basis for the transplantation of the

of proliferation and differentiation, acquiring

limbus in the management of limbal SC defi-

functional characteristics of the tissue or organ.

ciency. A brief account of the causes and effects

The same balance can be maintained if the two

of SC deficiency is provided as background to

daughter cells from one SC proceed down the

the indications and different techniques of lim-

path of differentiation and the two daughter

bal SC transplantation. The surgical techniques

cells of another SC stay back in the niche as stem

are elaborated together with postoperative

cells [57, 61].

management and any adjunctive procedures

The daughter cell(s) that step outside the

that may complement limbal transplantation.

stem cell pool are destined to divide and differ-

Ex vivo expanded limbal stem cells on amni-

entiate with the acquisition of features that

otic membrane or other substrates can also be

characterise the specific tissue. Such a cell is

used in ocular surface reconstruction. This con-

called a ‘transient amplifying cell’ (basal corneal

stitutes a distinct method of putative limbal stem

epithelial cells) and is less primitive than its

cell transplantation and is the subject of another

parent stem cell. It is believed in some quarters

chapter in this book. This technique of preparing

that there exists a window of opportunity dur-

the tissue construct and stem cell transplantation

ing which some of these cells (‘transient cells’)

is therefore omitted from this chapter.

[54, 55] can revert to the SC pool as SC. Transient

amplifying cells divide more frequently than

stem cells but have a limited proliferative poten-

3.2

tial and are considered the initial step of a path-

Stem Cells

way that results in terminal differentiation.

They differentiate into ‘postmitotic cells’ (wing

3.2.1

cells) and finally to ‘terminally differentiated

Definition

cells’ (superficial squamous cells). Both postmi-

totic and terminally differentiated cells are in-

Stem cells are progenitor cells that are responsi-

capable of cell division. All cells except stem

ble for cellular replacement and tissue regener-

cells have a limited life span and are destined to

ation. They are the ultimate source cells from

die [45, 66, 84].

which arise almost all other cells that constitute

Potency and plasticity are two key attributes

a given organ served by the SC. SC can be found

of SC. SC have the potential to give rise to differ-

in both embryonic and adult tissues and repre-

ent cell lineages. This potency is, however, not

sent only a very small proportion (0.01-10 %) of

uniform and there exists amongst SC a hierar-

the total cell mass [1, 32, 44].

chy of potential. SC can be totipotent, pluripo-

tent, multipotent, or unipotent. The zygote that

can form the entire embryo and part of the pla-

3.2.2

centa is an example of a totipotent cell. Cells of

Characteristics of Stem Cells

the inner cell mass from which most tissues that

arise from the three germ layers can be derived,

Stem cells are poorly differentiated or undiffer-

but not components of the placenta, are consid-

entiated, long-lived, slow cycling but highly

ered pluripotent. Most tissue specific SC are

clonogenic cells that have a high capacity for

multipotent, capable of producing lineages that

3.3

Limbal Stem Cells

can differentiate in two, three or more different

Summary for the Clinician

cell types with functional attributes of the organ

in which they reside. Some SC, in their natural

∑

Stem cells:

environment, may have only limited potential

- Undifferentiated

with the ability to generate only one specific cell

- Long lived

type. These SC are labelled unipotent SC or

- Slow cycling

committed progenitors. SC of the epidermis and

- Clonogenic

the corneoscleral limbus are considered to be

- Asymmetric division

examples of this category [1, 3, 68].

- Potency: usually pluripotent

The ‘plasticity’ of SC refers to their ability to

or multipotent

transdifferentiate. Some SC when relocated to a

- Plasticity: transdifferentiation

different site (tissue) can assume the role that

- Niche: SC microenvironment

supports the structure and function of the new

∑

SC progeny:

site, thus aiding in regeneration, repair and

‘Transient cells’

maintenance of the cell population at the new

– Transient amplifying cells -

site. Stem cell potential and plasticity are both

basal epithelium

more pronounced in embryonic SC compared

- Postmitotic cells - wing cells

to adult SC. Embryonic SC have virtually an un-

- Terminally differentiated cells -

limited potential for self-renewal and differenti-

superficial squamous cells

ation. Given the right microenvironment and

the right signals, adult and embryonic SC can

(theoretically) be made to follow a desired path

3.3

of differentiation or propagated indefinitely, in

Limbal Stem Cells

an undifferentiated state. Herein lies the im-

mense therapeutic potential of SC [3].

3.3.1

The Clinical Evidence

3.2.3

During corneal epithelial wound healing and

The Stem Cell ’Niche’

normal epithelial turnover, cell migration and

migration of sheets of epithelium [20] have

The microenvironment in which the SC reside is

been shown to occur in a centripetal manner

referred to as their ‘niche’ [60]. SC are usually

from the corneoscleral limbus towards the cen-

confined to their ‘niche’ where the microenvi-

tre of the cornea [4, 5, 51]. Large corneal epithe-

ronment supports and maintains the stemness

lial wounds, where the wound edge is closer to

of SC and affords a degree of protection. In sol-

the limbus, heal at a faster rate than smaller

id organs, where cell migration commences at

wounds [59]. Repeated denudation of the cen-

one point and progresses until the cells are shed

tral corneal epithelium shows that the healing

at a distant point(s), the SC niche is usually lo-

rate of the second wound is more rapid than

cated at the point of commencement. The ‘niche’

that of the first. This suggests that rapidly divid-

represents the collective influence of other local

ing younger cells of the periphery have moved

matrix cells, the extracellular matrix, its vascu-

to more central areas after the first trauma and

larity, basement membrane characteristics and

respond readily to the second [80].

prevalent growth factors and other cytokines. In

Human corneal epithelial defects with par-

the intestinal mucosa, for example, it is believed

tial limbal involvement demonstrate a preferen-

that the pericryptal fibroblasts/subepithelial

tial circumferential migration of a population of

myofibroblasts may serve as niche cells [60, 79]

cells along the limbus, from both ends of the

and in the epidermis, beta 1 integrin mediated

remaining intact limbus [21] (Fig. 3.1 A). Com-

adhesion to its ligand, type IV collagen, is

plete epithelial cover for the corneal surface is

shown to influence behaviour of epidermal SC

not established until limbal re-epithelialization

[44]. The niche also affords protection to the all-

is first complete, suggesting that the circumfer-

important SC [79, 95].

Chapter 3

Transplantation of Limbal Stem Cells

Fig. 3.1. A Healing of corneal epithelial wound in-

B ‘Columnar keratopathy’ is the name given by the

volving the limbus showing a preferential circumfer-

author to this presentation of alternating columns of

ential migration of tongue-shaped sheets of limbal

fluorescein stained epithelium and normal corneal

epithelial cells arising from either end of the remain-

epithelium. These correspond to the limbal palisades

ing intact epithelium. (Slit lamp anterior segment

and represent an early sign of limbal stem cell defi-

photograph with fluorescein dye) (with permission

ciency

from Br J Ophthalmol: Dua et al. 2001; 85:1379-1383).

Fig. 3.2. Slit lamp photograph of the limbus showing the palisade (of Vogt) structure with: A pigment columns

migrating into peripheral cornea and B fluorescein staining of columnar migration in response to a central

abrasion

entially migrating population of cells probably

palisade rete ridges provide a unique structure

represents in part the healing response of limbal

to the limbus (Fig. 3.2). The structure of the pal-

stem cells. In patients with limbal abnormali-

isades and the rete ridges, their vascularity and

ties, alternating columns of normal and fluores-

pigmentation are all analogous to repositories

cein staining cells often corresponding to limbal

of stem cells in the monkey palm epidermis

palisades - columnar keratopathy - have been

[9,

86,

88]. It has also been reported that

noted to extend from the limbus towards

hemidesmosomes of peripheral cells of normal

the centre in radial or curvilinear rows [22]

and healing mouse corneas are arranged in ra-

(Fig. 3.1 B). The palisades of Vogt and the inter-

dial rows, leading to the interpretation that this

3.3

Limbal Stem Cells

orientation represents centripetal migration of

cells

(H.S. Dua, unpublished observations,

epithelial cells

[5]. Very recently, a unique

Fig. 3.3A, B).

anatomical structure, termed the limbal epithe-

Several other attributes that are unique to the

lial crypt [27], has been discovered at the pe-

limbal epithelium (Table 3.1) have also been de-

ripheral end of the interpalisade rete ridges,

scribed. These include the presence of alpha-

numbering approximately five to seven per hu-

enolase [101, 102], EGF receptors [100, 103], pig-

man cornea. This has features consistent with

ment [9], cytokeratin profile (CK3/12 negative)

those of a SC repository or ‘niche’.

[7, 73], presence of vimentin [53-55], CK19 and

specific basement membrane characteristics

[34, 35, 85]. Vimentin and CK19 positive, CK3

3.3.2

negative clusters of cells with unique electron

The Scientific Evidence

microscopic morphology have been demon-

strated [54, 55]. Connexin 43 (Cx43), a gap junc-

Basic research has identified a number of char-

tion protein, has been noted in human corneal

acteristics that are unique to the limbal basal

but not limbal basal epithelium [12, 58, 96]. It has

epithelial cells and set them apart from the rest:

been proposed that absence of Cx43 segregates

Mitosis rates are highest at the limbus, both in

cells from adverse events generated in neigh-

the normal physiological state and following

bouring cells and helps preservation of SC in

stimulation [31, 37, 8]. Limbal epithelial cells

their microenvironmental niche [96].

have the greatest proliferative potential in vitro,

Zhao et al. [98] have recently reported that

compared to any other part of the cornea

limbal epithelial cells cultured in the presence of

[28-30]. Limbal basal cells lack the epithelial

mitogens express neural progenitor markers,

cell differentiation cytokeratin CK3 [18, 56, 73,

specifically nestin. A transcriptional factor, p63

101]. Impression cytology examination of the

involved in morphogenesis, has been proposed

human limbus shows that, morphologically, the

to identify keratinocyte stem cells at the limbus

limbal cells are smaller, more densely packed

[63], but its role as a marker of limbal SC is con-

and have a greater nucleus to cytoplasm ratio

troversial [25, 46]. Similarly, well defined mark-

compared to adjacent corneal and conjunctival

ers of haematopoietic SC, namely CD34 and

Fig. 3.3. A Impression cytology specimen of the hu-

man limbus from an eye bank donor eye. The limbal

cells are smaller, tightly packed and show a greater

nuclear-cytoplasmic ratio. B Montage of the human

limbus, peripheral cornea and conjunctiva

Chapter 3

Transplantation of Limbal Stem Cells

Table 3.1. Differences between epithelial cells of the limbus and central cornea (CK, cytokeratin; CX,

connexin; EGFR, epithelial growth factor receptor)

Limbus

Central cornea

CK 5/14+ve

CK 5/14-ve

CK 3/12-ve

CK 3/12+ve

CK 19+ve

CK 19-ve

P63+ve

P63-ve but see results in this paper

CX 43-ve

CX 43+

Vimentin+ve

Vimentin-ve

Intrinsic melanogenesis

Cytochrome oxidase and ATpase+ve

Alpha-enolase+ve

Beta-1-integrin+ve

EGFR+ve (strong)

EGFR+ve

ABCG2+ve

ABCG2-ve

The above data strongly supports the notion

that progenitor cells exist at the corneoscleral

limbus. Whether these are truly SC as defined in

other organ systems remains to be established.

There is evidence to suggest that SC or progeni-

tor cells for the conjunctival epithelium reside

maximally in the fornices and for goblet cells and

perhaps for conjunctival epithelium may also be

scattered throughout the epithelial surface.

Summary for the Clinician

Fig. 3.4. Limbal epithelial crypt: representing a solid

cord of cells extending from the undersurface of a

∑ Evidence for corneal epithelial (limbal)

limbal palisade. These cells are positive for the puta-

stem cells:

tive stem cell marker ABCG2. Haematoxylin stained

cryo section, ¥100

∑ Clinical:

- Unique palisade architecture

- Centripetal migration from limbus

- Circumferential migration along limbus

CD133, have failed to demonstrate any unique

- Pigment and other deposits migrating in

subpopulation of cells at the limbus [25, 47]. An

columnar manner from limbus

ATP-binding cassette transporter protein,

- Larger corneal epithelial wounds

ABCG2, is believed to be a marker of a side pop-

(closer to limbus) heal faster

ulation of cells that have the ability to efflux

- Second wounds heal faster

Hoechst 3342 dye [99]. Side population cells that

- Relative resistance of limbus epithelium

contain this transporter protein are believed to

to denudation

be stem cells [36]. Limbus epithelial cells have

- Columnar keratopathy

been shown to express ABCG2 [94] and these

- Limbal deficiency allows conjunctivali-

may represent the subpopulation that contain

sation of cornea and persistent epithelial

the stem cells. The limbal epithelial crypt re-

defects

cently demonstrated by Dua et al. [27] contains

∑ Scientific:

cells that predominantly stain positive for

- Different morphology of limbal cells

ABCG2, indicating that the crypt may provide

- Increased hemidesmosomes at limbus

the niche for corneal epithelial SC (Fig. 3.4).

basal epithelium

3.4

Limbal Stem Cell Deficiency

- Increased mitosis rates at limbus

– Medicamentosa including preservatives

– Increased proliferative potential

- Idiopathic

of limbal basal cells

- Absence of cytokeratin 12 in limbal

basal cells

3.4.2

- Absence of gap junctions in limbal

Effects of Limbal Stem Cell Deficiency

basal cells

(Modified from Dua et al. [25])

- Presence of certain enzymes such

as alpha-enolase and ABCG2

The hallmark of limbal stem cell deficiency is

- Different basement characteristics

‘conjunctivalisation’ of the cornea and the most

at limbus compared to central cornea

significant clinical manifestation is a persistent

- Presence of limbal epithelial crypts

corneal epithelial defect.

(niche)

The clinical symptoms of limbal deficiency

may include decreased vision, photophobia,

tearing, blepharospasm, and recurrent episodes

3.4

of pain (epithelial breakdown), as well as a his-

Limbal Stem Cell Deficiency

tory of chronic inflammation with redness.

Depending on the extent of limbal involve-

3.4.1

ment, SC deficiency can be partial or total. Par-

Causes of Limbal Stem Cell Deficiency

tial SC deficiency may vary in extent to involve

the pupillary area, when intervention is usually

Stem-cell deficiency can be congenital or ac-

required, or exclude the visual axis when none

quired. Congenital SC deficiency occurs as a re-

or minimal intervention with topical medica-

sult of hereditary aplasia of limbal stem cells as

tion may be required. Further, partial SC defi-

occurs in aniridia and congenital erythrokera-

ciency may vary in severity from mild, when

todermia. More often though, stem cell defi-

only an abnormal epithelial sheet covers a vari-

ciency is acquired as a result of extraneous in-

able area of the cornea, to severe when a part of

sults that acutely or chronically destroy limbal

the cornea, usually including the pupillary area,

stem cells. These include chemical or thermal

is covered by a thick fibrovascular pannus.

injuries, ultraviolet and ionising radiation,

The clinical features of SC deficiency, from

Stevens-Johnson syndrome, advanced ocular

mild to severe, include the following [13, 14, 18,

cicatricial pemphigoid, multiple surgery or

21, 22, 24, 43, 64, 88]: (a) loss of limbal anatomy,

cryotherapy, contact lens wear, or extensive/

(b) irregular, thin epithelium, (c) stippled fluo-

chronic microbial infection such as trachoma.

rescein staining of the area covered by abnor-

Keratitis associated with multiple endocrine de-

mal epithelium, (d) unstable tear film, (e) fila-

ficiencies, neurotrophic (neural and ischaemic)

ments and erosions, (f) superficial and deep

keratopathy and chronic limbitis also lead even-

vascularisation, (g) persistent epithelial defects

tually to SC deficiency but are less common [13,

leading to ulceration, melting and perforation,

18, 24, 33, 41, 42].

(h) fibrovascular pannus, and (i) scarring, kera-

tinisation and calcification.

Summary for the Clinician

1. Loss of limbal anatomy: The normal limbal

∑ Causes of limbal stem cell deficiency:

architecture with rows of palisades and the

∑ Congenital: aniridia, erythrokeratodermia

perilimbal vascular arcade is usually best de-

Acquired:

fined at the superior and inferior limbus. The

- Chemical and thermal burns

architecture may vary depending on age of

- Chronic inflammatory disorders

the individual. With increasing age the defi-

- Progressive cicatrisation conditions -

nition of palisades becomes less distinct

OCP, SJS

nasally and temporally. Pigmentation of the

- Prolonged contact lens wear

limbal palisades is a feature in some races.

– Multiple ocular surface surgery

Alterations in limbal anatomy include con-

Chapter 3

Transplantation of Limbal Stem Cells

Fig. 3.5. A Signs of mild limbal stem cell deficiency - peripheral conjunctivalisation highlighted with fluo-

rescein staining. The junction of corneal and conjunctival phenotypes of epithelia is marked with arrows.

B Peripheral vascularisation with loss of limbal architecture

tiguous or patchy fluorescein staining of

conjunctiva derived cells at the limbus and

extending onto the peripheral cornea, seg-

mental limbal hyperaemia indicating chron-

ic inflammation, thickening of limbal epithe-

lium, vascularisation of peripheral cornea

and scarring (Figs. 3.1B, 3.5A, B).

Irregular, thin epithelium: When the initial

injury is mild and superficial or the disease

process leading to stem cell deficiency is

slowly progressive, loss of a segment of

limbal epithelium may occur without signif-

icant damage to the substratum. A sheet of

Fig. 3.6. Peripheral conjunctivalisation with pooling

conjunctival/metaplastic epithelium conse-

of dye and stippled staining of the abnormal epitheli-

quently covers the cornea without any no-

um, between 12 and 3 o’clock

table vascularisation. This epithelium is usu-

ally thin and irregular as can be seen by the

pooling of fluorescein dye at the junction of

over it and areas of negative and positive flu-

the abnormal and remaining normal epithe-

orescein staining.

lium (Fig. 3.6, see also Fig. 3.11A) [14].

5. Tags of loose epithelium, filaments with mu-

Stippled fluorescein staining of the area cov-

cus and recurrent erosions are other features

ered by abnormal epithelium: The abnormal

associated with the abnormal epithelial cov-

conjunctival/metaplastic epithelium readily

er on the cornea.

takes up fluorescein dye [43], allowing easy

6. Superficial and deep vascularisation: In

visualisation of the abnormal cells and their

moderate to severe cases of stem cell defi-

pattern of distribution. The abnormal fluo-

ciency, superficial and/or deep vascularisa-

rescein-staining

‘conjunctivalised’ epitheli-

tion of the cornea occurs. It is largely re-

um may take on the pattern of columns,

stricted to the area of stem cell deficiency

whorls or wedges with the broad base to-

and may affect a segment of the limbus or the

wards the limbus and the narrow curving

entire circumference may become involved

apex toward the corneal centre (Figs. 3.5A,

(Fig. 3.7).

3.6) [22].

7. Persistent epithelial defects (Fig. 3.8): Chron-

Unstable tear film: The abnormal epithelium

ic non-healing ulceration of the corneal

demonstrates a rapid tear film break up time

epithelium or cycles of repeated breakdown

3.4

Limbal Stem Cell Deficiency

Fig. 3.7. Superficial and deep vascularisation with a

Fig. 3.8. Persistent epithelial defect and fibrovascu-

fibrovascular pannus encroaching on the cornea fol-

lar pannus on cornea related to total stem cell defi-

lowing chemical burn in which 9.5 clock hours of the

ciency following unilateral alkali

(cement) burn

limbus and 60 % of the conjunctiva were involved

(clinical grade 12/65 %) (with permission from Br J

(clinical grade - 9.5/60 %) (with permission from Br J

Ophthalmol: Dua HS and Azuara-Blanco A 2000;

Ophthalmol: Dua et al. 2001; 85:1379-1383)

84:273-278)

Fig. 3.9. A Right eye of patient with 10 clock hours of

volvement (clinical grade 10/70 % RE, 12/90 % LE).

limbus and 70 % conjunctival involvement following

Scarring, vascularisation, adhesions and some kera-

a chemical (alkali) burn. B Left eye of same patient

tinisation are present. The lids on both sides were also

with 12 clock hours of limbs and 90 % conjunctival in- severely damaged

followed by healing, associated with a chron-

ing thickness (Figs. 3.7, 3.8) [49]. This tissue

ic low grade inflammation, is a feature of lim-

supports the thickened multilayered con-

bal stem cell deficiency. These defects are li-

junctiva derived epithelium.

able to lead to deep stromal infiltrates that

9. Scarring, keratinisation and calcification:

may or may not be related to infection. The

The end stage of the aftermath of limbal stem

edges of the epithelial defect have a distinct

cell deficiency, whatever the cause, is scar-

rolled-up or heaped appearance. Over time,

ring and eventually calcification of the

progressive melting of the corneal stroma

affected tissue. Usually by this stage the in-

with perforation can occur.

flammation has subsided and the eye is com-

8. Fibrovascular pannus: In moderate to severe

paratively comfortable. In patients who have

cases of stem cell deficiency, epithelial cover

associated severe dry eyes the covering ep-

of the denuded cornea is associated with en-

ithelium becomes totally or partially kera-

croachment of fibrovascular tissue of vary-

tinised (Fig. 3.9 A, B).

Chapter 3

Transplantation of Limbal Stem Cells

dye tends to pool along the junction of the

Summary for the Clinician

sheets of corneal and conjunctival epithelial cell

∑ Effects of limbal stem cell deficiency

phenotypes.At this junction, the corneal epithe-

- Mild Æ severe

lial sheet shows tiny processes or undulations

- Loss of limbal anatomy

that give the junction its characteristic appear-

- Conjunctival epithelial ingress onto

ance.

cornea - stippled fluorescein staining

Loss of architecture of the limbal palisades of

- Columnar keratopathy

Vogt and vascularisation are other common fea-

- Unstable tear film over affected area

tures. When damage is extensive, vascularisa-

- Frank conjunctivalisation

tion occurs in the form of fibrovascular pannus,

- Corneal vascularisation -

which increases the thickness of the affected

superficial and deep

area of the cornea. However, the underlying

- Fibrovascular pannus covering corneal

corneal stroma may be considerably thinned by

surface

the initial insult of disease process.

- Persistent epithelial defect

The presence of goblet cells on impression

- Stromal melting

cytology specimens taken from the corneal sur-

- Perforation, scarring, calcification

face or in biopsy specimens of the fibrovascular

- Keratinisation

pannus covering the cornea is pathognomonic

of conjunctivalisation of the cornea (Fig. 3.10 A)

[25, 69]. Biopsy specimens also demonstrate a

3.4.3

multilayered, at times keratinised epithelium

Diagnosis of Stem Cell Deficiency

overlying dense fibrous and vascular tissue

(Fig. 3.10 B). Intraepithelial lymphocytes, which

The diagnosis of stem cell deficiency remains

are a feature of conjunctival epithelium, are also

essentially clinical. On slit lamp biomicroscopic

seen on conjunctivalised corneal epithelium.

examination, the conjunctivalised cornea pres-

These are predominantly CD8+/*HML-1 + cells

ents a dull and irregular reflex. The epithelium

(cytotoxic T lymphocytes expressing the hu-

is of variable thickness and translucent to

man mucosal lymphocyte antigen) [23, 25]. Fea-

opaque. Conjunctival epithelium on the cornea

tures of squamous metaplasia or loss of cornea

appears to be more permeable than corneal ep-

specific cytokeratins (CK 3/12) on immunohis-

ithelium and takes up fluorescein stain in a stip-

tology are other effects noted on biopsy speci-

pled or punctate manner. In cases of partial

mens.

conjunctivalisation of the cornea, fluorescein

Fig. 3.10. A Impression cytology from surface of cornea with stem cell deficiency and a fibrovascular pannus

showing goblet cells. PAS stain, ¥400. B Biopsy of fibrovascular pannus showing multilayered epithelium,

vascularisation and intraepithelial lymphocytes along the basal layers

3.5

Limbal Transplant Surgery

sheet derived from any existing sector of limbus

Summary for the Clinician

in the affected eye. This can be achieved by

∑ Diagnosis of limbal stem cell deficiency

SSCE (see below) [14, 15], especially if the cornea

- Essentially clinical

is partially covered by a layer of thin, metaplas-

- Impression cytology - goblet cells on

tic, conjunctivalised epithelium. If no healthy

cornea pathognomic

sector of limbus is available in the affected eye

- Biopsy - multilayered epithelium,

and if the other eye is normal with a positively

intraepithelial lymphocytes, vessels

documented absence of involvement in the

- Vimentin and CK 19 positive cells in

original injury, autologous limbal transplanta-

central cornea (normally present

tion should be considered. If the other eye is

in peripheral cornea and limbus)

also affected or the underlying condition is a

systemic illness such as Stevens-Johnson syn-

drome, allografts from a living related donor or

3.5

from a cadaver donor should be considered. In

Limbal Transplant Surgery

the acute stage of limbal stem cell deficiency, for

example acute chemical burns, auto-limbal or

3.5.1

living related donor limbal transplants should

Principles

be avoided at all costs. The chances of the trans-

planted material becoming caught up in the in-

Management of stem cell deficiency can be con-

flammatory and scarring process are high with

sidered in the following steps:

loss of a valuable resource for future recon-

struction. Use of auto or living related donor tis-

After Acute Injury. When a patient presents

sue, if available, should be attempted in quiet

after an acute insult it should be ascertained

eyes. All the above procedures can be comple-

whether the involvement of the limbus is partial

mented with amniotic membrane transplanta-

or total. This can be done by use of fluorescein

tion. Penetrating keratoplasty may be combined

stain and slit lamp examination. If partial,

with or following any of these procedures.

appropriate medication required for the under-

Limbal transplantation involves taking a

lying cause and to control inflammation should

lamellar strip of limbal tissue, usually with

be initiated. The eye should be examined at 24-

some adjacent peripheral cornea and/or con-

or 48-h intervals and the process of re-epithe-

junctiva and transplanting it to a suitably pre-

lialisation observed. If this is occurring from the

pared bed in the host eye. Sutures are usually

remaining intact limbal epithelium [21], this

required to keep the donor graft in place.

should be encouraged and any attempt at re-ep-

ithelialisation from the conjunctival epithelium

should be discouraged by sequential sectoral

3.5.2

conjunctival epitheliectomy (SSCE, see below)

Preoperative Considerations

[14, 15]. If total, allow the cornea to be covered by

conjunctival epithelium, if possible, before con-

All associated lid abnormalities, intraocular

templating surgical intervention. This may take

pressure problems and presence of cataract

several days. The guiding principle should be

should ideally be dealt with prior to undertaking

that corneal epithelial cover for cornea and con-

ocular surface restorative surgery. Symble-

junctival epithelial cover for conjunctiva is the

pharon correction with amniotic membrane or

ideal end result but conjunctival epithelial cov-

buccal mucosa graft should also precede stem

er for cornea is preferable to no epithelial cover

cell grafting. At times, if a corneal graft proce-

to cornea.

dure is being contemplated at the time of stem

cell grafting, it can be combined with cataract

In Established Cases. The principles underly-

extraction and lens implantation. When an intu-

ing surgical procedures involving limbal stem

mescent cataract is associated with raised pres-

cells are firstly to expand the corneal epithelial

sure, corneal grafting may become a necessity if

Chapter 3

Transplantation of Limbal Stem Cells

a dense fibrovascular pannus or corneal scar

∑

In established cases:

precludes visualisation of the interior of the eye.

Treat eye lid problems, glaucoma and

Patients with limbal SC deficiency and con-

conjunctival adhesions first

junctivalised corneal surface tend to manifest

Partial or total

persistent chronic inflammation. Stem cell

–

Partial:

grafts do not perform well in the presence of in-

a) Visual axis not involved: sympto-

flammation and can be destroyed by the inflam-

matic, lubricants of SSCE

matory and scarring processes. Ideally inflam-

b) Visual axis involved: SSCE

mation should be controlled and the eye

c) Dense fibrovascular pannus:

rendered as quiet as possible with the use of

sector limbal transplant

topical and systemic steroids or other immuno-

Total:

suppressants which may become necessary in

a) Unilateral: auto-limbal transplant

some conditions such as Stevens-Johnson syn-

b) Ex vivo expansion of autologous

drome and ocular cicatricial pemphigoid.

limbal cells

Most stem cell grafts do not survive in a dry

c) Bilateral: allo-limbal transplant

(eye) environment. At times the injurious insult

d) Ex vivo expansion of cells (living

resulting in stem cell deficiency also results in a

related, living non-related, cadaver)

severe dry eye state. In such situations, if topical

e) Amniotic membrane and autologous

lubricants including autologous serum drops,

serum drops as adjuncts

punctal occlusion and buccal mucosa grafts do

f) Allo-transplants require systemic

not restore adequate moisture to the ocular sur-

immunosuppression

face, a keratoprothesis procedure should be

considered.

Summary for the Clinician

3.6

∑

Treatment algorithm

Surgical Techniques

∑

General principles:

- Manage underlying factors, e.g.,

3.6.1

chronic inflammation, contact lens wear,

Sequential Sector Conjunctival

topical medications

Epitheliectomy (SSCE) [14, 15]

- Topical lubrication

(Figs. 3.11, 3.12)

- All associated problems, e.g., raised

pressure, conjunctival adhesions,

In cases with partial, mild to moderate conjunc-

lid malpositions, should be addressed

tivalisation of the cornea, without significant fi-

before undertaking ocular surface

brovascular pannus, removal of the conjuncti-

reconstruction

valised epithelium is all that is required. This

- Limbal transplants do not perform well

can be achieved at the slit lamp under topical

in dry eyes

anaesthesia, using a crescent blade or a surgical

∑

In acute limbus injury:

knife. It is important to remove all conjunctival

- If partial, i.e. some limbus is surviving -

epithelium, especially along its line of contact

allow corneal epithelialisation to occur

with the remaining corneal epithelium. Follow-

from limbus derived cells - SSCE

ing removal of conjunctival epithelium from the

- If total:

corneal surface, it is important to closely moni-

a) Allow conjunctival epithelium

tor the patient to ensure that the denuded sur-

to grow onto cornea

face is re-epithelialised by cells derived from the

b) Transplant sheet of ex vivo expanded

remaining corneal epithelial sheet, i.e. limbal

limbal epithelial cells

derived cells and not by conjunctival cells. This

c) Avoid use of autologous or living

can be effected by repeatedly debriding (se-

related donor tissue until acute

quential epitheliectomy) any conjunctival ep-

inflammation is well under control

ithelium that encroaches upon the limbus until

3.6

Surgical Techniques

Fig. 3.11 A-D. Sequential sector conjunctival ep-

lium too has started to re-encroach on the cornea.

itheliectomy (SSCE, H.S. Dua). A Conjunctivalisation

D After complete healing, the visual axis is now

of the cornea involving the visual axis following

covered by healthy corneal epithelium. A new line of

chemical injury. The demarcation between the two

contact between conjunctival and corneal epithelium

phenotypes of cells is clearly visible. B Appearance

is established (fluorescein stained anterior segment

immediately after removing the abnormal epithelium

photographs). The patient’s vision improved from

(epitheliectomy). C The corneal epithelial sheet is mi-

3/18 to 6/9 (with permission from Br J Ophthalmol:

grating across the surface but the conjunctival epithe- Dua HS 1998; 82:1407-1411)

the limbus and corneal surface is re-populated

has the theoretical advantage of not overstress-

by limbal epithelium derived cells.

ing the small remaining sector of limbal ‘stem’

In cases where only 1 or 2 clock hours of lim-

cells. This technique of SSCE can also be useful-

bal epithelium is surviving, it may be appropri-

ly combined with limbal transplant to allow

ate to attempt re-epithelialisation of the visual

cells derived from transplanted limbal tissue

axis only, with limbal derived cells. An area cor-

(auto or allo) to re-populate the host corneal

responding to the visual axis is debrided off its

surface without ‘contamination’ from conjuncti-

conjunctival epithelial cover and re-epitheliali-

val epithelium (see below).

sation with limbal derived cells is achieved. This

Chapter 3

Transplantation of Limbal Stem Cells

3.6.2

Auto-limbal Transplantation

(Figs. 3.13, 3.14)

In patients where total stem cell deficiency

affects only one eye, an auto-limbal transplant

procedure is the ideal option [6, 19, 42, 48, 49, 88,

89]. It is important, however, to be absolutely

certain that the donor eye was not involved at

the time of the initial injury. In unilateral mani-

festations of systemic diseases, harvesting tissue

from the apparently normal eye is not recom-

mended.

The surgical technique consists of the follow-

ing steps (the author’s [19] modified technique

is described): (a) a 16-mm Flieringa ring is su-

tured in place when the procedure is to be com-

bined with a corneal graft (and lens extraction

with implant). A 360° peritomy is first per-

formed in the recipient eye. (b) The fibrovascu-

lar pannus covering the corneal surface is dis-

sected off at a suitable plane. Any bleeding

points are individually cauterised with light

diathermy. (c) The donor tissue consisting of

corneal-limbal-conjunctival explants is har-

vested from the contralateral normal eye. Two

explants, corresponding to

2 clock hours

(11-1 o’clock and 5-7 o’clock) and consisting of a

very narrow strip (1 mm or less) of peripheral

cornea, limbus and 3 mm of bulbar conjunctiva,

are harvested. The conjunctival area to be re-

Fig. 3.12.

Conjunctivalisation

the

superior

moved is marked with a surgical marker pen.

cornea involving the visual axis. B, C After SSCE with-

The conjunctiva is incised superficially with a

out and with fluorescein stain, respectively. The visu-

pair of scissors and dissected in a superficial

al axis is now clear

plane up to the limbus.An angled bevelled blade

Fig. 3.13 A, B. Diagrammatic representation of autologous limbal transplantation. A Positioning of explants

on recipient limbus at the 12 and 6 o’clock positions without or with (B) a corneal graft (with permission from

Br J Ophthalmol: Dua HS, Azuara-Blanco A 2000; 84:273-278)

3.6

Surgical Techniques

is used to (lamellar) dissect the corresponding

limbal area extending into peripheral cornea to

just inside (central) to the vascular arcade. (d)

Suitable beds may be prepared at the superior

and inferior limbus of the recipient eye by using

the excised explants as templates to mark the

area to be prepared. This is not always essential.

(e) The donor tissue is then sutured onto the re-

cipient eye with two interrupted 10-0 nylon su-

tures at the corneal margin and two along the

scleral edge of the explant. Care should be taken

not to bury the knots in the explant tissue as this

could strip the explants off when attempting to

remove the sutures in the postoperative period.

At times the knots may be left unburied to facil-

itate removal. The conjunctiva of the recipient

eye is then approximated to the donor conjunc-

tiva with interrupted 8-0 Vicryl sutures (ab-

sorbable), taking a bite into episclera. (f) When

a penetrating keratoplasty is also required, this

is performed after the limbal explants are first

sutured in place. (g) A bandage contact lens is

placed on the cornea and subconjunctival an-

tibiotics and corticosteroids are injected at the

end of the procedure.

Adjunctive use of amniotic membrane can

be made either as a graft to provide a suitable

bed for limbal explant derived epithelial cells to

grow on the cornea and/or as a patch to prevent

conjunctival epithelial cells from extending

onto the cornea and admixing with the limbal

explant derived cells (see below).

3.6.3

Allo-limbal Transplantation

Fig. 3.14 A-C. Auto-limbal transplantation. A Pre-

3.6.3.1

operative persistent epithelial defect following an al-

Living Related Donor

kali (cement) burn as shown in Fig. 3.8. B Postopera-

tive status after auto-limbal transplants at the 6 and

When a living related donor, who is tissue

12 o’clock positions. The patient’s eye is stable over

5 years postoperatively (with permission from Br J matched to the recipient, is available, tissue is

Ophthalmol: Dua HS, Azuara Blanco A

2000;

harvested from one donor eye and used on the

84:273-278. C Donor site for autologous limbal trans-

recipient eye exactly as described above for

plant, stained with fluorescein. Note that the central

auto-limbal transplantation [10, 70].

edge of the removed tissue needs to extend just cen-

tral to the limbal vascular arcade

Chapter 3

Transplantation of Limbal Stem Cells

Fig. 3.15 A-E. Diagrammatic representation of allo-

the donor explant is placed slightly peripheral to the

limbal transplantation. A Injection of air to firm the

recipient limbus, more than one donor may be re-

donor globe. B Harvesting the limbal circumference

quired as shown. E Combined allo-limbal transplant

from the donor globe. C Positioning of explants on re-

and corneal graft (with permission from Br J Oph-

cipient limbus without C or with D a corneal graft. As thalmol: Dua HS, Azuara-Blanco A 1999; 83:414-419

3.6.3.2

corneal diameter (i.e., average of vertical and

Cadaver Donor

horizontal corneal diameter) is used to trephine

the central donor cornea to one-fourth to one-

In most instances, limbal tissue is obtained

fifth of the stromal depth

(approximately

from cadaver donor eyes [16, 41, 42, 48, 81-83, 88,

150 mm). Proper centration is important to en-

89, 91]. In such an event, tissue matching is not

sure that a uniform width of peripheral cornea

usually practical. In the author’s protocol, a pair

is obtained. (c) Superficial lamellar dissection

of ‘fresh’ donor eyes is used within 48 h of death.

of the peripheral cornea is then carried out us-

Donor eye retrieval should be done within 24 h

ing an angle bevelled blade, and extended into

of death and surgery within the next

24h.

the sclerocorneal junction and 1 mm beyond,

Donor age of less than 50 years is preferred.

into sclera. Approximately 1-2 mm of donor

‘Fresh’ and ‘young’ donor eyes are preferred be-

conjunctiva, if present, is maintained. The dis-

cause the success of the procedure depends on

sected tissue is divided at one point and exci-

the transplantation of healthy limbal stem cells.

sion completed with a curved scissors, by cut-

The surgical technique consists of the follow-

ting along the outer circumference of the

ing steps (the author’s technique [16] is de-

dissected tissue. The limbal tissue to be grafted

scribed) (Fig. 3.15): Donor limbus tissue is pre-

thus consists of an open ring of peripheral

pared before the patient is anaesthetised. (a)

corneal and limbal epithelium (and conjuncti-

The donor eyeball is inflated with air (1-2 ml),

val epithelium at places), and superficial

injected through the stump of the optic nerve,

corneal, limbal and scleral stroma. (d) Prepara-

to make the globe firm. (b) The globe is fixed on

tion of the recipient eye is similar to that de-

a Tudor Thomas stand. A vacuum (or manual)

scribed for auto-limbal transplantation except

trephine with a diameter 3 mm smaller than the

that a ‘bed’ is not prepared to receive the limbal

3.6

Surgical Techniques

ring explant. The ‘open ring’ of donor tissue is

corticosteroids are injected at the end of the

placed on the host limbus and sutured with in-

procedure.

terrupted 10-0 nylon sutures at the corneal and

Adjunctive use of amniotic membrane can

scleral margin. Six to eight sutures are first

be made either as a graft to provide a suitable

passed along the inner (corneal) edge of the

bed for limbal explant derived epithelial cells to

donor tissue and partial thickness of host

grow on the cornea and/or as a patch to prevent

corneal stroma. A similar number of sutures are

conjunctival epithelial cells from extending

then passed directly opposite to the inner su-

onto the cornea and admixing with the limbal

tures, along the outer (scleral) edge of the donor

explant derived cells (see below).

tissue. These are anchored to the superficial

sclera of the host. The tension on these sutures

determines the final tension on the inner su-

3.6.4

tures. The knots are trimmed and buried. (e)

Adjunctive Surgery

This method invariably leaves a small gap (ap-

proximately 5-8 mm) between the two ends of

3.6.4.1

the donor tissue ring (superiorly). This is filled

Amniotic Membrane Grafts

with a piece of donor limbal tissue, cut to size,

harvested from the other eye of the same donor.

The amniotic membrane serves as a useful ad-

This piece usually requires a couple of addition-

junct to stem cell grafting [2, 17, 26, 50, 77, 90]. It

al sutures along either edge. (f) The host con-

is commonly deployed to provide a suitable

junctiva is approximated to the scleral edge of

substratum for the transplanted limbal graft

the transplanted limbal ring with interrupted

derived epithelial cells to migrate on and form

8-0 Vicryl sutures (absorbable). (g) A penetrat-

adhesion complexes. After excision of the fibro-

ing keratoplasty if required at the time of sur-

vascular tissue, if the underlying host bed is

gery is performed after the limbal ring is su-

found to be irregular and scarred, use of a 9- or

tured in place. The donor graft for penetrating

10-mm disc of amniotic membrane, epithelial

keratoplasty

(usually

7-7.5 mm) is obtained

side up, can provide a suitable substratum for

from the central cornea of the donor whole

the transplanted limbal derived epithelial cells

globe. (h) A bandage contact lens is placed on

to migrate upon. The amniotic membrane can

the cornea and subconjunctival antibiotics and

also be deployed as a biological bandage to the

Fig. 3.16 A, B. Use of double amniotic membrane to

and the outer membrane, epithelial side down, acts as

prevent admixture of conjunctival and corneal ep-

a patch. B Regenerating cells from the peritomised

ithelium on the corneal surface. A The inner mem-

conjunctiva are seen growing on the outer mem-

brane disc is sutured with the epithelial surface up to

brane. In the absence of the outer membrane (patch)

act as a graft and substrate for the cells to grow on,

these would have encroached on the corneal surface

Chapter 3

Transplantation of Limbal Stem Cells

denuded corneal stroma, allowing epithelialisa-

ganathan, submitted to Br J Ophthalmol 2005).

tion to occur beneath it whilst trapping inflam-

This offers the opportunity to carry HLA typing

matory cells and downregulating inflammation

and matching and also allows for depletion of

and scarring at the same time. Two amniotic

antigen presenting Langerhans cells. It should

membranes, one inner one serving as a graft

therefore be possible to use organ culture pre-

and one outer membrane serving as a patch, can

served corneoscleral discs for simultaneous

be simultaneously applied. The outer mem-

allo-limbal transplant and keratoplasty with

brane is sutured such that its edges are tucked

reduced risk of immune mediated rejection.

under the peritomised conjunctiva. Conjuncti-

va derived epithelium then grows on the outer

membrane and is prevented from admixing

3.6.5

with limbus-derived epithelium that is spread-

Postoperative Treatment

ing onto the corneal surface. This technique was

developed by the author and is regularly em-

Topical preservative-free antibiotic drops such

ployed [26] (Fig. 3.16). It avoids the need for

as chloramphenicol 0.5 % are used four times a

SSCE postoperatively. The outer membrane falls

day for the first month. Topical preservative-

off or can be removed in 10-14 days. For further

free steroid drops such as prednisolone acetate

details on amniotic membrane, see Chap. 2 on

1 % are used four times a day for the first

amniotic membrane transplantation.

8-12 weeks, and slowly tapered during the ensu-

ing weeks. A low dose of topical corticosteroids

3.6.4.2

(one drop per day) is maintained unless eleva-

Ex Vivo Expansion of Limbal Cells

tion of intraocular pressure occurs. Autologous

serum eyedrops (20 %) [52, 92, 93] are given

Limbal ‘stem cell’ transplantation can also be

hourly until the epithelialsation is complete,

carried out by ex vivo expansion of limbal ep-

usually in 7-10 days. Preservative free artificial

ithelial cells, either directly or on a substrate of

tears are then instituted. It is important to close-

fibrin, collagen or amniotic membrane [26, 62,

ly monitor the re-epithelialisation process until

74, 75, 87].

completed. Any attempt by conjunctiva derived

cells to encroach onto the corneal surface

3.6.4.3

should be thwarted by SSCE, until the periphery

Corneal Grafts

(limbus) of the host cornea is re-epithelialised

by limbus derived cells.

Lamellar or full thickness corneal grafts can be

All patients undergoing allo-limbal trans-

combined with auto- or allo-limbal transplanta-

plantation also need systemic immunosuppres-

tion. This may be necessary when the cornea

sion. Besides steroids, azathioprine, cyclosporin

damage is severe and when it is considered that

A, rapamycin, mycophenolate mofetil and

the host corneal bed will not support a healthy

tacrolimus (FK506, Prograf) have been used [11,

epithelium despite use of an amniotic mem-

72, 78, 97]. Theoretically, immunosuppression

brane. In general terms, a definitive corneal

should be continued almost indefinitely. The

graft for visual purposes should be deferred un-

author has used cyclosporin A and of late FK506

til ocular surface epithelial integrity has been

up to 18 months postoperatively [78]. Attempts

restored by limbal transplantation. However, in

to reduce or stop the drug have resulted in lim-

a recent study using limbal tissue remaining

bal and/or corneal graft rejection episodes. For-

after keratoplasty from organ cultured cor-

tunately, the dose required to prevent or control

neoscleral discs, we have shown that such tissue

rejection episodes is very low

(2-8 mg/day,

(where the death to enucleation time and the

maintaining a blood trough level of 1-12 mg/l).

time lapse between enucleation to placement in

Serious side effects, though they occur, are not

organ culture is short and where the donor is

very common, but require constant monitoring

relatively young) retains good proliferative ca-

of patients and measures of kidney and liver

pacity for up 30 days in storage (V. Shanmu-

functions. It is good practice to involve a clinical

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Limbal Stem Cell Culture

José L. Güell, Marta Torrabadella, Marta Calatayud, Oscar Gris,

Felicidad Manero, Javier Gaytan

Core Messages

4.1

∑ The stem cells of the corneal epithelium

Introduction

seem to be located in the limbal basal layer

and are the ultimate source of constant

The stem cells of the corneal epithelium seem to

epithelial renewal

be located in the limbal basal layer and are the

∑ Ex vivo expansion of limbal epithelial stem

ultimate source of constant corneal epithelial

cells has been developed mainly to circum-

renewal. A new strategy of treating limbal stem

vent potential complications, for both the

cell deficiency is to transplant a bioengineered

recipient and the living donor, arising from

graft by expanding limbal epithelial stem cells

standard conjunctival limbal transplanta-

ex vivo on amniotic membrane.

tion

Ex vivo expansion of limbal epithelial stem

∑ With the appropriate procurement and

cells has been developed to circumvent poten-

preservation, human amniotic membrane

tial complications both for the recipient and the

can be used as a biological substrate with-

donor arising from conjunctival limbal auto-

out viable and proliferative active cells but

graft transplantation. This technique expands

with the advantages of basement mem-

limbal epithelial progenitor cells from a small

brane and as a source of beneficial bio-

biopsy using a 3T3 fibroblast feeder layer [13] or

logical factors

amniotic membrane. Using the amniotic mem-

∑ To examine the epithelial phenotype, im-

brane to constitute such a composite graft, suc-

munostaining techniques are used with a

cessful reconstruction of the normal corneal

panel of monoclonal antibodies to mucins

surface has been achieved in several human

and keratins

studies with partial or total limbal stem cell

∑ The preparation of human amniotic mem-

deficiencies [8, 9].

brane and the culture of explanted tissue

As previously described, amniotic mem-

must be performed by specially trained

brane, the innermost layer of the fetal or placen-

personnel in a stem-cell management

tal membrane, consists of an epithelial mono-

laboratory

layer, a thick basement membrane, and an

∑ Preliminary clinical experience is highly en-

avascular stroma (Fig. 4.1). With appropriate

couraging, not only regarding final clinical

procurement and preservation, amniotic mem-

outcome but also because of the flexibility

brane can be used as a biological substrate with-

of the technique, the increase in possible

out viable and proliferative active cells [8]. It is

cases to be treated and the reduced need

thus non-immunogenic and therefore does not

for systemic immunosuppression

require immunosuppression when used for

∑ Ocular surface reconstruction is becoming

transplantation. The amniotic membrane stro-

a much more staged than artistic approach,

ma also contains quantities of growth factors,

and we are of the opinion that its final clini-

various antiangiogenic and anti-inflammatory

cal results will improve strongly in the near

proteins and natural inhibitors to various pro-

future especially with the help of the new

teases.

cell bioengineering technique

Chapter 4

Limbal Stem Cell Culture

body, which recognizes K3 keratin, stains the

suprabasal limbal epithelium and the full thick-

ness of the central corneal epithelium, but not

the conjunctival epithelium. AE5 antibody

stains suprabasal human limbal epithelial cells

(HLEC) cultured on amniotic membrane for

13-21 days. Immunostaining for K12 keratin

by AK2 antibody is also positive for limbal

suprabasal epithelial cells and for the full thick-

ness of the corneal epithelium, but negative for

the conjunctival epithelium in vivo. HLEC on

amniotic membrane are negative for AK2. K14

keratin is expressed in the basal and suprabasal

cell layers of the conjunctival limbal and pe-

Fig. 4.1. Human amniotic membrane. Cross-section

ripheral corneal epithelium, but is found pre-

reveals a cuboidal epithelial monolayer (1), a thick

dominantly in the basal epithelial cells of the

basement membrane (2) and an avascular stroma (3)

central corneal epithelium. HLEC cultured on

amniotic membrane showed full thickness

Recently, Grueterich et al. identified the cul-

staining to K14 keratin after 13-21 days of cul-

ture environment that will favor the mainte-

turing. MUC5AC (Mucina 5AC) recognizes con-

nance of the stem cell containing the limbal

junctival goblet cell secreting mucins and stains

epithelial phenotype [9]. This is achieved by

conjunctival goblet cells in vivo. MUC5AC do

culturing limbal explants on an intact human

not stain any cells cultured on amniotic mem-

amniotic membrane, which retains the devital-

brane. Collectively, these results indicate that

ized amniotic epithelium, without the use of a

the resultant phenotype of HLEC grown on am-

3T3 fibroblast feeder layer. The expanded ep-

niotic membrane retains a limbal origin, is pre-

ithelium on intact amniotic membrane adopts a

dominantly basal epithelial cells, and remains

limbal epithelial phenotype whereas that on

undifferentiated (Table 4.1) [9].

denuded amniotic membrane reveals a corneal

epithelial phenotype.

4.3

Preparation of Human Amniotic Membrane

4.2

Epithelial Phenotype

Amniotic membrane tissue can be obtained,

processed, and preserved frozen as reported by

To examine the epithelial phenotype, immunos-

Lee and Tseng [9] at the Eye Bank. The amniot-

taining techniques are used with a panel of

ic membrane measuring 5¥5 cm, after thawing

monoclonal antibodies to mucins and keratins.

and washing, is tightened on a 3-cm culture

In normal ocular surface epithelia, AE5 anti-

plate with the basement membrane side up.

Table 4.1. Immunostaining expression of keratins K3, K12 and K14, and MUC5AC of corneal epithelium, con-

junctival epithelium, limbal epithelium and HLEC grown on amniotic membrane cultures

Immunostaining

Corneal

Conjunctival

Limbal epithelium

HLEC

epithelium

K3 keratin

Positive

Negative

Positive suprabasal

K12 keratin

Positive

Negative

Positive suprabasal

Negative

K14 keratin

Positive basal

Positive

MUC5AC

Negative

Positive

Negative

4.5

Tissue Procurement

4.4

Culture of Explanted Tissue

A piece of limbal tissue measuring 1¥2 mm con-

taining epithelial cells and part of the corneal

stroma is obtained for ex vivo culture. The tis-

sue source is the contralateral eye in autologous

transplantation or a living donor in related

allotransplantation. Cadaveric donor is also a

source of limbal tissue for conditions in which

both eyes are affected and no living donor is

available.

The obtained tissue is placed with Ham’s F12

medium containing 50 mg/ml gentamicin and

1.25 mg/ml amphotericin B until it is processed.

Limbal tissue is exposed for 5 min to Dispase II

(1.2 U/ml in Mg²⁺ and Ca²⁺ free Hank’s balanced

salt solution, HBSS) at 37 °C under humidified

5 % CO₂. The explants are then cultured in

DMEM medium, which is a 1:1 mixture of

DMEM and Ham’s F12 medium containing

5 ng/ml epithelial growth factor (EGF), 5 mg/ml

insulin,

5 mg/ml transferrin, 5 ng/ml sodium

selenite,

0.5 mg/ml hydrocortisone,

30 ng/ml

cholera toxin A, 0.5% dimethylsulfoxide (DMSO),

Fig. 4.2 A, B.

Ex vivo expansion of limbal stem cells

50 mg/ml gentamicin, 1.25 mg/ml amphotericin

from limbal biopsy. A Culture plate with the tightened

B and 5 % autologous serum, at 37 °C under 5 %

amniotic membrane and a limbal biopsy placed in the

CO₂ and 95 % humidity. The medium is renewed

center (arrows). B Phase-contrast microscopy of the

expanded cells reveals a monolayer of epithelial cells

every 2-3 days [2]. For allogeneic related trans-

of small and uniform size, ¥400

plantation, donor serum is used and for allo-

geneic non-related transplantation AB tested

blood bank serum is employed. The limbal ep-

ithelial cell explants are plated onto the base-

ment-membrane side of the amniotic mem-

4.5

brane, placed in the center. The extent of each

Tissue Procurement

outgrowth is monitored with a phase contrast

microscope. During the expansion phase the

The safety of biological medicinal products re-

limbal epithelial outgrowth exhibits a compact

lies on rigorous control of each of their com-

and uniform cell layer (Fig. 4.2).

ponents. Human tissue should be handled

The culture is maintained for 2-3 weeks, by

according to European Commission Directive

which time the epithelial cells have grown and

2003/63/EC [8]. Corneoscleral tissue from hu-

spread to form a cell layer that covers an area

man donor eyes is obtained after proper in-

2-3 cm in diameter. Every week bacteriological

formed consent in the case of a living donor or

testing is performed to assess microorganism

from an authorized Eye Bank for cadaveric

contamination. The mycoplasma content test

donors. Human amniotic membrane should be

and Gram’s test are performed

24h before

obtained after elective cesarean delivery when

transplantation.

blood-borne microorganisms such as human

immunodeficiency virus, hepatitis virus type B

and C, and syphilis have been excluded by sero-

Chapter 4

Limbal Stem Cell Culture

logic tests. Hepatitis virus type C and human

oratory over a piece of amniotic membrane and

immunodeficiency virus should additionally be

using them in a pathologic eye to restore the

excluded by means of PCR.

corneal epithelium. We prefer amniotic mem-

brane because its basal membrane is very simi-

Summary for the Clinician

lar to corneal epithelial basal membrane [7, 11],

∑ Immature corneal cells are located in the

and the epithelium of the amniotic membrane

epithelial basal layer of the limbus

contains several growth factors that promote

∑ Ex vivo expansion of limbal epithelial cells

cellular proliferation [7]. The main advantage is

is a technique that avoids potential compli-

that in nearly all cases we avoid immunosup-

cations to the donor and the recipient eye

pression, and cause minimal injury to the donor

∑ Immunohistochemical features allow us to

eye, so there is no risk of iatrogenous limbal de-

distinguish immature from mature cells on

ficiency in that eye.

the cultured cells

Limbal deficiency must have been confirmed

∑ Human amniotic membrane provides

previously by clinical examination, impression

the limbal epithelium with an adapted

cytology and, if possible, immunofluorescence

microenvironment and a solid basal layer

on the affected eye.

∑ Amniotic membrane epithelium favors

The process starts with, after informed con-

limbal stem cell growth

sent, the extraction of a limbal biopsy from the

∑ Biological medicinal products rely on

donor eye (the healthy eye of the same patient

rigorous control of each of their compo-

or a relative or cadaver donor eye). It must be

nents and should be handled according to

done in the operating room, under sterile condi-

the specific laws of each country

tions and with great care being taken to avoid

the conjunctival tissue, because we could ex-

pand other cells than corneal epithelial stem

4.6

cells. The size of the biopsy must be as small as

Preliminary Clinical Experience

possible, but large enough to ensure cellular

growth in culture (2 mm²), with a depth of

In standard ocular surface reconstruction, am-

100 mm, without blood vessels or conjunctival

niotic membrane transplantation in the cornea

tissue. Once we have the biopsy, it must be taken

and conjunctiva provides a basement mem-

to the laboratory as soon as possible, where it is

brane, especially when the ocular surface is

treated as mentioned above, and then we need

highly irregular, and a source of biological fac-

to wait for 2 or 3 weeks, depending on the cellu-

tors to enhance and improve reepithelialization

lar growth observed, before we can implant the

[3-7]. Once the amniotic membrane is sutured,

amniotic membrane with the expanded cells on

we may proceed with our standard limbal trans-

the eye. Every process which involves any ma-

plantation over it.

nipulation of the tissue must be done under

Because sclerocorneal limbal tissue is highly

sterile conditions.

vascularized with a high antigenic weight, we

need to prescribe long-term systemic immuno-

suppression in most limbal transplantation cas-

4.6.1

es (cadaver or relative donor).

Principles for Taking the Biopsy

Limbal stem cell culture is a new technique

with obvious advantages over the other tech-

1. Avoid or eliminate the conjunctiva. Conjunc-

niques used to restore ocular surface. The aim is

tival epithelial cells grow easily in culture

to cause as little damage to the ocular surface as

and interfere with corneal epithelium prolif-

possible. Ex vivo expansion of corneal stem cells

eration.

is a technique described previously [7, 8, 11, 13],

2. Perform the biopsy under sterile conditions.

and has been developed by several groups. It

3. Take a biopsy as small as

1-2 mm² and

basically consists of taking a small piece of the

100 mm in depth.

donor eye limbus, expanding the cells in the lab-

4.7

Case Report

4. Take a biopsy from the superior limbus if

On the other hand, one limitation is preopera-

possible, to ensure immature cells are includ-

tive cell type determination. To assess limbal

ed on it (the superior limbus contains more

stem cell transplant success we do not manipu-

stem cells) [13]

late the culture. Rather we implant the central

area, where the graft shows the best growth, and

We usually implant the tissue after a lamellar

we verify the particular biological and im-

keratectomy is performed to eliminate any re-

munohistochemical characteristics of the cul-

mains of conjunctival tissue over the cornea, su-

tured cells with the remaining piece of tissue

turing the graft, with the small biopsy included,

after the surgery. In all cases limbal stem cell

over the corneal surface, then placing a thera-

immunophenotyping is demonstrated.

peutic contact lens over the graft to preserve it

Summary for the Clinician

and avoid blinking-trauma during the first

2 weeks. Postoperative treatment consists of

∑

Amniotic membrane transplantation on the

topical steroids and antibiotics, autologous

cornea and conjunctiva contributes, in

serum drops to promote epithelial growth, and

standard ocular surface reconstruction,

immunosuppressive agents if the biopsy is per-

as a basement membrane and as a source

formed on a relative or unknown donor.

of biological factors

In our series of seven patients transplanted,

∑

In most standard limbal transplantation

the only complication detected in the immedi-

cases, we all need to prescribe long-term

ate postoperative period was corneal melting

systemic immunosuppression

with perforation, which was solved with pene-

∑

The use of amniotic membrane as our

trating keratoplasty (PKP).

“cell transporter” was decided upon

The most important purpose of this tech-

because of our wide and long-term experi-

nique is to expand immature cells from the pro-

ence of using it in our standard ocular

liferative compartment of the limbus and keep

surface reconstructive techniques

them active for as long as possible, which en-

∑

The most important purpose of our tech-

sures a healthy ocular surface, and allows us

nique is to expand immature cells from the

to perform other procedures to restore visual

proliferative compartment of the limbus

acuity such as PKP or lamellar keratoplasty

and keep them active for as long as possible

∑

We must keep in mind the main conceptual

advantages of limbal stem cell culture in

4.6.2

clinical practice

Advantages of Limbal Stem Cell Culture

1. In many cases our patient has enough

4.7

healthy limbal area in at least one of the eyes

Case Report

for a small biopsy to be taken but not for a

large (90°) standard biopsy to be done for

A 31-year-old Caucasian male with a story of

standard transplantation.

bilateral caustication came to us in July 2002. At

2. Many close relatives (HLA matched if possi-

the first exploration of the eyes in early Septem-

ble) will be amenable to a small biopsy but not

ber 2002, the right eye showed visual acuity

to a large one with the known associated risks.

(VA), light perception, diffuse corneal conjunc-

In both (1) and (2), we might eliminate or

tivalization with central perforation and exten-

strongly reduce systemic immunosuppres-

sive superior symblepharon; in the left eye the

sion

VA was count fingers 10 cm, and diffuse corneal

3. In those cases where the donor tissue is from

conjunctivalization with extensive inferior

a cadaver, the main advantage of the culture

symblepharon was seen. Our case notes then

is the density of viable cells at the time of

read as follows:

transplantation compared with the fresh

12.09.02

Emergency PKP plus amniotic mem-

original piece of tissue.

brane as a patch OD

Chapter 4

Limbal Stem Cell Culture

10.12.02

Inferior symblepharon surgery OD

25.02.03

Superior symblepharon surgery OD

26.08.03

Inferior symblepharon surgery OS

08.03.04 Impression cytology OS

(Fig. 4.5):

(Fig. 4.3) only 2 h (10-12 o’clock) with healthy

limbal tissue; healthy limbal area

biopsy for culture on amniotic mem-

brane

25.03.04

Lamellar corneal scar dissection and

cultured cells on amniotic membrane

sutured on the cornea - conjunctival

surface OS

29.04.04 PKP + standard amniotic membrane

(Fig. 4.6) transplantation + lateral temporary

Fig. 4.3. Specimen from a limbal deficiency eye. Pre-

operative image, Papanicolaou’s stain

tarsorrhaphy

19.10.04 Sectorial conjunctival epitheliectomy

(Fig. 4.7) as described by Dua [7] (area from

6 o’clock to 8 o’clock) and selective

suture removal

Fig. 4.4 A, B. Slit-lamp appearance of both eyes in

March 2004. A left eye; B white cornea with vessels,

right eye

Fig. 4.5 A, B. Operating room impression cytology

of the left eye. Note the presence in all the specimens of

goblet cells characteristic of conjunctival epithelium

Chapter 4

Limbal Stem Cell Culture

Gris O, Wolley-Dod C, Güell JL et al. (2002) Histo-

4.8

logical findings after amniotic membrane graft in

the human cornea. Ophthalmology 109:508-512

Future Standard Staging Approach

Gris O, del Campo Z, Wolley-Dod C et al. (2003)

for Ocular Surface Reconstruction

Conjunctival healing after amniotic membrane

graft over ischemic sclera. Cornea 22(7):675-678

1. IOP control: shunt tube if necessary

Grueterich M, Espana E, Tseng SC (2002) Con-

2. Lids and conjunctival “cul de sac” recon-

nexin 43 expression and proliferation of human

structive surgery

limbal epithelium on intact and denuded amniot-

3. Limbal reconstruction: limbal stem cell cul-

ic membrane. Invest Ophthalmol Vis Sci 43:63-71

Koizumi N, Inatomi T, Suzuki T et al. (2001) Cul-

ture will definitely improve the actual results

tivated corneal epithelial stem cell transplanta-

of corneal reconstruction. We must remem-

tion in ocular surface disorders. Ophthalmology

ber that the presence of new conjunctival

108:1569-1574

cells is also necessary in some cases such as

Kruse FE, Joussen AM, Rohrschneider K et al.

pemphigoid or Stevens-Johnson syndrome

(2000) Cryopreserved human amniotic mem-

4. PKP or deep anterior lamellar keratoplasty if

brane for ocular surface reconstruction. Graefes

necessary

Arch Clin Exp Ophthalmol 238:68-75

10.

Lee SH, Tseng SCG (1997) Amniotic membrane

5. Keratoprothesis in those cases where stage 3

transplantation for persistent epithelial defects

and 4 fails

with ulceration. Am J Ophthalmol 123:303-312

11.

Meller D, Pires RTF, Tseng SC (2002) Ex vivo

preservation and expansion of human limbal ep-

References

ithelial stem cells on amniotic membrane cul-

tures. Br J Ophthalmol 86:463-471

Dua HS, Azuara-Blanco A (2000) Limbal stem

12.

Rama P, Bonini S, Lambiase A et al. (2001) Autol-

cells of the corneal epithelium. Surv Ophthalmol

ogous fibrincultured limbal stem cells perma-

44:415-425

nently restore the corneal surface of patients with

Dua HS, Saini JS, Azuara-Blanco A, Gupta P

total limbal stem cell deficiency. Transplantation

(2000) Limbal stem cell deficiency: concept, aeti-

72:1478-1485

ology, clinical presentation, diagnosis and man-

13.

Tsai RJF, Li L-M, Chen J-K (2000) Reconstruction

agement. Ind J Ophthalmol 48:83-92

of damaged corneas by transplantation of autolo-

Dua HS (1998) The conjunctiva and corneal ep-

gous limbal epithelial cells. N Engl J Med 343:

ithelial wound healing. Br J Ophthalmol 82:1407-

86-93

1411

4. Gris O, Guell JL, del Campo Z (2000) Limbal-con-

junctival autograft transplantation for the treat-

ment of recurrent pterygium. Ophthalmology

107: 270-273

Deep Anterior Lamellar Keratoplasty

Gerrit R.J. Melles

dissection depth during surgery, and the avail-

Core Messages

ability of various lasers may provide new possi-

∑ With current techniques, the clinical out-

bilities for the management of anterior corneal

come of a lamellar keratoplasty may be

disorders. In fact, we may be currently witness-

similar to that after penetrating keratoplasty

ing the most dramatic change in the concept of

∑ Compared to conventional penetrating

keratoplasty from being a conventional pene-

keratoplasty, new lamellar keratoplasty

trating procedure towards that of custom-made

techniques provide safer “closed-system”

corneal tissue replacements. Below, the most

surgeries with less morbidity and better

recent developments are summarised and the

clinical outcomes

most important issues concerning DALK are

∑ The scope of surgical tools for lamellar

discussed.

keratoplasty has been expanded to provide

feasible and adjustable techniques

∑ Since postoperative treatment may be

5.2

the most challenging aspect in kerato-

Main Drawbacks of Conventional DALK

plasty surgery, careful patient selection

and psychologic preparation are important

Since the early 1900s, penetrating keratoplasty

in achieving good results

has been the procedure preferred by most sur-

∑ Especially in the patient population eligible

geons for the treatment of corneal disorders. On

for lamellar keratoplasty, the procedure

average, the overall clinical result of penetrating

may have major advantages for both the

keratoplasty seems rather poor and is often

surgeon and the patient, such as longer

complicated by a high degree of astigmatism,

graft survival, less aftercare and less

suture related complications, and incomplete

dependency on the health care system

wound healing. Despite its advantages, i.e. less

∑ Lamellar keratoplasty surgery may be

risk of intraocular complications and allograft

slowly tending towards the use of custom-

rejection, a lamellar procedure is often consid-

made transplants

ered troublesome, mainly because of the risk of

perforation during surgery, the development of

interface haze, and the time-consuming, tedious

surgical technique.

5.1

Introduction

5.3

In the past few years, deep anterior lamellar ker-

Different Concepts

atoplasty (DALK) has seen renewed interest as

an alternative to conventional penetrating ker-

To overcome these problems, several techniques

atoplasty. The introduction of several new dis-

are now available with which to perform a con-

section techniques, the optical visualisation of

trolled deep anterior lamellar keratoplasty pro-

Chapter 5

Deep Anterior Lamellar Keratoplasty

cedure at a planned corneal depth with minimal

disorder may also have a compromised en-

risk of perforation and interface haze develop-

dothelium, due to combined disease, the dis-

ment.

ease itself or prolonged medication (for ex-

∑

The creation of an optical interface by filling

ample, combined dystrophies or herpes

the anterior chamber with air allows the sur-

simplex virus keratitis/endotheliitis).

geon to visually control the dissection depth

∑

Penetrating keratoplasty bad prognosis. In

during the entire surgery. This allows the

patients in whom a conventional penetrating

surgeon to choose the desired dissection

procedure is contraindicated, complete visu-

depth. In cases in which a penetrating

al rehabilitation is often not the primary

keratoplasty may have a bad prognosis, for

goal. In these cases, a relatively shallow dis-

example a patient with Down syndrome or

section depth (70-80 %) may be considered,

recurrent herpes simplex virus keratitis, the

which greatly reduces the risk of perforation

surgeon may aim for a relatively shallow dis-

during surgery, for example, extreme kerato-

section depth to complete the procedure

conus in Down syndrome or long-standing

with a minimal risk of perforation.

recurrent herpes keratitis.

∑

Instead of removing the anterior corneal tis-

∑

Patient age. Young patients with an isolated

sue layer by layer, the cornea may be dissect-

corneal disorder like keratoconus may bene-

ed to the desired depth at the first go. This

fit the most from a lamellar procedure. The

saves time, and if a perforation occurs, it is in

endothelial cell loss after lamellar kerato-

the very early phase of the surgery, so the

plasty has been found to show a similar pat-

procedure can be quickly converted to a

tern to that of the physiological cell loss in a

penetrating keratoplasty.

virgin cornea, which may significantly im-

∑

Instead of performing a blunt dissection,

prove the long-term expectation for graft

several instruments and medical devices as

survival. Since the integrity of the globe is

well as various lasers are currently available

better preserved in a lamellar procedure and

with which to obtain a regular and smooth

the risk of wound rupture or dehiscence may

dissection plane.

be relatively low, a lamellar keratoplasty may

give fewer restrictions to sports and other

Summary for the Clinician

daily activities that are relatively important

∑

New lamellar keratoplasty techniques allow

to young people. With a lamellar procedure,

for a single stromal dissection at an optical-

the risk of allograft rejection may also be

ly controlled depth

greatly reduced, which may give fewer re-

∑

Improved instrument designs and various

strictions to people travelling to or living in

lasers allow the creation of a smooth recipi-

countries with a less sophisticated health

ent stromal bed

care system.

∑

Atopic constitution. The long-term results of

any type of keratoplasty procedure may be

5.4

relatively poor in patients with atopic disease

Important Preoperative Considerations

or a concurrent facial skin disorder. In our

series, the occurrence of relatively serious

Careful patient selection and patient prepara-

and long-standing complications such as

tion may in part determine whether the out-

persistent epithelial defects, suture infil-

come of a deep anterior lamellar keratoplasty

trates, and partial melts proved relatively fre-

procedure is considered succesful both by the

quent in these cases and difficult to manage.

surgeon and the patient. The main parameters

∑

Alternative treatments. Several procedures

for patient selection are:

are currently available that may allow for a

∑ Endothelial cell density. The good condition

visual outcome similar to a deep anterior

of the recipient endothelium is a prerequisite

lamellar keratoplasty, but that are more pa-

for a lamellar procedure. Although relatively

tient friendly. For example, amniotic mem-

infrequent, corneas with an anterior corneal

brane transplantation with subsequent con-

5.6

Choice of DALK Surgical Technique

tact lens fitting often provides a useful visual

eligible for deep anterior lamellar keratoplasty.

acuity. Phototherapeutic keratectomy can be

A fairly large number of these patients may have

effective in the treatment of epithelial dys-

a long history of uncomplicated contact lens

trophies. Intrastromal ring segments may

wear up to the moment that the steepening

give fairly good results in corneal thinning

corneal contour causes unacceptable contact

disorders. The femtosecond laser may also

lens discomfort. Since none of the currently

broaden the possibilities for replacement of

available DALK procedures allows for a con-

specific corneal layers.

trolled restoration of the corneal contour, and

postoperative contact lens fitting often greatly

Summary for the Clinician

improves the final visual outcome, it may be rec-

∑ Important considerations prior to lamellar

ommended to inform the patient that the goal

keratoplasty include assessment of the con-

of the surgery is to flatten the cornea and enable

dition of the recipient endothelium, overall

contact lens wear rather than to restore the

prognosis, patient age, presence of an atopic

corneal surface contour.

constitution, and less invasive treatments

Summary for the Clinician

∑ Patient preparation for lamellar kerato-

5.5

plasty may include a downgrading of

Psychological Preparation of the Patient

expectations and a definition of the

intended goal of surgery

Once the decision to perform a deep anterior

lamellar procedure has been taken, it is recom-

mended that the expectations of the patient are

5.6

appropriately modified. Since most patients are

Choice of DALK Surgical Technique

familiar with the outcome of cataract surgery,

the surgeon may explain to the patient that cur-

Given the indication, the presence of a con-

rent keratoplasty surgical techniques on average

traindication for penetrating keratoplasty, and

do not provide similar visual results.

the anatomy of the individual patient, the sur-

Since a lamellar procedure always bears the

geon may first want to consider whether intra-

risk of perforation and the need for conversion

operative perforation is unacceptable and what

to a penetrating procedure, it should be ex-

dissection depth is desired. In most cases, a dis-

plained to the patient that a lamellar keratoplas-

section depth of >90 % or a separation of De-

ty will be attempted but that in the surgery has

scemet’s membrane from the recipient posteri-

to be completed as a conventional penetrating

or stroma will give the best postoperative result.

procedure. Although the risk of perforation

The surgeon may also want to consider what

with some techniques may be as low as 5 %, the

perforation might occur and/or how it should

patient then is less likely to perceive the surgery

be dealt with during the surgery.

as ‘a failure’ in the event of a perforation, i.e.

∑ Air- or viscodissection of Descemet’s mem-

conversion to a penetrating procedure. The pa-

brane. Separation of Descemet’s membrane

tient should also be informed that secondary

from the recipient posterior stroma by using

surgery may be necessary shortly after the ker-

air dissection (big bubble technique) or vis-

atoplasty to position the donor properly. If the

codissection has the advantage that a near

anterior chamber needs to be filled with air to

anatomical donor to host interface can be

manage a perforation, the patient anticipates

obtained with a minimal risk of interface

on surgical aftercare, whereas such a minor

haze development. The dissection method is

secondary treatment may otherwise alarm the

indirect (not manually controlled but de-

patient as it is quickly perceived as emergency

pendent on the pressure built by air or vis-

surgery to save the eye.

coelastic at the cleavage plane above De-

In the Netherlands, keratoconus is the indi-

scemet’s membrane) and, as a result, if a

cation in approximately half of the patients

perforation occurs, the perforation tends to

Chapter 5

Deep Anterior Lamellar Keratoplasty

be paracentral and large, often requiring

conversion to a penetrating graft. For that

5.7

reason, the surgeon may choose to have

Clinical Results (Figs. 5.1-5.3)

donor tissue available with good quality en-

dothelium.

In conventional anterior lamellar keratoplasty,

∑

Manual corneal dissection at a visually con-

the recipient anterior corneal tissue was re-

trolled depth. To monitor the dissection

moved ‘layer by layer’. The most common tech-

depth during surgery, the anterior chamber

nique was that of lifting the tissue, stretching

should be filled with air to create an optical

the fibres at the dissection plane and cutting the

reference plane. Using an optical reflex, the

fibres with a crescent knife. The donor tissue

surgeon can perform a dissection of up to

was usually dissected using a blunt spatula to

90-95 % depth, removing most of the dis-

avoid perforation. Although the approach ap-

eased corneal stroma. Visualisation of the

peared effective, the dissection methods may

depth of the dissection instruments greatly

have been the major cause for the development

reduces the risk of perforation to less than

of interface haze, the major drawback of a

5 %. If a perforation occurs, the perforation is

lamellar procedure, since it affected the clinical

usually small and located at the 12 o’clock

outcome significantly.

surgical position or in the far periphery. If

Physically the normal cornea is milky white,

necessary, the perforation can be sealed by

but within the spectrum of visible light it ap-

dissecting slightly shallower ‘over’ the perfo-

pears transparent through ‘constructive inter-

ration site, so that the hole is closed by a

ference’, i.e. the incoming light rays will bounce

self-sealing stromal flap. The number of cas-

between the layered collagen fibres until they

es requiring conversion to a penetrating pro-

have crossed the cornea. As a result, any disor-

cedure may therefore be low, so that the

ganisation of the layered fibrous structure may

availability of a donor cornea with good

be expected to degrade the ability of the light to

quality endothelium is not mandatory.

pass the cornea. The scattered light is clinically

∑

Laser dissections. Several groups have evalu-

observed as opaque areas at the donor-to-host

ated the use of the excimer laser to create a

interface, or interface haze.

host bed for a deep lamellar graft. The cur-

rent equipment and software allows for the

input of topography and pachymetry values,

so that recipient corneas with an irregular

surface or thinned stroma can be managed

with a topo-linked and pachy-linked deep

excimer ablation. With the introduction of

the femtosecond laser, the precision of the

ablation/dissection plane created may fur-

ther improve, but not all software currently

allows dissections over 400 mm in depth.

Summary for the Clinician

∑

The lamellar keratoplasty surgical tech-

nique should be chosen taking into consid-

eration the desired dissection depth, man-

agement of inadvertent perforations and

availability of donor tissues

Fig. 5.1. Slit-lamp photograph of a deep anterior

lamellar keratoplasty on the first postoperative day.

Note that the organ cultured full-thickness donor

button positioned onto the lamellar bed is still

swollen

5.7

Clinical Results

of Descemet’s membrane the anterior De-

scemet’s will be exposed, excimer laser ablation

will provide a smooth host bed, and if the dis-

section is made manually it may be recom-

mended to use sharp instruments rather than a

blunt dissection knife. When the host bed is suf-

ficiently deep, a full-thickness donor button can

be positioned onto the host bed. Descemet’s

membrane may be stripped off the donor

cornea, which leaves a perfectly smooth surface

of posterior corneal stroma.

As a result, current techniques for a deep an-

terior lamellar keratoplasty are not associated

with significant interface haze development,

and multiple investigators have found the final

visual performance to be similar in eyes with a

Fig. 5.2. Slit-lamp photograph of a deep anterior

lamellar or penetrating keratoplasty. The final

lamellar keratoplasty performed with manual dissec-

tion at an optically controlled corneal depth

astigmatic error may be lower with lamellar

procedures, especially when a large graft diam-

eter is used. The visual outcome does vary with

the indication for surgery. For example, eyes

grafted for keratoconus on average achieve a

much better visual acuity than those grafted for

recurrent herpes simplex.

Thus, with the main drawback in performing

a lamellar transplant eliminated, there may be

few arguments to prefer a penetrating to a

lamellar procedure. First, because the endothe-

lial cell density after lamellar keratoplasty may

show a pattern of cell loss as in virgin corneas,

the long-term survival of a lamellar graft may

be expected to be much better than after. Sec-

ond, the risk of allograft rejection is minimised.

Although stromal rejections with secondary

keratic precipitates onto the recipient endothe-

Fig. 5.3. Slit-lamp photograph of a deep anterior

lium do occur, such rejections are most often

lamellar keratoplasty performed using viscodissec-

less severe and easily managed with a topical

tion of Descemet’s membrane. Note the remnant re-

steroid pulse therapy. Third, a lamellar proce-

cipient stromal fibres at the level of Descemet’s mem-

dure may provide the patient with much more

brane that are typical for the procedure

social freedom.The integrity of the eye is better

preserved, sutures can be removed much soon-

er, medication can be tapered quicker, and the

overall aftercare necessary to maintain a func-

From LASIK treatments we learned that

tional graft is greatly reduced. Since deep ante-

corneal dissections do not per se induce inter-

rior lamellar keratoplasty indications are limit-

face haze. Since histologically interface haze can

ed to anterior corneal disorders, and these

be correlated with interface scarring, it seems

disorders are usually found in young people,

essential to obtain a smooth interface. With all

long-term graft survival as well as less depend-

the approaches mentioned above a smooth host

ency on the health care system is important in

bed can be obtained.With air or viscodissection

this patient population.

Chapter 5

Deep Anterior Lamellar Keratoplasty

12.

Bhojwani RD, Noble B, Chakrabarty AK, Stewart

Summary for the Clinician

OG (2003) Sequestered viscoelastic after deep

lamellar keratoplasty using viscodissection.

∑ The clinical outcome of a lamellar kerato-

Cornea 22:371-373

plasty with current techniques may be simi-

13.

Bilgihan K, Ozdek SC, Sari A, Hasanreisoglu B

lar to that after penetrating keratoplasty

(2003) Microkeratome-assisted lamellar kerato-

∑ Especially in the patient population eligible

plasty for keratoconus: stromal sandwich.

for lamellar keratoplasty, the procedure

J Cataract Refract Surg 29:1267-1272

may have major advantages for both the

14.

Bodenmueller M, Goldblum D, Frueh BE (2003)

surgeon and the patient, such as longer

Penetrating keratoplasty in Down’s syndrome.

Klin Monatsbl Augenheilkd 220:99-102

graft survival, the need for less aftercare

15.

Chau GK, Dilly DA, Sheard CE, Rostron CK (1992)

and a lower dependence on the health care

Deep lamellar keratoplasty on air with lyo-

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16.

Coombes AG,Kirwan JF,Rostron CK (2001) Deep

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Trimarchi F, Poppi E, Klersy C, Piacentini C (2001)

lamellar keratoplasty using visco-dissection.

Deep lamellar keratoplasty. Ophthalmologica

Cornea 19:427-432

215:389-393

39.

Melles GRJ, Eggink FAGJ, Lander F, Pels E,

53.

Vajpayee RB, Bhartiya P, Sharma N (2002) Central

Rietveld FJR, Beekhuis WH, Binder PS (1998) A

lamellar keratoplasty with peripheral intralamel-

surgical technique for posterior lamellar kerato-

lar tuck: a new surgical technique for kerato-

plasty. Cornea 17:618-626

globus. Cornea 21:657-660

40. Melles GRJ, Lander F, Beekhuis WH, Remeijer L,

54.

Xie L, Shi W, Liu Z, Li S (2002) Lamellar kerato-

Binder PS (1999) Posterior lamellar keratoplasty

plasty for the treatment of fungal keratitis.

for a case of pseudophakic bullous keratopathy.

Cornea 21:33-37

Am J Ophthalmol 127:340-341

55.

Yang YF, Matheson M, Dart JK, Cree IA (2001)

41.

Melles GRJ, Lander F, van Dooren BTH, Pels E,

Persistence of acanthamoeba antigen following

Beekhuis WH (2000) Preliminary clinical results

acanthamoeba keratitis. Br J Ophthalmol 85:277-

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280

gy 107:1850-1857

Corneal Transplant Rejection

T.P.A.M. Slegers, M.K. Daly, D.F.P. Larkin

factors for rejection [18, 53]. In one-third of all

Core Messages

corneal grafts that are deemed failed, signs of a

∑ Allograft rejection is the commonest single

destructive attack by the immune system have

cause of corneal graft failure

been observed [61].

∑ Corneal transplant antigen recognition in

A rejection episode results in loss of donor

most cases is almost exclusively mediated

endothelial cells, critical for maintenance of

by recipient antigen-presenting cells.

corneal transparency. As human endothelial

CD4⁺ T-lymphocytes have the central role

cells do not repair by mitosis to any meaningful

in the alloreactive cell population

extent, the consequence is that donor corneal

∑ Endothelial rejection episodes can be

transparency is lost if cell density falls below the

reversed by intensive topical steroid in

threshold necessary for prevention of stromal

most patients. Poor outcomes result from

swelling. Endothelial decompensation results

delay in presentation and/or initiation in

either

(1) from the time of an irreversible

treatment

episode of acute graft rejection or (2) at an in-

∑ Patients with recipient corneal vascularisa-

terval following one or more episodes of rejec-

tion, a previously rejected ipsilateral trans-

tion which have been reversed by therapy. En-

plant and inflammation at the time of

dothelial cells are thus the critical target in the

transplantation, are at highest risk of rejec-

allogeneic response.

tion and have very poor graft survival

While, on the one hand, reversal of acute

∑ Little information is available from ran-

graft rejection episodes does not present such

domised trials on prophylaxis by transplan-

challenges in cornea as in other transplanted

tation antigen matching or immunosup-

tissues, effective prophylaxis in corneal graft re-

pression in this patient group

cipients identified at high risk of rejection is

much less evidence-based. Thus the impact of

graft rejection continues to justify high priority

in corneal research. Although the first success-

6.1

ful penetrating corneal graft was reported in

Introduction

1906, it took another half a century before the

first description of opacification of a previously

Despite the relative immune privilege of the

clear corneal graft was published. Paufique

cornea as a transplant tissue and both the recip-

named this event “maladie du greffon” (disease

ient corneal bed and anterior chamber being an

of the graft) and suggested that this clinical

immune privileged site [35, 49], the most com-

finding was caused by sensitisation of the donor

mon cause of corneal graft failure in all reports

by the recipient [37]. This description followed

is allogeneic rejection. In first graft recipients

the experiments reported by Medawar a few

with no vascularisation of the recipient corneal

years previously, in which differences were ob-

bed, 2-year survival rates exceed 90%; this de-

served between rabbit skin grafts of donor and

creases to 35-70 % in recipients with high risk

recipient origin, giving rise to the term “histo-

Chapter 6

Corneal Transplant Rejection

compatibility”

[32]. Maumenee subsequently

confirmed this suggestion in a rabbit model of

corneal transplantation in which he showed

that donor corneas could induce an immune re-

action [31]. The development of corneal trans-

plantation models in rat [59] and mouse [46] fa-

cilitated study of rejection in inbred donor and

recipient animals with a wide range of inves-

tigative immunological reagents.

6.2

Fig. 6.1. Subepithelial infiltrates in a penetrating

Incidence

corneal allograft. The appearance is similar to that

seen in adenovirus keratitis, involving the donor

cornea only

In reports from large cohorts of corneal graft

recipients, the proportion undergoing a rejec-

tion episode at some stage post-transplant

ranges from 18 % to 21 % [12, 22, 60]. In those

Table 6.1. Risk factors for rejection

graft recipients in whom rejection occurs, re-

Preoperative

Deep vascularisation

ported rates of successful reversal of the rejec-

of two or more quadrants

tion episode range from 50 % to 90 % [21, 34].

Previously rejected ipsilateral

Allograft rejection occurs most commonly in

graft

the second 6 months postgrafting, and it has

Active corneal inflammation

been reported that more than 10% of the ob-

at time of graft

served reactions can take place as late as at least

Paediatric graft recipient

4 years after surgery [23, 34, 39]. This indicates

that all corneal grafts need long-term surveil-

Large diameter graft

lance and are at risk practically indefinitely.

Graft proximity to the limbus

Postoperative

Loosening of sutures

Removal of sutures,

6.3

wound dehiscence

Factors Predisposing

Graft inflammation

to Corneal Graft Rejection

HSV infection recurrence

Preoperative characteristics of the graft recipi-

Non-viral graft infection

ent eye can be clearly identified in many pa-

tients to indicate significantly high risk of graft

failure. Proposed graft recipient corneas

(1)

with two or more quadrants of deep vasculari-

predisposing to failure due to rejection with ad-

sation, (2) bearing a previously rejected graft

ditional clinical features that confer significant

(Fig. 6.1) and (3) that are inflamed at the time of

risk of graft failure due to other complications,

transplantation are at significantly higher risk

such as glaucoma or ocular surface disease [26,

of rejection [2, 6, 30, 57, 63, 62]. There is less

41]. These preoperative clinical features must be

robust evidence in the published literature that

evaluated carefully in the decision whether to

grafts in children, large diameter donor corneas

proceed with corneal transplantation.

and proximity of donor cornea to the recipient

Once transplantation is successfully com-

limbus are at higher risk (Table 6.1) [30, 41, 54,

pleted, care must be taken to prevent postoper-

56]. Clearly more than one of these factors may

ative events which predispose to rejection, such

be operational in one patient. There may also be

as vascularisation of recipient cornea (Fig. 6.2)

association of one or more of the above factors

or graft wound, suture loosening, or graft infec-

6.5

Histopathology

tion by bacteria or recurrent herpes simplex

virus (HSV).

6.4

Clinical Features

Epithelial rejection, diagnosed by a linear opac-

ity which stains with fluorescein, comprised up

to 10 % of all rejection episodes in one series

and occurs on average 3 months after grafting

[1].Although dead donor epithelial cells are rap-

Fig. 6.2. Endothelial rejection line, keratic precipi-

idly replaced by recipient epithelial cells and no

tates and folds in Descemet’s membrane in rejection

scarring occurs, the presence of this type of

rejection reflects that the recipient is now sensi-

tized to the donor and can progress to stromal

and/or endothelial rejection. Stromal rejection

is characterised by nummular subepithelial

infiltrates (Fig. 6.1), identical to those found in

adenovirus keratitis. Patients with both epithe-

lial and stromal types of rejection may be

asymptomatic or have mild ocular discomfort

only. In contrast, patients with endothelial re-

jection will usually present with visual distur-

bance and iritis symptoms. If examined early

after rejection symptom onset, anterior cham-

ber cell infiltration without flare or graft abnor-

mality will be seen.At later times after symptom

Fig. 6.3. Almost total loss of endothelial cells in

onset, the signs in succession are (1) aggregated

corneal graft specimen removed at graft replacement

alloreactive cells adherent to graft endothelium

6 months following rejection onset

evident as keratic precipitates, (2) an endo-

thelial line with precipitates and (3) localised

oedema corresponding to a rejection line or

total graft oedema (Fig. 6.2). Visible graft pre-

cipitates on slit-lamp biomicroscopy imply focal

6.5

and variable but irreversible endothelial cell

Histopathology

loss, compromising endothelial pump function

and resulting in stroma oedema in those grafts

Descriptions of the pathological features of

with severe inflammation or low endothelial cell

corneal transplant rejection result from exami-

density prior to rejection onset. Pachymetry is

nation of grafts replaced following irreversible

helpful in detecting an increase in oedema and

failure. Therefore these specimens illustrate late

also deturgescence following the start of steroid

changes in end-stage corneal opacification,

treatment. In one study it was found that next

usually some months at least following treat-

to the preoperative diagnosis, graft thickness

ment of rejection. Characteristic findings in

during rejection, as objectively measured by

stroma are vascularisation with mononuclear

pachymetry, is a prognostic sign for reversibili-

cell infiltration and keratocyte loss; few if any

ty of a rejection episode [34]. Risk factors for

endothelial cells remain (Fig. 6.3) [28]. Several

significant endothelial cell loss are delay in ini-

studies have shown increased numbers of HLA

tiating anti-rejection treatment more than 1 day

class II positive cells infiltrating stroma in sec-

and recipient age greater than 60 years [13].

tions of rejected grafts [38, 58].

Chapter 6

Corneal Transplant Rejection

include the paucity of donor-derived major his-

6.6

tocompatibility complex (MHC) class II⁺ APC,

Immunopathological Mechanisms

and corneal epithelial and endothelial expres-

sion of Fas ligand [7, 16], interaction of which

6.6.1

with Fas on alloreactive effector cells leads to

Immune Privilege and Its Breakdown

death of the infiltrating leukocyte.

Corneal grafts at high risk of rejection are

Immune privilege is a dynamic phenomenon

identified by several risk factors, most of which

in which the destructive effect of a “normal”

reflect breakdown of facets of immune privi-

immune response to particular antigens is

lege. Prospective clinical outcome studies iden-

either altered or absent in order to protect the

tify the most significant of these to be recipient

microanatomy of highly organised tissues in

corneal vascularisation, corneal inflammation

the eye. In corneal transplantation, both (1) the

at the time of transplantation, which induces

recipient corneal bed and anterior chamber and

APC infiltration in the recipient cornea prior

(2) the transplanted tissue have features of

to surgery, and a previously rejected ipsilateral

immune privilege.

graft.

Several features of the anterior chamber con-

tribute to immune privilege.There are mechan-

ical barriers that impair immune cell access to

6.6.2

the anterior chamber and transplanted cornea.

Afferent Arm of the Allogeneic Response

One barrier is the lack of blood and lymphatic

vessels in a normal cornea. While experimental

In circumstances where the immune privileged

and clinical studies have clearly shown that

features of the cornea are bypassed by the im-

transplants are much more likely to be rejected

mune system, the first stage in rejection is

in vascularised corneas, the stimuli to vascular-

recognition of the presence of non-self tissue.

isation are likely also to induce lymph vessel

There are two routes of allorecognition. By the

growth. Following transplantation, it is in lymph

indirect pathway, recipient APC enter the graft

vessels that antigen-presenting cells (APC) mi-

to capture and process donor antigens, migrat-

grate from the graft to lymphoid organs for pres-

ing to the lymphoid system to present the anti-

entation of graft antigens to T lymphocytes. An-

gen in context with self MHC class II molecules

other route for alloreactive cells to reach the

to T cells. Most experimental evidence points to

anterior chamber and donor corneal endotheli-

the neck lymph glands as the location for anti-

um is closed by the tight junction barrier formed

gen presentation [40, 45, 65].

between non-pigmented epithelial cells and

Recent identification in the central cornea

non-fenestrated iris vessels [10].

of a population of dendritic cells, which can be-

In the event that leukocytes enter the anteri-

come MHC II⁺ and migrate to the draining

or chamber, mechanisms are available to either

lymph nodes [11, 17, 29], and MHC class II⁺

deviate or blunt a potentially harmful immune

macrophages [9] indicates that direct allore-

response. For example the aqueous humour

cognition of the corneal graft antigens is possi-

contains immunosuppressive molecules as trans-

ble. By this pathway, donor APC bearing allo-

forming growth factor

(TGF)-b, vasoactive

antigens migrate from the graft and activate T

intestinal polypeptide

(VIP), a-melanocyte

lymphocytes via their own non-self MHC class

stimulating hormone (MSH), and calcitonin

II molecules. Direct allorecognition would be

gene related protein (CGRP), which contribute

more prominent in the occasional clinical cir-

to induction by an allograft of deviated sys-

cumstance in which a donor cornea is trans-

temic delayed-type hypersensitivity [35, 52].

planted which has an increased population of

In addition to lack of blood and lymphatic

APCs, such as after viral infection. However, in

vessels, cornea allografts have been shown in

most circumstances it is assumed that corneal

laboratory studies to have additional features

allorecognition is predominantly by the indi-

which contribute to immune privilege. These

rect pathway.

6.8

Prevention of Rejection

Evidence from cell kinetic studies in murine

is likely to be due to delay in recognition and

corneal grafts demonstrates that within several

initiation of treatment, with resulting signifi-

hours of transplantation the graft is infiltrated

cant donor endothelial cell loss [13]. In others,

by granulocytes and macrophages [27]. From

failure to reverse rejection may be due to failure

macrophage depleting studies evidence has

of topical steroid to reverse effector compo-

been provided that these cells play a crucial role

nents of the allogeneic response. In respect of

in the afferent phase of graft rejection [47].

additional systemic steroid, a single dose of in-

travenous methylprednisolone was found to be

more effective than oral steroid in patients with

6.6.3

endothelial rejection who presented within

Efferent Arm of the Allogeneic Response

8 days of onset [20]. A second pulse of intra-

venous methylprednisolone at 24 or 48 h gave

When T-helper cells have identified the present-

no benefit when compared to a single dose at

ed antigen as non-self, effector mechanisms are

initial diagnosis [19]. However, a subsequent

generated against donor tissue. Cytokines in-

randomised trial demonstrated no significant

cluding particularly tumour necrosis factor [42]

benefit of intravenous methylprednisolone in

and interferon-g [25] have been clearly identi-

addition to topical steroid, in respect of graft

fied in aqueous humour and the cornea prior to

survival or interval to a subsequent rejection

observed endothelial rejection onset. After

episode within a 2-year follow-up period [21]. In

corneal transplantation it has been shown that

the same study, endothelial rejection was re-

alloantibody, cytotoxic T lymphocytes and de-

versed in 33 of 36 patients treated, indicating

layed type hypersensitivity responses are com-

that steroid-resistant rejection is uncommon.

ponents of the effector response. Experimental

Other studies examining the efficacy of topical

studies, using CD4+ knockout mice and mono-

or oral cyclosporin administered in combina-

clonal antibodies directed against CD4+ T cells,

tion with intravenous steroid have reported

have pointed to the central role of this lympho-

similar outcomes, with irreversible rejection in

cyte subpopulation [3, 64]. The mechanism by

a small proportion of patients [66, 67].

which corneal endothelial cells are killed is not

yet clear. At time of graft destruction increasing

levels of natural killer (NK) cells, known to be

6.8

able to lyse corneal endothelial cells, are detect-

Prevention of Rejection

ed in the aqueous humour of grafted rats [14].

There is additional evidence that nitric oxide

6.8.1

could mediate in destruction of donor endo-

Immunosuppression

thelial cells [8, 44, 51].

In patients without risk factors for graft rejec-

tion identified prior to surgery, typical postop-

6.7

erative immunosuppression comprises steroid

Treatment of Rejection

drops such as dexamethasone 0.1 % four times

daily for the first 2-3 months, reducing gradual-

The objective of treatment is to reverse the re-

ly to zero by 6 months post-transplant. Regimes

jection episode at the earliest possible time, in

vary from centre to centre. There is much less

order to minimise donor endothelial cell loss

consensus on which additional measures to take

and preserve graft function. With the anatomi-

as prophylaxis in patients at high risk of rejec-

cal advantage that corneal transplants are

tion (Table 6.1), in whom topical steroid alone

superficial, intensive administration of topical

is insufficient to prevent rejection. The result

corticosteroid, such as dexamethasone 0.1 %,

of a continuing shortage of large comparative

treatment is successful in reversing most endo-

prospective studies is that immunosuppression

thelial rejection episodes. In most cases in

protocols in current use result from individual

which topical steroid fails to reverse rejection, it

clinical experience, with some influence from

Chapter 6

Corneal Transplant Rejection

experimental evidence and small uncontrolled

HLA-A, and -B but not HLA-DR. The possible

and/or retrospective clinical studies. However,

benefit of planned -DR mismatching in a setting

ophthalmologists are cautious about adminis-

of known class I histocompatibility is at present

tering potentially toxic systemic immunosup-

being investigated in an ongoing prospective

pressive agents, even in those patients in whom

trial, the outcome of which is awaited with in-

a surviving graft would allow vision in the only

terest. In 1996, a randomised although retro-

eye. The subject of immunosuppression in pre-

spective study reported a beneficial effect of

vention of corneal graft rejection is discussed in

DRB1 matching in recipients at high risk on ac-

another chapter in this text.

count of vascularisation and/or retransplanta-

tion

[4]. Subsequently a beneficial effect of

HLA-DPB1 matching in high-risk corneal trans-

6.8.2

plantation with a significantly higher rate of

HLA Matching

1-year rejection-free graft survival compared to

those without matching was shown [33].

In vascularised organ allotransplantation there

In corneal transplantation therefore the ef-

is robust evidence supporting HLA matching of

fect of HLA matching is less than clear and the

donor and recipient, with the data of Opelz and

data are most ambiguous for class II matching.

others demonstrating stratification of the risk

Resolution of this clinically important issue is

of rejection according the number of class I and

not simple. In contrast to solid organs, results of

especially class II mismatches [36]. HLA match-

matching for cornea are likely to be influenced

ing is in routine use internationally in cadaver-

by the facts that: (1) allorecognition is predomi-

ic renal and other organ transplantation. In

nantly by the indirect pathway in most patients

corneal transplantation by contrast, for recipi-

[5], and

(2) minor transplantation antigens,

ents at high risk of rejection HLA class I and

shown to have a significant effect on graft sur-

class II -DR matching is routinely done in some

vival in untreated rodent recipients [24, 48, 50]

countries, whereas in other countries no match-

and presented by the indirect pathway, remain

ing takes place at all. Roelen suggested a benefit

unmatched in HLA-matched recipients. It is

for HLA-A and -B matching in high-risk corneal

also worth noting here that the effects of HLA

allograft recipients based on his findings that

matching on corneal graft outcome have not yet

primed, donor-specific cytotoxic T cells were

been investigated in the setting of systemic im-

present in rejected corneas but absent in donors

munosuppression prophylaxis. Studies in solid

with good graft function [43]. However, the ben-

organ transplantation have shown that more

efit of histocompatibility matching in corneal

effective rejection prophylaxis can override an

transplantation has been disputed and is cer-

HLA matching effect in unsensitised recipients.

tainly less clear than for solid organ grafts, even

in corneal recipients at perceived high risk of

graft rejection. Two large prospective studies on

6.9

HLA-A, -B, or HLA-DR antigen matching high-

Future Prospects

risk recipients have reported divergent findings.

The Collaborative Corneal Transplant Studies

Reducing the impact of allograft rejection is a

Research Group reported that matching of these

major challenge in corneal disease. It can be

antigens did not decrease the risk of corneal

expected that following developments in tech-

graft failure secondary to rejection [53]. In con-

niques of lamellar keratoplasty, wider use of this

trast the Corneal Transplant Follow-up Study

type of surgical procedure,particularly for stro-

found there was increased risk of graft rejection

mal corneal pathology, will reduce the impact of

with mismatch of HLA class I antigens (relative

endothelial rejection. However, the presence of

risk 1.27 per mismatch), but decreasing risk of

a group of patients with no alternative to pene-

rejection with -DR mismatches (relative risk

trating keratoplasty, a high risk of rejection and

0.58 per mismatch) in high risk patients [55].

no alternative prophylactic intervention justi-

This study therefore supported matching at

fies clinical trials of novel treatment strategies

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Sagoo P, Chan GL, Larkin DFP, George AJT (2004)

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Maguire MG, Stark WJ, Gottsch JD, Stulting RD,

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She SC, Steahly LP, Moticka EJ (1990) A method

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Maumenee AE (1951) The influence of donor-

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32.

Medawar PB (1944) The behaviour and fate of

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Slegers TPAM, Broersma L, Van Rooijen N, Hooy-

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Munkhbat B, Hagihara M, Sato T, Tsuchida F, Sato

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tion 77:1641-1646

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48.

Sonoda Y, Streilein JW (1992) Orthotopic corneal

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Naacke HG, Borderie VM, Bourcier T, Touzeau O,

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Streilein JW (2003) New thoughts on the im-

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Taylor AW (1999) Ocular immunosuppressive mi-

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New Aspects of Angiogenesis in the Cornea

Claus Cursiefen, Friedrich E. Kruse

Core Messages

7.1

∑ Corneal angiogenesis is associated with the

Introduction

most common forms of corneal blindness

both worldwide as well as in industrialized

Corneal avascularity is of paramount impor-

countries

tance in maintaining corneal transparency, the

∑ Corneal angiogenesis is primarily caused

latter being essential for good visual acuity.

by inflammatory diseases of the cornea

Therefore, in all higher animals depending on

(most commonly keratitis), corneal hypoxia

good vision, the cornea normally is devoid of

(contact lens wear) and limbal antiangio-

blood and lymphatic vessels [12, 13, 21, 30]. Nev-

genic barrier defects (most commonly

ertheless, several diseases and surgical manipu-

aniridia, chemical burns)

lations can lead to corneal (hem)angiogenesis

∑ In corneal inflammation, (hem)angiogene-

(i.e., ingrowths of blood vessels from the limbal

sis (i.e., outgrowth of pathologic blood

vascular arcade into the cornea) and lymphan-

vessels into the cornea) is usually accompa-

giogenesis (i.e., ingrowths of lymphatic vessels

nied by lymphangiogenesis (outgrowth of

from the limbal vascular arcade into the cornea

lymphatic vessels)

[12, 13, 30]). Corneal hem- and lymphangiogen-

∑ Pathologic corneal lymphatic vessels are

esis can cause a significant reduction in visual

invisible at the slit-lamp, but can be visual-

acuity and blindness as well as render these

ized using specific immunohistochemical

corneas high risk in the case of a subsequent

markers in explanted vascularized corneas

penetrating keratoplasty [12, 13, 30]. In fact,

∑ Preexisting blood and lymphatic vessels

corneal angiogenesis is associated with the

are strong risk factors for immune rejec-

most common cause of corneal blindness

tions after keratoplasty

worldwide (trachoma) as well as the most com-

∑ In addition, about 50 % of patients under-

mon form of infectious blindness in western

going low-risk keratoplasty also develop

countries (herpetic keratitis [12, 13, 30]). Where-

corneal angiogenesis postoperatively. In

as the animal cornea has been used as in vivo

animal models of corneal transplantation,

model to study the mechanisms of angiogenesis

this postoperative mild combined hem-

for decades, the molecular pathways responsible

and lymphangiogenesis significantly

for maintaining normal avascularity of the hu-

increases the risk for immune rejections

man cornea (“angiogenic privilege”) have only

∑ Novel antiangiogenic and antilymphangio-

started to evolve in recent years [15]. The same is

genic therapies can improve graft survival

true for the role of lymphatic vessels growing

after both low-risk and high-risk keratoplas-

into the cornea in inflammatory corneal dis-

ty by reducing the incidence of immune

eases (lymphangiogenesis [3, 4, 11, 16]). Corneal

rejections (novel therapeutic concept)

lymphangiogenesis has recently been shown to

∑ Novel, directly antiangiogenic drugs for

be of essential importance in the induction of

application against corneal angiogenesis

immune responses after corneal transplanta-

will be available medium term as a spin-off

tion, so that novel antihem- and antilymphan-

of antiangiogenic cancer treatments

Chapter 7

New Aspects of Angiogenesis in the Cornea

giogenic therapies are starting to emerge as new

with angiogenesis. This active maintenance of

tools to improve graft survival in both the low-

corneal avascularity has been termed “corneal

risk and the high-risk setting of corneal trans-

angiogenic privilege” [12, 13, 15]. Corneal angio-

plantation [3, 17].

genic privilege is not only essential for good vi-

sual acuity but is also responsible for the excel-

lent survival of corneal grafts placed into

7.2

avascular low-risk recipient beds, since in these

“Angiogenic Privilege of the Cornea”

cases the graft is physically separated from both

or “How Does the Normal Cornea Maintain

the afferent (lymphatic) and the efferent (blood

Its Avascularity?”

vascular) arm of a so-called immune-reflex arc,

leading to immune rejection after keratoplasty

Although the cornea - due to its anatomically

(Fig. 7.1A [3, 13, 17, 30]). Whereas the cornea has

exposed position - is in constant contact with

served as the in vivo model system for the study

numerous minor inflammatory and thereby an-

of the mechanisms of angiogenesis for decades

giogenic stimuli, the normal cornea remains

[in fact, as early as the 1940s Michaelson hy-

avascular [12, 13, 15]. Even after more severe

pothesized the existence of a soluble angiogenic

trauma - such as refractive surgery - the cornea

factor mediating corneal angiogenesis (which

in contrast to other tissues does not respond

later turned out to be primarily the angiogenic

Fig. 7.1. A Schematic diagram

of the so-called “immune reflex

arc” leading to immune rejec-

tions after keratoplasty. The

afferent arm consists of lym-

phatic vessels allowing exit of

antigen presenting cells from

the donor cornea, the central

processing unit is the regional

cervical lymph nodes [initiation

of production of immune effec-

tor cells], and the efferent arm

consists of blood vessels allow-

ing entry of immune effector

cells to the graft [with kind

permission from Streilein JW

(1999) Immune responses and

the eye. Karger, New York, p 17].

B Immune reflex arc in a vascu-

larized high-risk cornea (with

kind permission from [13]). The

donor cornea has direct access

both to the afferent lymphatic

arm (1, 2) and thereby to the re-

gional lymph node (3) as well as

to the efferent blood vascular

arm (4) of an immune-reflex

arc. This explains the much

higher rate of immune rejec-

tions occurring in vascularized

high-risk eyes

7.3

Corneal (Hem)angiogenesis

Table 7.1. Angiogenic and antiangiogenic factors (selection)

Angiogenic growth factors

Antiangiogenic factors

VEGF (VEGF-A, VEGF-C, VEGF-D)

Thrombospondins (TSP 1 and TSP 2)

FGF (bFGF, aFGF)

PEDF

Interleukin-1

Angiostatin

TGF (alpha, beta)

Endostatin

growth factor VEGF)], the strategies used by the

infectious) stimuli for angiogenesis which

cornea for the normal maintenance of its avas-

threaten the integrity of the whole eye or even

cularity are only partly understood [15, 19]. In

the whole body [15].

general, angiogenesis or inhibition of angiogen-

Summary for the Clinician

esis depend on a balance between proangio-

genic factors (such as the VEGF family growth

∑ Cornea and cartilage are the only avascular

factors VEGF-A, -C and -D) and antiangiogenic

tissues of the human body

factors (such as the thrombospondins; Table 7.1

∑ Corneal avascularity is actively maintained,

[1, 28]). If the balance tips more to proangio-

e.g., after refractive surgery (“corneal

genic factors, angiogenesis starts [1, 28]. In the

angiogenic privilege”)

cornea, normally the balance is shifted towards

∑ Corneal angiogenesis is associated with and

antiangiogenic factors to maintain avascularity.

potentially causative of the most common

Indeed, several antiangiogenic factors (such as

causes of corneal blindness worldwide

PEDF, thrombospondins 1 and 2, antiangiogenic

(trachoma) and the most common form of

matrix cleavage products such as angiostatin

infectious corneal blindness in industrial-

and endostatin, IL1RA) have already been iden-

ized countries (herpetic keratitis)

tified in the cornea [2, 12, 13]. It seems that these

antiangiogenic factors are strategically located

at the inner and outer linings of the cornea

7.3

(Descemet’s membrane and epithelial basement

Corneal (Hem)angiogenesis

membrane) to counteract angiogenic stimuli

both from inside (e.g., high concentrations of

7.3.1

angiogenic growth factors in the aqueous hu-

General Mechanisms

mor during proliferative diabetic retinopathy)

of Corneal (Hem)angiogenesis

and from outside

(e.g., against angiogenic

growth factors from the tear film [2, 12, 13]).

According to Folkman, a balance between an-

Animal experiments using mice deficient in

giogenic and antiangiogenic factors in each tis-

one or more antiangiogenic factors (such as

sue and situation determines whether angio-

thrombospondins 1 and 2) have shown that the

genesis occurs or not. If the balance is tipped

corneal angiogenic privilege is redundantly or-

towards angiogenic growth factors, vessel out-

ganized so that the absence of one or two factors

growth starts (“angiogenic switch”), whereas if

does not cause spontaneous ingrowths of limbal

inhibitors prevail, angiogenesis is prohibited.

blood vessels [15]. This is in contrast to other

Several angiogenic growth factors [primarily

intraocular tissues such as the iris, where the

growth factors from the VEGF family (VEGF-A,

absence of these factors causes increased

VEGF-C, VEGF-D), FGF, IL-1, etc.] as well as in-

vascularity [15]. This demonstrates that evolu-

hibitors of angiogenesis have been identified in

tionarily the cornea has acquired a robust

recent years (PEDF, thrombospondins, angio-

and redundant antiangiogenic system normally

statin, endostatin, etc.; see Table 7.1

[1,

28]).

maintaining avascularity unless it is overrun by

Pathologic angiogenesis (to clearly separate this

overwhelmingly strong (usually inflammatory/

process from lymphangiogenesis, we will subse-

Chapter 7

New Aspects of Angiogenesis in the Cornea

Table 7.2. Common causes of corneal angiogenesis

Pathomechanism

Diseases/conditions

1. Corneal inflammation/infection

Keratitis (most commonly herpetic keratitis,

but also bacterial and fungal)

Graft rejection

Autoimmune diseases affecting the cornea/sclera

2. Corneal hypoxia

Contact lenses with low Dk values

(especially extended wear)

3. Defects of the limbal antiangiogenic barrier

Inherited defects (e.g., aniridia)

Acquired limbal defects (e.g., after chemical cautery)

4. Secondary/iatrogenic

After keratoplasty

After corneal wound repair

quently refer to it as “hemangiogenesis”) and

also of the limbal antiangiogenic barrier sup-

lymphangiogenesis into the cornea mainly oc-

ports this concept.

cur in settings of an inflammatory “insult” to

the cornea, corneal hypoxia or limbal barrier

defects, all overriding the angiogenic privilege

7.3.2

of the cornea, which is actively maintained [2,

Common Causes

12, 13, 15]. Clinical conditions most commonly

of Corneal (Hem)angiogenesis

associated with corneal neovascularization in-

clude keratitis (herpetic and bacterial in na-

The most common causes for corneal angiogen-

ture), contact lens wear as well as inherited or

esis are enlisted in Table 7.2 [6, 24]. According

acquired limbal deficiency states

(primarily

to the mechanisms leading to angiogenesis

chemical burns [2, 12, 13, 15]). Growth factors of

outlined above, these corneal diseases fall into

the VEGF family have been identified as key

three general categories: (1) diseases leading to

players in both inflammation-driven hem- and

strong inflammation within the cornea (auto-

lymphangiogenesis into the normally avascular

immune or infectious; most commonly herpet-

cornea [7, 12, 13].

ic keratitis);

(2) diseases leading to hypoxia

Release of angiogenic growth factors gener-

within the cornea

(most commonly contact

ally is induced primarily by two factors: (1)

lenses with low Dk values) and (3) diseases with

inflammation and inflammatory cytokines (at

inherited (e.g., aniridia) or acquired (e.g., after

the cornea, e.g., keratitis) and (2) hypoxia (at

chemical cautery) defects of the limbal “anti-

the cornea, e.g., contact-lens induced). The gen-

angiogenic” barrier [2]. In addition, “second-

eral process of sprouting angiogenesis follows

ary” corneal angiogenesis can occur after

the following steps: (1) vasodilatation, (2) degra-

surgical manipulations at the cornea, which pri-

dation of extracellular matrix, (3) mitotic acti-

marily involve placement of corneal sutures

vation of endothelial cells and (4) chemotactic

(e.g., after corneal wound repair, after corneal

migration of endothelial cells out of the pre-

transplantation, after block excision [9]).

existing vessels towards an angiogenic stimulus.

The precise mechanisms whereby the sharp

limbal border between hem- and lymphvascu-

7.3.3

larized conjunctiva and avascular cornea is

Clinical Consequences

maintained are unclear. It is possible that limbal

of Corneal Hemangiogenesis

stem cells located at niches in that area con-

tribute to the antiangiogenic barrier of the lim-

Corneal angiogenesis can lead to reduced visu-

bus. Clinically the fact that destruction of limbal

al acuity not only by the physical presence of

stem cells, e.g., by cautery, causes destruction

blood vessels itself, but also due to leakage of

7.3

Corneal (Hem)angiogenesis

thermore, as outlined in Sects. 7.3.4 and 7.4.2,

corneal angiogenesis impairs the prognosis of

corneal grafts placed into vascularized high-

risk corneas. In fact, the Collaborative Corneal

Transplantation Study

[24]

(and numerous

other clinical and experimental studies [30])

revealed preexisting corneal blood vessels as

the strong(est) risk factor for subsequent im-

mune rejections.

Summary for the Clinician

∑ Corneal angiogenesis starts when the

balance between proangiogenic and anti-

angiogenic factors in the cornea is shifted

towards angiogenic growth factors

∑ The most common clinical conditions

associated with corneal angiogenesis are

corneal inflammation (keratitis), hypoxia

(contact lens) and limbal barrier defects

(chemical burns)

∑ Corneal angiogenesis leads to reduced

visual acuity by the physical presence of

vessels itself, but also by leakage of water,

lipids and erythrocytes

∑ Preexisting corneal blood vessels are a

strong risk factor for subsequent immune

rejections after keratoplasty

7.3.4

Corneal Hemangiogenesis

After Keratoplasty

Preexisting corneal blood vessels - as men-

tioned above - have long been identified as

strong risk factors for immune rejection after

Fig. 7.2 A-C. Complications of corneal angiogenesis

keratoplasty [24]. But, until very recently the

leading to reduced visual acuity: A lipid keratopathy

role of the mild angiogenesis (and - as we will

(arrows); B intrastromal hemorrhage; C secondary

discuss in Sect. 7.4.2 - parallel lymphangiogen-

stromal edema due to leakage from immature blood

esis) occurring after keratoplasty in preopera-

vessels. In addition, corneal angiogenesis makes such

a cornea a high-risk recipient bed in the case of sub-

tively avascular recipient beds was unclear

sequent keratoplasty (with kind permission from [12])

[8-10].

7.3.4.1

Corneal Hemangiogenesis

products from immature corneal blood vessels

After Low-Risk Keratoplasty

(Fig. 7.2). This includes corneal edema due to

water leakage, corneal lipid keratopathy due to

The exact pathomechanism for postkeratoplas-

lipid leakage and intrastromal or subepithelial

ty neovascularization is unknown. The interac-

hemorrhage (e.g., in contact lens patients). Fur-

tion of suture material with corneal epithelium/

Chapter 7

New Aspects of Angiogenesis in the Cornea

Fig. 7.3 A, B. Secondary

corneal angiogenesis after

low-risk keratoplasty (A with

kind permission from [12]).

New capillaries develop in every

second patient, are usually

oriented towards the outer

suture turning point and then

grow centripetally along the

suture track (arrows). In 10 % of

patients they reach the donor-

host junction or grow further

into donor tissue. The most

common location of postkerato-

plasty angiogenesis is at the

6 o’clock and 12 o’clock posi-

tions

stroma as well as wound healing processes in

ciently, further underlining the need for more

the interface seem to be important. Indeed,

specific antiangiogenic therapies (see Sect. 7.5.2

comparing corneal neovascularization within

[9, 10]).

the first postoperative year between patients

A retrospective semiquantitative analysis of

having undergone mechanical (more intense

136 patients having undergone low-risk kerato-

wound healing) versus nonmechanical

(ex-

plasty (primarily patients with keratokonus and

cimer laser: less wound healing) keratoplasty

Fuchs’ dystrophy) revealed that more than 50 %

showed that the incidence of corneal angiogen-

of patients after low-risk keratoplasty

(with

esis was lower in the nonmechanical (48 %)

preoperatively avascular corneas) postopera-

compared to the mechanical trephination group

tively develop corneal angiogenesis within the

(75 %; p<0.01 [10]). This indicates that in the

first year [8, 9]. New vessels are primarily locat-

low-risk setting, development of postoperative

ed in the 6 o’clock and 12 o’clock positions and

corneal neovascularization seems to be affected

tend to grow towards the outer suture turning

by the trephination technique and subsequent

points (Fig. 7.3). Thereafter, capillaries usually

wound-healing response. Support for this con-

follow the suture track towards the interface [9].

cept comes from experimental data where post-

In about 10 % of patients these new vessels actu-

operative corneal hem- and lymphangiogenesis

ally reach donor tissue. Risk factors for postop-

within the first week do not differ between allo-

erative neovascularization include: embedding

geneic and syngeneic grafts in the mouse mod-

of the suture knots in the host stroma, active

el of corneal transplantation

[17]. Since in

blepharitis, and a large recipient bed. Experi-

the syngeneic model by definition there is no

ments in the mouse model of low-risk kerato-

immune response possible because donor and

plasty have recently shown that these capillaries

host are immunologically identical, postopera-

are always accompanied by biomicroscopically

tive hem- and lymphangiogenesis really seem

invisible lymph vessels

(lymphangiogenesis;

to be triggered by surgery and wound healing

Fig. 7.4; see Sect. 7.4.2 [16, 17]). Therefore, even if

responses [17]. This establishes surgery itself

it clinically appears only mild, this combined

and the degree of wound healing after kerato-

angiogenesis and lymphangiogenesis after low-

plasty as novel risk factors for the induction of

risk keratoplasty provides access for both arms

immune responses after keratoplasty. Conven-

(afferent lymphatic as well as efferent blood vas-

tional steroid therapy is not able to stop or

cular) of an immune reflex arc towards the graft

prevent this postoperative angiogenesis suffi-

(Fig. 7.1). Indeed, experiments in the mouse

7.3

Corneal (Hem)angiogenesis

Fig. 7.4 A-I. The mouse model of low-risk kerato-

with kind permission from [17]). Whereas there are

plasty demonstrates early and parallel outgrowths of

no blood or lymphatic vessels immediately postoper-

both blood and lymphatic vessels after low-risk ker-

atively in the low-risk recipient corneal bed, after

atoplasty (left column slit-lamp aspect, middle column

3 days both vessel types clearly grow out from the lim-

corneal whole mounts, right column detail showing

bal arcade and at day 7 in this model reach the host-

limbus at left and interface at right; blood vessels

graft interface

stained in green, lymphatic vessels stained in red;

model of low-risk keratoplasty recently identi-

7.3.4.2

fied postkeratoplasty neovascularization as a

Corneal Hemangiogenesis

risk factor for subsequent immune rejections

After High-Risk Keratoplasty

[16, 17]. An antihem- and antilymphangiogenic

therapy significantly improved graft survival

Even after high-risk keratoplasty, preexisting

after low-risk keratoplasty

(Fig. 7.5). Studies

blood vessels tend to increase (Cursiefen et al.,

are under way to evaluate whether this also

unpublished observation,

2004). Only after

holds true for the human low-risk keratoplasty

keratoplasty for herpetic keratitis does anecdot-

setting.

al evidence suggest that removal of the angio-

Chapter 7

New Aspects of Angiogenesis in the Cornea

Fig. 7.5. Inhibition of heman-

giogenesis and lymphangio-

genesis after low-risk kerato-

plasty using a VEGF-A specific

cytokine trap (VEGF Trap) in

the mouse model of corneal

transplantation significantly

reduces immunological graft

rejections (p<0.05; with kind

permission from [17])

genic stimulus leads to a reduction in corneal

angiogenesis [33]. Animal experiments recently

7.3.5

clearly demonstrated that even after high-risk

Corneal Angiogenesis Due

keratoplasty there is a significant further in-

to Contact Lens Wear

crease in both hem- and lymphangiogenesis. In

addition, inhibition of these processes even

Prevalence of contact lens-associated corneal

after high-risk keratoplasty (in the mouse mod-

angiogenesis varies widely in the literature, but

el) could improve subsequent graft survival

is generally estimated to be within a range of

(Cursiefen et al., submitted).

11-23 % of contact lens wearers. The intensity

also can vary from some small capillaries usual-

Summary for the Clinician

ly at the 6 o’clock and 12 o’clock positions to

∑ Corneal angiogenesis postoperatively

deep stromal mature blood vessels with second-

occurs in about 50 % of patients after

ary scar formation. The cause seems to be a re-

low-risk keratoplasty in preoperatively

duced oxygenation of corneal epithelium and

avascular recipient beds

stroma, not primarily due to reduced diffusion

∑ Postoperative angiogenesis reaches donor

through the contact lens, but due to a reduced

tissue in more than 10 % of patients

exchange of the sub-lens tear film which leads

∑ Animal experiments suggest that angiogen-

to reduced oxygenation of this tear film by lid

esis after keratoplasty is accompanied by

capillaries and thereby corneal hypoxia. This in

clinically invisible lymphangiogenesis

turn leads to upregulation of proinflammatory

∑ Postoperative hem- and lymphangiogenesis

cytokines, VEGF-A and then angiogenesis

have been identified as risk factors for

[5-7]. Since even the mild hem- and lymphan-

immune rejection after keratoplasty

giogenesis occurring after low-risk keratoplasty

(mouse model)

have been identified as risk factors for immune

∑ Inhibition of postkeratoplasty angiogenesis

rejections after keratoplasty - in analogy - care-

and lymphangiogenesis seem to improve

ful attention should be given to contact lens-in-

graft survival both in the low-risk and

duced corneal angiogenesis occurring in kera-

high-risk setting (mouse experiments)

toconus patients, since these patients might go

∑ Surgery itself and the degree of wound

on to keratoplasty and one might create a high-

healing after keratoplasty are novel risk

er-risk scenario in the case of later keratoplasty

factors for the induction of immune

[16, 17].

responses after keratoplasty

7.4

Corneal Lymphangiogenesis

∑ Angiogenesis seems to play a pathogenic

7.3.6

role in herpetic stromal keratitis; anti-

Angiogenesis as a Cause

angiogenic therapy should be helpful in

of Disease Progression, not a Sequel

these patients

(Herpetic Keratitis)

∑ Fine-needle diathermy is an easy to per-

form, quick and cheap approach for the

Corneal angiogenesis not only can follow in-

temporary occlusion of larger corneal

flammatory and infectious diseases of the

vessels prior to corneal surgery

cornea, but may also be pathogenetically rele-

vant for the induction of certain corneal dis-

eases. Recent work by Rouse and coworkers

7.4

demonstrated that inhibition of angiogenesis in

Corneal Lymphangiogenesis

animal models of herpetic keratitis could pre-

vent or diminish the intensity of herpetic stro-

Lymphangiogenesis, i.e., the development of

mal keratitis [33]. This suggests that efferent

new lymph vessels, has recently gained wide

blood vessels may be essential in the pathogen-

interest for its important role in tumor metasta-

esis of stromal herpetic keratitis by providing

sis and induction of alloimmunity after or-

CD4+ lymphocytes an entry site into the

gan transplantation [26]. Antilymphangiogenic

corneal stroma. Novel emerging antiangiogenic

strategies have improved survival in animal

therapies (see Sect. 7.5.2) may become part of

tumor models by reducing tumor metastasis.

the pharmacologic armamentarium to treat or

Furthermore, antihem- and antilymphangio-

prevent herpetic stromal keratitis [33].

genic strategies have improved graft survival af-

ter organ transplantation in the mouse model of

corneal transplantation (see below in Sect. 7.4.2

7.3.7

[3, 17]). On the other hand, pro-lymphangio-

Surgery in Vascularized Corneas

genic treatment is desirable for patients with

congenital or acquired lymphedema.

Surgery in vascularized corneas necessitates

special approaches. Whereas refractive surgery

in heavily vascularized eyes cannot be recom-

7.4.1

mended since intraoperative bleeding causes

Mechanisms of Corneal

changes in the ablation profile, LASIK, e.g., can

Lymphangiogenesis

be performed in eyes with minor peripheral

blood vessels in the cornea. Care should be tak-

Whereas it has been known for more than

en not to cause bleeding and if so to carefully

100 years that the normally avascular cornea

stop bleeding and keep the ablation zone free of

can be invaded by blood vessels (hemangiogen-

erythrocytes.

esis), it was unclear until very recently whether

For penetrating keratoplasties in heavily

the normally alymphatic human cornea could

vascularized corneas it may be advisable to

be invaded by lymphatic vessels from the lym-

preoperatively - functionally - occlude larger

phatic arcade at the limbus (lymphangiogenesis

vessels, e.g., using fine-needle diathermy [27].

[2, 12, 13]). The main reasons for that unclarity

Alternative approaches are discussed below in

were: (1) the fact that lymph vessels - in contrast

Sect.1.5.2.

to erythrocyte-filled blood vessels - are not de-

tectable biomicroscopically using the normal

Summary for the Clinician

slit-lamp magnification and (2) the lack of spe-

∑ Care should be taken with contact-lens in-

cific markers for lymphatic endothelium. The

duced corneal angiogenesis in keratoconus

latter has changed in the last 5-10 years with the

patients, since that may compromise the

advent of several specific markers of lymphatic

success of a subsequent keratoplasty due to

endothelium (such as LYVE-1, podoplanin and

increased risk of immune rejections

VEGF receptor 3 [26]). These novel markers

Chapter 7

New Aspects of Angiogenesis in the Cornea

Fig. 7.6 A, B. Lymphatic vessels in vascularized hu-

man corneas. A Immunohistochemistry with a novel

marker specific for lymphatic endothelium (LYVE-1)

clearly separates blood vessels

(stained here in

green) from non-erythrocyte-filled lymphatic vessels

(stained in red). B Electron microscopy reveals the

large, non-erythrocyte-filled lumen of a thin-walled

lymphatic vessel in a vascularized human cornea (top

panels). In contrast, erythrocyte-filled blood vessels

have a thick, multilayered basement membrane

(lower panels; with kind permission from [11]; Lu

lumen, EN endothelial cell, Pe pericyte, ECM extracel-

lular matrix)

enabled for the first time the precise identifica-

sels are invisible at slit-lamp magnifications,

tion of lymphatic vessels in vascularized human

they might not be as detrimental for corneal

corneas (Fig. 7.6 [11]). Lymphatic vessels were

transparency as blood vessels are. In fact, ani-

significantly more common in corneas with a

mal experiments suggest that the “antilymph-

short history of corneal inflammation (usually

angiogenic privilege” of the cornea is not

keratitis or trauma) and also were significantly

redundantly organized.

more common in heavily vascularized corneas

Although previous studies especially by

[11]. Therefore the chance of having both patho-

Collin and coworkers from the 1970s suggested

logical blood and clinically invisible lymphatic

the existence of lymphatic vessels in vascular-

vessels present is strongly correlated with the

ized animal corneas [4], only very recently has it

degree of corneal angiogenesis, which can be

again become possible unequivocally to identi-

judged by slit-lamp evaluation. Furthermore,

fy corneal lymphangiogenesis, e.g., in the

recent work suggests that it is possible to

mouse model of corneal transplantation using

demonstrate lymphatic vessels in vivo in the

the above-mentioned markers (Fig. 7.7 [3, 16,

cornea using confocal microscopy (HRT II us-

17]). Using the mouse model of corneal neovas-

ing the Rostock module). Since lymphatic ves-

cularization, we were recently able to demon-

7.4

Corneal Lymphangiogenesis

Fig. 7.7 A-D. Pathologic corneal blood vessels

blood and lymphatic vessels after an inflammatory

(stained in green with CD31) and lymphatic vessels

stimulus (suture) in segments from a corneal whole

(stained in red with LYVE-1) originate from the lim-

mount (limbus at bottom, central cornea with suture

bal arcade (A). Time course of parallel outgrowth of at the top; B blood vessel, L lymphatic vessel)

strate that after an inflammatory stimulus to the

below, this supports the clinical practice of not

cornea, there is usually parallel and very early

performing penetrating keratoplasties in fresh-

(within 48 h) outgrowth of both blood and lym-

ly inflamed eyes, but of waiting until inflamma-

phatic vessels. Both originate from the limbal

tion has calmed down to improve graft survival

vascular arcade (Fig. 7.7 [16]). The cornea there-

[18]. Lymphangiogenesis is mediated by the

fore is also an excellent model system with

VEGF family growth factors VEGF-A, -C and -D

which to study the mechanisms not only of an-

as well as by FGF and PDGF [26]. Stimuli for the

giogenesis but also lymphangiogenesis and test

release of the main lymphangiogenic growth

pharmacologic compounds for the relative inhi-

factor VEGF-C are primarily inflammatory in

bition of both processes in the animal model

nature, explaining the clinical observation that

[19]. Compared to blood vessels, lymphatic ves-

human corneal lymphangiogenesis is more

sel tend to regress much more quickly and more

common shortly after keratitis [11, 26].

completely after an inflammatory challenge to

Summary for the Clinician

the cornea [18]. For example, after a short,

2-week-long inflammatory stimulus

(corneal

∑ During corneal inflammation there are

sutures), all lymphatic vessels in the mouse

parallel outgrowths of both blood and

cornea are completely regressed after 6 months,

lymphatic vessels from the limbus into the

whereas blood vessels persist (partly as non-

cornea (combined hemangiogenesis and

perfused ghost vessels) indefinitely. As outlined

lymphangiogenesis)

Chapter 7

New Aspects of Angiogenesis in the Cornea

∑ Novel immunohistochemical studies pro-

also enhance speed and amount of antigenic

vide unequivocal evidence for lymphangio-

material or APCs traveling to the regional

genesis in vascularized human corneas,

lymph node. This induces alloimmunization at

although lymphatic vessels are not visible

the lymph node and production of alloreactive

using slit-lamp magnifications in vivo

effector cells, which then travel via the efferent

∑ Lymphatic vessels are more common in

blood vascular arm to the donor cornea and

heavily vascularized human corneas and

induce graft rejection (Fig. 7.1 B).

are more common shortly after a corneal

The relative importance of lymphatic vessels

inflammation (keratoplasty, keratitis,

(representing an exit route for APCs) versus

immune rejection, etc.)

blood vessels (representing an entry route for

∑ In vivo confocal microscopy seems to be a

effector cells) in vascularized corneas in rela-

technique for the visualization of lymphatic

tion to graft rejections is not fully understood.

vessels in vivo in the cornea

But since clinically detectable corneal blood

vessels are neither necessary nor sufficient for

immune rejection of experimental corneal

7.4.2

grafts, an important role of the afferent lym-

Importance of Lymphangiogenesis

phatic pathway mediated by lymphatic vessels is

for Induction of Alloimmunity

likely [20, 25, 30-32]. The relatively higher im-

After Keratoplasty

portance of the afferent lymphatic arm of the

immune response in dictating the outcome of

Normal corneas lack both blood and lymphatic

corneal graft survival has recently been demon-

vessels. This corneal avascularity is not only es-

strated by several elegant studies: Indefinite

sential for corneal transparency but also con-

survival of both fully mismatched orthotopic

tributes to the enhanced prognosis of low-risk

non-high-risk grafts [31] and 90 % survival of

keratoplasty compared to other solid organ

fully mismatched high-risk corneas

[32] in

transplantation by suppressing both “arms” of a

BALB/c mice was achieved by removal of cervi-

potential “immune reflex” that could lead to

cal lymph nodes by cervical lymphadenectomy.

transplant rejection after keratoplasty [29, 30].

Furthermore, pharmacologic strategies inhi-

However, both secondary to a variety of diseases

biting (angiogenesis and) lymphangiogenesis

and after surgical manipulations, the cornea

after low-risk keratoplasty (see Sect. 7.5.2) and

can be invaded by new blood and lymphatic

even after high-risk keratoplasty can signifi-

vessels outgrowing from limbal blood vessels, as

cantly improve corneal graft survival. In sum-

outlined above. This implies that, e.g., in vascu-

mary, interference with both the afferent lym-

larized high-risk corneas after keratoplasty the

phatic and the efferent blood vascular arm of an

graft has direct contact both to the blood and to

immune reflex arc after both low-risk and high-

the lymphatic system (Fig. 7.1 B). Whereas the

risk keratoplasty is a novel and interesting strat-

blood vessels provide a route of entry for im-

egy to improve graft survival. All this supports

mune effector cells (CD4⁺ alloreactive T-lym-

the novel concept that antiangiogenic therapies

phocytes, macrophages, etc.), corneal lymphatic

can modulate immune responses after kerato-

vessels provide a drainage pathway for both

plasty and thereby improve graft survival.

antigenic material (cells, cellular debris) and

antigen-presenting cells (APCs) from the graft

to the regional lymph node. In addition, im-

7.4.3

munomodulatory cytokines, present in high-

Non-immunological Effects

risk beds, or induced after surgical manipula-

of Corneal Lymphangiogenesis

tion of the graft, could travel to the lymph node

as could memory T cells and hyaluronic acid

Whereas the normal human cornea is devoid

(HA) breakdown products that are known to

of HA, upregulation of HA expression in all

activate dendritic cells [12, 13, 29]. Besides en-

corneal layers can consistently be observed in

abling the transport itself, lymphatic vessels

inflammatory corneal diseases, after trauma

7.5

Antiangiogenic Therapy at the Cornea

and keratoplasty. Increased amounts of HA in

All three approaches need to be modified ac-

the cornea, e.g., after refractive procedures, are

cording to whether only blood, only lymphatic

associated with reduced corneal transparency

or both vessel types are to be targeted. So far, no

(“haze”). This might suggest that HA causes

specific antiangiogenic therapy for application

local shifts in water content in the corneal

to the cornea is available. But a lot of specific an-

wound and thereby also local shifts in trans-

tiangiogenic drugs have already entered phase

parency due to interference with the fine-tuned

II and III clinical trials in cancer and, e.g., AMD

spacing of corneal collagen fibrils. In extraocu-

treatment, so that there is a realistic chance that

lar tissues, most of the inflammation-associated

as a “spin-off ” of antitumor treatment, specific

HA deposited is transported to and metabo-

antihem- and antilymphangiogenic agents will

lized in regional lymph nodes and the liver.

be available (preferably in topical formulations)

LYVE-1, one of the specific lymphendothelial

for use at the cornea medium-term.

markers, is an HA receptor, and is thought to

mediate HA uptake into lymphatic vessels and

to facilitate transport to regional lymph nodes.

7.5.1

Since LYVE-1 is expressed on corneal lymph

Established

vessels, these could be involved in transport of

and Novel Antiangiogenic Therapies

pathologic corneal HA from the cornea to re-

gional lymph nodes. Lymphangiogenesis into

The drugs available so far to inhibit angiogene-

the cornea in this setting might be beneficial for

sis in the cornea only have indirect antiangio-

removal of surplus HA, which would otherwise

genic effects. Topical steroids and cyclosporin A

interfere with corneal transparency [12].

suppress the inflammatory component induc-

ing angiogenesis. Since they have no strong di-

Summary for the Clinician

rect antiangiogenic mechanism, their effect is

∑ Since lymphatic vessels are the essential

limited [9]. But since there is so far no alterna-

and required afferent arm of immune

tive, they are still the mainstay of topical antian-

rejections after keratoplasty, antilymph-

giogenic treatment at the cornea. Amniotic

angiogenic pharmacologic strategies can

membrane transplantation and even amniotic

improve graft survival

membrane supernatant have been shown to

∑ New concept: Antiangiogenic therapy

exhibit antiangiogenic effects.Whether this also

modulates immune responses

covers an antilymphangiogenic effect is un-

known. Much more attractive for inhibiting

corneal angiogenesis and lymphangiogenesis

7.5

are direct antiangiogenic agents. Numerous of

Antiangiogenic Therapy at the Cornea

these have already entered phase II and III

clinical trials for anticancer indications

(see

Antiangiogenic therapeutic approaches at the

www.cancer.gov/clinicaltrials/developments/an

cornea can be broadly divided into three cate-

ti-angio-table.html). The only antiangiogenic

gories [21-23]:

agent with FDA approval so far is Avastin

1. Angiostatic/antiangiogenic, i.e., to stop the

(Genentech). Other candidates in trials include

outgrowths of new vessels (classical antian-

the cytokine trap VEGF Trap

(Regeneron),

giogenic approach)

VEGF aptamers (Macugen, Eyetech), antiangio-

2. Angioregressive (meaning regression of al-

genic steroids (anecortave acetate, Alcon) and

ready established pathologic vessels, which is

many more. We recently demonstrated a very

especially important, e.g., in prevascularized

potent antiangiogenic effect of the VEGF-A

high-risk eyes)

cytokine trap (Regeneron) on both inflamma-

3. Angio-occlusive [meaning the (temporary)

tion-induced corneal hemangiogenesis and

functional occlusion of blood vessels, usual-

surprisingly also on lymphangiogenesis

ly prior to corneal surgery]

(Fig. 7.8 [16]). This nearly complete inhibition

shows the much higher potency of direct

7.5

Antiangiogenic Therapy at the Cornea

Fig. 7.8 G.

(continued)

antiangiogenic agents compared for, e.g., steroids

(although no formal comparison has been per-

formed so far). Since all of these compounds are

in trial for systemic applications in tumor and

posterior eye diseases, some time will evolve

until topical formulations for treatment at the

cornea become available. Nevertheless, the dra-

matic developments in the antiangiogenic can-

cer field will definitively provide useful spin-off

products for the anterior eye segment.

Angioregressive therapies would allow the re-

gression of preformed pathologic corneal blood

vessels. Whereas the regression of novel, newly

Fig. 7.9 A, B. Fine-needle diathermy is an easy and

outgrown blood vessels (in the so-called “prun-

quick method for the functional occlusion of larger

ing phase” [14]) can be achieved by removal of

corneal stromal blood vessels, e.g., prior to kerato-

angiogenic agents such as VEGF, older and more

plasty (with kind permission from [27]). Either the

vessel is coagulated directly or a needle is passed

mature and pericyte-covered vessels no longer

through/adjacent to the vessel and the cautery ap-

depend on angiogenic signaling [14]. Induction

plied to the needle

of regression of these mature vessels is more

complex, and would have to involve anti-VEGF

strategies combined with agonists of the vascu-

lar endothelial TIE2 receptor (angiopoietin 2).

atoplasty in vascularized high-risk eyes or to

The regression phase for immature vessels

stop leakage into the cornea out of these blood

again is very short, so that, e.g., removal of a

vessels. Besides the more experimental ap-

loose suture or a hypoxia-inducing contact lens

proach of corneal photodynamic therapy, fine-

needs to be performed very early after the onset

needle diathermy is a reliable, cost-effective and

of vessel outgrowths to cause regression of the

quick treatment option. Corneal vessels are

new vessels. Since the mechanisms responsible

either directly cautered or a suture needle is

for the maintenance of lymphatic vessels are

placed into/next to the vessel and the needle tip

poorly understood, so far no approach for

is then cautered (Fig. 7.9 [27]).

the regression of lymphatic corneal vessels is

known. Fortunately, lymphatic vessels in the

cornea seem to regress spontaneously after the

(inflammatory) stimulus subsides [18].

Angio-occlusive approaches are useful, e.g.,

to prevent intraoperative bleeding during ker-

Chapter 7

New Aspects of Angiogenesis in the Cornea

Conners MS, Stoltz RA, Davis KL et al. (1995) A

Summary for the Clinician

closed eye contact lens model of corneal inflam-

mation. Part 2: Inhibition of cytochrome P450

∑ Antiangiogenic treatments fall into three

arachidonic acid metabolism alleviates inflam-

categories: angiostatic, angioregressive

matory sequelae. Invest Ophthalmol Vis Sci 36:

and angio-occlusive

841-850

∑ Mainstays so far are topical steroids and

Cursiefen C, Küchle M, Naumann GOH (1998)

cyclosporin A eyedrops

Angiogenesis in corneal diseases: Histopathology

∑ Novel (topical) antiangiogenic drugs will

of 254 human corneal buttons with neovascular-

dramatically improve the potential to

ization. Cornea 17:611-613

Cursiefen C, Rummelt C, Küchle M (2000) Im-

inhibit corneal angiogenesis effectively

munohistochemical localization of VEGF, TGFa

and TGFb₁ in human corneas with neovascular-

ization. Cornea 19: 526-533

7.5.2

Cursiefen C, Wenkel H, Langenbucher A et al.

Novel Antihemangiogenic

(2001) Standardisiertes Beurteilungsschema zur

and Antilymphangiogenic Therapies

semiquantitativen Analyse der kornealen Neo-

to Improve Graft Survival After Keratoplasty

vaskularisation mittels projizierter Hornhaut-

photographien: Pilotstudie zur Analyse der

kornealen Neovaskularisation nach Nicht-Hoch-

Using one of the novel direct antiangiogenic

risiko-Keratoplastik vor anschließender Im-

agents

[the VEGF-A specific cytokine trap

munreaktion. Klin Monatsbl Augenheilkd 218:

(VEGF Trap) from Regeneron], it was shown re-

484-491

cently that inhibition of hemangiogenesis and

Cursiefen C, Wenkel H, Martus P et al. (2001) Pe-

lymphangiogenesis after low-risk keratoplasty

ripheral corneal neovascularization after non-

improves corneal graft survival significantly

high-risk keratoplasty: influence of short- versus

longtime topical steroids. Graefes Arch Clin Exp

(Fig. 7.5

[17]). Furthermore, pharmacologic

Ophthalmol 239:514-521

strategies targeting the VEGF receptor 3, medi-

10.

Cursiefen C, Martus P, Nguyen NX et al. (2002)

ating primarily lymphangiogenesis and partly

Corneal neovascularization after nonmechanical

hemangiogenesis, were also able significantly to

versus mechanical corneal trephination for non-

improve graft survival postkeratoplasty

[3].

high-risk keratoplasty. Cornea 21:648-652

This establishes postkeratoplasty neovascular-

11.

Cursiefen C, Schlötzer-Schrehardt U, Küchle M et

ization in the low-risk bed as a novel risk factor

al. (2002) Lymphatic vessels in vascularized hu-

man corneas: immunohistochemical investiga-

for subsequent immune rejections and supports

tion using LYVE-1 and Podoplanin. Invest Oph-

the novel concept of modulating immune re-

thalmol Vis Sci 43:2127-2135

sponses after corneal grafting by antihem- and

12.

Cursiefen C, Seitz B, Dana MR et al. (2003) Angio-

antilymphangiogenic therapies.

genesis and lymphangiogenesis in the cornea:

pathogenesis, clinic and treatment. Ophthal-

mologe 100:292-299

References

13.

Cursiefen C, Chen L, Dana MR et al. (2003)

Corneal lymphangiogenesis: Evidence, mecha-

nisms and implications for transplant immunol-

Carmeliet P (2003) Angiogenesis in health and

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disease. Nat Med 9:653-660

14.

Cursiefen C, Rummelt C, Küchle M et al. (2003)

Chang JH, Gabison EE, Kato T et al. (2001)

Pericyte recruitment in human corneal angio-

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mol 12:242-249

15.

Cursiefen C, Masli S, Ng TF et al. (2004) Roles of

Chen L, Hamrah P, Cursiefen C et al. (2004) Vas-

thrombospondin 1 and 2 in regulating sponta-

cular endothelial growth factor receptor-3 (VEG-

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FR-3) mediates dendritic cell migration to lymph

and iris. Invest Ophthalmol Vis Sci 45:1117-1124

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16.

Cursiefen C, Chen L, Borges L et al. (2004) Via

transplants. Nature Med 10:813-815

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Cursiefen C, Maruyama K, Liu Y et al. (2004) In-

25.

Nguyen NX, Seitz B, Langenbucher A et al. (2004)

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Clinical aspects and treatment of immunological

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18.

Cursiefen C, Maruyama K, Jackson DG et al.

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Pepper MS, Tille JC, Nisato R et al. (2003) Lym-

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Pillai CT, Dua HS, Hossain P (2000) Fine needle

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Cursiefen C, Ikeda S, Nishina P et al. (2005) Spon-

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Saharinen P, Tammela T, Karkkainen MJ et al.

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1547

Histocompatibility Matching in Penetrating Keratoplasty

Daniel Böhringer, Rainer Sundmacher, Thomas Reinhard

Core Messages

8.1.1

∑ HLA matching reduces graft rejections in

Immune Reactions Constantly Threaten

normal- as well as in high-risk keratoplasty

Graft Survival

∑ Most patients can be served with an HLA

compatible graft within well below a year,

Despite the anterior eye chamber immune priv-

even on a monocenter waiting list

ilege, graft rejections are a major complication

∑ Waiting time for a histocompatible graft

of penetrating keratoplasty as they facilitate

can be predicted and discussed with each

subsequent graft failure. Application of topical

patient in advance

corticosteroids for several months has com-

∑ The HLAMatchmaker algorithm can balance

monly been thought to be sufficiently protec-

waiting time and histocompatibility for

tive. On average, 18 % of patients with normal-

patients with rare HLA phenotype

risk keratoplasty [13] and up to 75 % in high-risk

∑ The HLA-A1/H-Y minor antigen equals

cases

[15] nevertheless experience immune

immunogenicity of HLA mismatches:

reactions. Life span of affected grafts is signifi-

allocating male HLA-A1 positive donors

cantly reduced from an endothelial graft reac-

for female recipients should be avoided

tion. Immune reactions thus increase the inci-

∑ Long-term graft survival will improve upon

dence of re-keratoplasties due to failed grafts in

routinely matching major and selected

the long run.

minor histocompatibility antigens.

Graft reactions can be reduced by means of

This strategy will outweigh the cost of

intensified and prolonged prophylaxis with top-

HLA typing in the long run

ical corticosteroids and with systemic immuno-

suppression [14]. The protective effect, however,

ceases upon discontinuation of the immuno-

suppressive regimen. Any long-term depend-

ence on immunosuppression is associated

with additional costs and potentially serious

side effects.

Antigen matching, that is avoiding grafts

8.1

bearing antigens that are foreign to the recipi-

Introduction

ent upon allocation, improves histocompatibili-

ty. Matching of human leukocyte antigens

In this chapter, the recent evolvements in histo-

(“HLA Matching,” Sect. 8.1.2) and more recently

compatibility matching for penetrating kerato-

of further antigen systems (“Minor Matching,”

plasty are presented and a strategy for clinical

Sect.8.1.3) has turned out to be a potent adjunct

practice is recommended.

to immunosuppression in the fields of allogene-

ic transplantation, i.e., in penetrating kerato-

plasty.

102

Chapter 8

Histocompatibility Matching in Penetrating Keratoplasty

HLA antibodies could be detected at that time.

Summary for the Clinician

Today, the serologic HLA typing assay is per-

∑ Unlike immunosuppression, HLA matching

formed using the complement-dependent cyto-

can permanently reduce graft rejections.

toxicity assay (CDC). For HLA typing, cells are

incubated against a battery of standardized an-

tibodies as defined by the International Histo-

8.1.2

compatibility Workshop (IHW). These IHW test

Major Transplantation Antigens (HLA)

sera define the HLA antigens that can be detect-

ed from the assay. The assay is further incubat-

Experimental transfer of tumors from one

ed with rabbit complement. Lysis is induced by

mouse strain to others led to the discovery of

complement activation in cells that were re-

the major histocompatibility complex (MHC).

cognized by a specific antibody. For detection of

In humans, these antigens were termed human

lysis, a dye is eventually added.

leukocyte antigens (HLAs). Antibodies against

The first generation of CDC test sera was un-

HLAs were first discovered after a transfusion

able to detect differences of only a few amino

reaction of a multiparous woman despite blood

acid residues between certain HLA alleles. Upon

group compatibility.

availability of monoclonal antibody techniques,

The HLA antigens are subdivided into three

the International Histocompatibility Workshop

classes according to their expression pattern

released a new generation set of test sera that

and their association with other molecules.

eventually was able to split most alleles known

Only HLA genes of class I and II are relevant to

at that time (“broads”) into further HLA alleles

transplantation medicine as they encode for

(“splits”).

polymorphic membrane bound molecules.

As the HLA nomenclature was already estab-

∑ Class I molecules are expressed on almost all

lished at that time, the newly discovered alleles

nucleated body cells. They comprise a heavy

were termed splits of the established broad anti-

chain, a light chain (b₂-microglobulin) and a

gens and sequentially assigned higher numbers

small peptide of nine amino acid residues.

than the established broad antigens.

Three gene loci of major importance have

Further improvements in HLA typing were

been identified for this system:HLA-A,HLA-

achieved by means of molecular techniques as

B and HLA-C.

discovery of new alleles with these molecular

∑ Class II molecules are homodimers. These

methods no longer depends on isolation of

molecules are exclusively expressed on cell

viable cells. Using the polymerase chain reac-

families that share the ability to present ex-

tion (PCR), small parts of the genome can be

tracellular antigens to the immune system

detected by means of specific primers and an

(e.g., monocyte and lymphocyte derived

amplification reaction. This is achieved either

strains). HLA class II loci of major immuno-

by sequence specific oligonucleotide priming

logic importance are HLA-DR and HLA-DQ.

where alleles are identified by characteristic

“fingerprint” sequences or by the more expen-

8.1.2.1

sive sequence typing of the whole allele. These

Methods for Typing HLA Antigens

molecular techniques led to further subdiffer-

entiation of most split alleles.

HLA antigens were discovered by means of

For example, the broad antigen HLA DR3

serological assays. In the beginning, only the

is dividable into the split antigens DR17 and

macroagglutination assay was available. For this

DR18, which in turn can be subdivided into

assay, patient serum was mixed with test cells

DRB1*0302 and DRB1*0303 at the molecular

bearing only the respective antigen. Any macro-

level.

scopically visible agglutination demonstrated

In corneal transplantation, blood for HLA

the presence of antibodies directed against the

typing is commonly collected up to 72h after

respective test cells. Depending on the availabil-

the donor’s death. From autolytic changes, a suf-

ity of appropriate test cells, only a subset of all

ficient amount of viable cells is often unavail-

8.1

Introduction

103

able for serologic analysis. In this situation,

analysis

[18]. The CCTS for example was

molecular methods can still detect the HLA

based on typing data that differed by 55 %

phenotype with excellent precision.

from retyping with modern techniques, in-

validating all conclusions drawn from that

Summary for the Clinician

particular investigation.

∑ Various HLA molecules are bound to the

Additionally, most studies suffered from

surface of all nucleated cells. These antigens

poor statistical power due to heterogeneity

are potentially targeted by the immune

of the study groups. Multiple centers, lack of

system unless they are tolerated as of birth.

standardization regarding surgical experi-

One or two different versions (alleles) of

ence and keratoplasty procedure as well as

each HLA locus can be produced by each

differences in immunosuppression regimens

cell. Over 20 alleles have been identified for

most likely also influenced outcome and thus

each of the HLA loci.

confounded or obscured the HLA effect.

∑ In penetrating keratoplasty, the alleles of

Finally, most studies were performed on

donor and recipients should be typed with

high-risk patients, who not only are at in-

immunogenetic techniques due to higher

creased risk of immune reactions but are

accuracy and precision, especially in blood

also at risk of graft failure from events other

samples collected up to 72 h after clinical

than graft rejection. When correlating total

death of the donor.

graft failures with HLA matching, any statis-

tical association might be obscured from

8.1.2.2

non-immunological graft failures such as

HLA Matching in Penetrating Keratoplasty

protracted elevated intraocular pressure.

In penetrating keratoplasty, contrary to other

8.1.2.2.2

fields of transplantation, the potentials of HLA

Current Evidence

matching are currently mostly unexploited as

the beneficial effect was demonstrated only re-

On the basis of modern and reliable HLA typing

cently. This calls for explanation as the human

techniques, recently four monocenter studies

cornea has been known for longer to express

from Europe were published. These well-de-

HLA antigens and these antigens are known tar-

signed investigations uniformly confirmed a

gets of cytotoxic T cells in the process of graft

beneficial effect of HLA compatibility in pene-

rejection [12].

trating keratoplasty [1, 13, 15, 18]. Each study

stresses additional aspects with respect to HLA

8.1.2.2.1

matching as follows:

Methodical Problems

∑ In 1,681 consecutive transplantations from

with Older Investigations

only one center, a benefit was found from

matching the class II locus HLA-DR addi-

Numerous studies performed in the past

tionally to the class I loci A and B [18].

2 decades came to contradictory results as to the

∑ A beneficial effect from class I matching

usefulness of HLA matching [17]. Three short-

alone was only observed when matching

comings of study design in these older investi-

is based on split rather than broad

(see

gations, however, compromised the power to

Sect.8.1.2.1) HLA alleles [1].

demonstrate any matching effect:

∑ A beneficial effect was demonstrated even

1. The major problem with almost all older

for normal risk (first keratoplasty for bullous

studies is the poor quality of HLA typing at

keratopathy, Fuchs’ endothelial dystrophy,

that time. The importance of highly accurate

keratoconus with centrally sutured graft or

HLA typing for successful HLA matching

avascular corneal scars) keratoplasty alone

was recently recognized. Even 5 % of faulty

when matching HLA A, B and DR broad

HLA DR typing obscures the beneficial effect

alleles (Fig. 8.1) [13].

as demonstrated in a recent simulation

104

Chapter 8

Histocompatibility Matching in Penetrating Keratoplasty

Fig. 8.1. Beneficial effect for

normal risk keratoplasty alone

when matching HLA A, B and

DR broad alleles [13]

concatenated to form the triplet-string for a

Summary for the Clinician

particular allele. The association of the triplet-

∑ A mounting body of evidence supports

string with a particular allele is exclusively de-

the beneficial effects of HLA matching in

fined for alleles at molecular typing resolution.

normal- as well as in high-risk keratoplasty.

Degree of matching is assessed by counting

∑ Accuracy and precision of HLA typing are

all triplets of the donor’s triplet-strings that are

crucial to HLA matching.

not identical to any of the corresponding

triplets of the recipient’s four triplet-strings

8.1.2.2.3

(two for HLA-A and -B each).

Variable Immunogenicity of Individual

HLA mismatches with zero to few mis-

HLA Mismatches

matched triplets are supposed to be fully histo-

compatible with regard to the antibody epi-

HLA mismatches differ in strength of immuno-

topes. Additionally, they are known not to cause

genicity and thus in deterioration of graft sur-

a deterioration of graft survival in kidney trans-

vival. This has been observed for longer in kid-

plantation

[10]. In penetrating keratoplasty,

ney transplantation [6] and in keratoplasty [5].

recently a beneficial effect of this algorithm was

For HLA class I loci, the structural basis of this

demonstrated (Fig. 8.2) [3].

phenomenon has recently been established [7, 8,

Summary for the Clinician

9, 10]. This paved the way for predicting “accept-

able” mismatches on an individual basis: the

∑ The HLAMatchmaker algorithm can help in

HLAMatchmaker algorithm defines nearly 50

reducing graft rejections for penetrating

omnipresent epitopes within the molecular

keratoplasty to a similar extent as conven-

structure of all HLA class I alleles. These epi-

tional HLA matching

topes are thought to be particularly exposed to

∑ This algorithm is crucial for providing his-

the immune system and partitioned into

tocompatible grafts to patients with rare

triplets of amino acid residues to account for

HLA phenotypes within a reasonable time.

thermodynamic characteristics of the antibody

Alternatively, waiting time can be traded for

recognition reaction. All triplets are formally

a better match grade

8.1

Introduction

105

8.1.3.1

Selected Minor Antigens

8.1.3.1.1

H-Y

Male grafts can be subject to alloimmune reac-

tivity in female recipients, as antigens of the

H-Y group are only expressed in male individu-

als and not in females. H-Y antigens are sup-

posed to occur in all tissues including the

human cornea.

Epitopes of the H-Y antigen family are ex-

pressed either in the context of HLA-A1 or HLA-

A2. The HLA-A1/H-Y antigen is located in the

Y-chromosome-encoded DFFRY protein, The

HLA-A*0201-restricted HLA-A2/H-Y antigen

contains a post-translationally modified cys-

teine that significantly affects T-cell recognition.

As to matching the HLA-A1/H-Y epitope, a

20 % reduction of graft rejections was recently

Fig. 8.2.

Beneficial

effect

the

HLAMatchmaker

demonstrated on

252 keratoplasties

(manu-

algorithm in penetrating keratoplasty [3]

script in preparation), whereas matching of the

HLA-A2/H-Y epitope did not affect graft sur-

vival.

8.1.3

From this observation, male HLA-A1 posi-

Minor Transplantation Antigens

tive donors should not be allocated to female

recipients. The prevalence of this setting is as

Graft rejections are observed even in HLA iden-

high as 13 % in the German keratoplasty popu-

tical allogeneic transplantations. These effects

lation.

are ascribed to disparities in minor histocom-

patibility (H) antigens. A single minor H mis-

8.1.3.1.2

match even exceeds the immunogenicity of a

HA-3

single MHC mismatch in a mouse-model for

high-risk keratoplasty.

Another HLA-A1 restricted H antigen expressed

H antigens are peptides derived from poly-

in all corneal layers is the HA-3 epitope. This

morphic proteins. Their immunogenicity arises

epitope is derived from the lymphoid blast cri-

as a result of their presentation on the plasma

sis (Lbc) oncoprotein. Two alleles, VTEPGTAQY

membrane in the context of HLA class I or II,

(HA-3T) and VMEPGTAQY

(HA-3M), have

where they are recognized by alloreactive HLA

been demonstrated. T-cell immune reactivity

restricted T cells. In animal models, antigen pre-

has only been observed in the direction of

senting cells (APCs) such as limbal Langerhans

HA-3T. The prevalence of the immunogenic

cells have been demonstrated to migrate from

setting is as low as 3 % in the German kerato-

the graft to the host spleen via the camero-

plasty population. HLA-A1/HA-3 matching can

splenic axis. The spleen might thus be the

thus be thought of as being of slight importance

source of a cytotoxic specific immune response

and HA-typing is not recommended for routine

directed against foreign graft H antigens pre-

use.

sented by graft APCs.

106

Chapter 8

Histocompatibility Matching in Penetrating Keratoplasty

8.1.3.1.3

to the long-standing reluctance towards HLA

Blood Group Antigens

matching in penetrating keratoplasty.

When these patients are identified in advance,

Blood group antigens are expressed on the

a randomly assigned graft with appropriate im-

cornea. In high risk situations, a significant re-

munosuppression can be opted for a priori or the

duction of graft reactions after penetrating ker-

stringency with respect to histocompatibility can

atoplasty was observed in retrospective investi-

be reduced, e.g., using the HLAMatchmaker algo-

gations [4, 11].

rithm (Sect. 8.1.2.2.3). An algorithm for predict-

Future research holds the prospect of

ing the waiting period is thus vital for informed

demonstrating an additional HLA restricted in-

consent on histocompatibility, which has to be

fluence of blood group antigens in normal risk

discussed with each patient individually. This

situations as well.

problem was solved recently with an algorithm

that can predict the expected time on the waiting

Summary for the Clinician

list on an individual basis.

∑ HLA-A1 positive grafts from male donors

should not be allocated to female recipients

∑ Matching blood group antigens reduces

8.2.2

graft rejections in high-risk keratoplasty

Algorithm for Predicting the Time

∑ The rapidly evolving field of minor anti-

on the Waiting List

gens is subject to ongoing and future

research in penetrating keratoplasty

An algorithm that is based on the HLA pheno-

type,a database of the most common haplotype

frequencies in the donor population and last

8.2

but not least parameters of the local cornea

Time on the Waiting List Associated

bank can robustly predict the estimated waiting

with Histocompatibility Matching

time for an HLA compatible graft. This algo-

rithm has been retrospectively validated against

8.2.1

an historical waiting list of almost 1,400 HLA

Waiting Time Variance Has Been

typed patients (Table8.1) [2]. The assumptions

a Barrier to Histocompatibility Matching

of this algorithm are summarized in the follow-

ing two sections.

Histocompatibility matching is associated with

additional time on the waiting list from refusing

8.2.2.1

all newly available grafts while waiting for

Percentage of HLA Compatible Grafts

the first that satisfies the histocompatibility re-

quirements. Due to the social and individual

Twenty-seven different HLA phenotypes match

costs of blindness, waiting periods exceeding

any recipient who is a heterozygote for the HLA

1 year are hardly reasonable.

loci A, B and DR. Only one phenotype, however,

This waiting period is highly variable, de-

matches an individual who is completely homo-

pending on the recipient’s histocompatibility

zygous at these loci. The total percentage of the

antigens: individuals with common HLA phe-

donor population matching the HLA phenotype

notypes can be routinely provided a matching

of any recipient is well approximated by the sum

graft within a few months as prevalence of com-

of all population frequencies (donor popula-

patible phenotypes is common in the donor

tion) of the compatible HLA phenotypes. The

population as well. On the other hand, individu-

frequency of any HLA phenotype is the product

als with a rare HLA phenotype commonly re-

of both haplotype frequencies. Haplotype fre-

main on the waiting list for years without being

quencies for the HLA loci A, B and DR can be

allocated a compatible graft. These patients,

retrieved from a common database comprising

waiting in vain for an HLA match, contributed

the respective donor population [16].

8.3

Recommended Clinical Practice

107

Table 8.1. Validation of algorithm against a historical waiting list of almost 1400 HLA typed patients [2]

Zero mismatches

One mismatch

Two mismatches

Predicted period

(only of recipients for which

a match was found below)

17±159

7±49

1±6

Simulated period

15±14

5±9

1±3

(29 %)

(71 %)

(83 %)

R=0.28; p<0.001

R=0.36; p<0.001

R=0.45; p<0.001

8.2.2.2

Actual Estimation of the Waiting Time

8.3

Recommended Clinical Practice

The daily rate of new HLA compatible grafts

equals the product of the daily rate of new HLA

According to current knowledge, HLA matching

typed grafts and the percentage of HLA com-

should be performed at least for the HLA loci A,

patible donors as described in the previous sec-

B and DR.

tion. Assuming a Poissonian distribution of

All corneal grafts should be typed for these

donors, expected waiting period is reciprocal to

HLA loci in order to increase the pool available

the daily rate of new HLA compatible grafts.

for histocompatibility matching. Molecular typ-

The algorithm is summarized in Eq. 8.1.

ing should be preferred over serologic methods

as molecular methods can still detect the HLA

365

phenotype with excellent precision when blood

(8.1)

for HLA typing is collected after up to 72 h post-

2GR∑GF

mortem. An additional benefit of molecular

where t [years] is expected waiting period, GF is

typing is the applicability of the HLAMatch-

total share of compatible HLA phenotypes and

maker algorithm (Sect. 8.1.2.2.3).

GR is local daily rate of new donors.

As for the recipient, all patients should be

A certain percentage of grafts are unsuitable

typed upon the keratoplasty being indicated,

for transplantation due to quality control. This

again preferably with immunogenetic tech-

can be adjusted using a local empiric constant

niques for the HLAMatchmaker algorithm.

for each cornea bank.

All patients awaiting normal risk keratoplas-

ties should be told their expected time on the

Summary for the Clinician

waiting list (Sect. 8.2). Improved prognosis from

∑ Most patients can be served with an HLA

HLA compatibility should be weighed against

compatible graft within well below a year,

the expected waiting time. This strategy needs

even on a monocenter waiting list

to be discussed with each patient individually.

∑ Patients that waited in vain for an HLA

Patients awaiting high-risk keratoplasty

match for a long time contribute to the

should be provided a histocompatible graft in

reluctance towards HLA matching

almost all cases. The HLAMatchmaker algo-

in penetrating keratoplasty

rithm can be applied to balance time on the

∑ Waiting time for a histocompatible graft

waiting list with the degree of histocompatibili-

can be predicted from the HLA phenotype

ty that is realistically achievable in patients with

and discussed with the patient in advance

rare HLA phenotype.

108

Chapter 8

Histocompatibility Matching in Penetrating Keratoplasty

Matching of HLA and additional antigen sys-

Duquesnoy RJ, Marrari M (2002) HLAMatch-

tems (e.g., H-Y/HLA-A1 and blood group anti-

maker: a molecularly based algorithm for histo-

compatibility determination. II. Verification of

gens), the HLAMatchmaker algorithm and wait-

the algorithm and determination of the relative

ing time prediction are only feasible with an

immunogenicity of amino acid triplet-defined

integrated highly specialized software package

epitopes. Hum Immunol 63:353-363

from professionalized high-volume institutions

10.

Duquesnoy RJ, Takemoto S, de Lange P, Doxiadis

responsible for allocation.

II, Schreuder GM, Persijn GG et al. (2003) HLA-

In summary, long-term graft survival will

matchmaker: a molecularly based algorithm for

improve upon routinely matching major and

histocompatibility determination. III. Effect of

matching at the HLA-A,B amino acid triplet level

certain minor histocompatibility antigens in

on kidney transplant survival. Transplantation

all keratoplasties. This policy will outweigh the

75:884-889

costs of HLA typing in the long run.

11.

Inoue K, Tsuru T (1999) ABO antigen blood-

group compatibility and allograft rejection in

corneal transplantation. Acta Ophthalmol Scand

References

77:495-499

12.

Niederkorn JY (2001) Mechanisms of corneal

Beekhuis WH, Bartels M, Doxiadis II, van Rij G

graft rejection: the sixth annual Thygeson Lec-

(2003) Degree of compatibility for HLA-A and -B

ture, presented at the Ocular Microbiology and

affects outcome in high-risk corneal transplanta-

Immunology Group meeting, October 21, 2000.

tion. Dev Ophthalmol 36:12-21

Cornea 20:675-679

Böhringer D, Reinhard T, Böhringer S, Enczmann

13.

Reinhard T, Böhringer D, Enczmann J, Kogler G,

J, Godehard E, Sundmacher R (2002) Predicting

Mayweg S, Wernet P et al. (2004) Improvement of

time on the waiting list for HLA matched corneal

graft prognosis in penetrating normal-risk ker-

grafts. Tissue Antigens 59:407-411

atoplasty by HLA class I and II matching. Eye

Böhringer D, Reinhard T, Duquesnoy RJ, Böh-

18:269-277

ringer S, Enczmann J, Lange P et al. (2004) Bene-

14.

Reinhard T, Reis A, Kutkuhn B, Voiculescu A,

ficial effect of matching at the HLA-A and -B

Sundmacher R

(1999) Mycophenolatmofetil

amino-acid triplet level on rejection-free clear

nach perforierender Hochrisiko-Keratoplastik.

graft survival in penetrating keratoplasty. Trans-

Eine Pilotstudie. Klin Monatsbl Augenheilkd 215:

plantation 77:417-421

201-202

Borderie VM, Lopez M, Vedie F, Laroche L (1997)

15.

Reinhard T, Spelsberg H, Henke L, Kontopoulos T,

ABO antigen blood-group compatibility in corneal

Enczmann J, Wernet P et al. (2004) Long-term re-

transplantation. Cornea 16:1-6

sults of allogeneic penetrating limbo-keratoplas-

Creemers PC, Kahn D, Hill JC (1999) HLA-A and

ty in total limbal stem cell deficiency. Ophthal-

-B alleles in cornea donors as risk factors for graft

mology 111:775-782

rejection. Transpl Immunol 7:15-18

16.

Schipper RF, Oudshoorn M, D’Amaro J, van der

Doxiadis II, Smits JM, Schreuder GM, Persijn GG,

Zanden HG, de Lange P, Bakker JT et al. (1996)

van Houwelingen HC, van Rood JJ et al. (1996)

Validation of large data sets, an essential pre-

Association between specific HLA combinations

requisite for data analysis: an analytical survey of

and probability of kidney allograft loss: the taboo

the Bone Marrow Donors Worldwide. Tissue

concept. Lancet 348:850-853

Antigens 47:169-178

Duquesnoy RJ (2002) HLAMatchmaker: a molec-

17.

Vail A, Gore SM, Bradley BA, Easty DL, Rogers CA,

ularly based algorithm for histocompatibility de-

Armitage WJ (1997) Conclusions of the corneal

termination. I. Description of the algorithm. Hum

transplant follow up study. Collaborating Sur-

Immunol 63:339-352

geons. Br J Ophthalmol 81:631-636

Duquesnoy RJ, Howe J, Takemoto S (2003) HLA-

18.

Völker-Dieben HJ, Claas FH, Schreuder GM,

matchmaker: a molecularly based algorithm for

Schipper RF, Pels E, Persijn GG et al. (2000)

histocompatibility determination. IV. An alterna-

Beneficial effect of HLA-DR matching on the

tive strategy to increase the number of compati-

survival of corneal allografts. Transplantation 70:

ble donors for highly sensitized patients. Trans-

640-648

plantation 75:889-897

Current Systemic Immunosuppressive Strategies

in Penetrating Keratoplasty

Alexander Reis, Thomas Reinhard

Despite the advantage that the transplanted

Core Messages

organ can directly (and not via the vascular sys-

∑ Immunologic rejection is the main cause

tem) be reached with topical steroids in ex-

of corneal graft failure

tremely high concentrations, thereby interfer-

∑ Acute rejection is mainly mediated by T

ing with the host’s immune system right at the

cells and can be prevented with steroids,

“battlefield” of graft rejection, this strategy is

IL-2 inhibitors (cyclosporine, tacrolimus),

only sufficient in a normal-risk situation.

mycophenolate mofetil and TOR inhibitors

The clonal expansion of alloreactive T cells

(everolimus, rapamycin)

occurs in lymphoid organs (i.e. lymph nodes

∑ Based on their risk of immunologic rejec-

and spleen): after the recognition of the foreign

tion, corneal transplants are rated as either

tissues by T cells, these specific T cells start to

normal-risk or high-risk transplants

proliferate and generate an immunological

∑ In a normal-risk situation the postoperative

army against the graft. It is therefore crucial not

application of topical steroids is sufficient

only to work with topical steroids but to employ

to prevent acute graft rejection in most

immunosuppressive substances systemically. In

cases

a high-risk situation you have to fight the inhos-

∑ In high-risk keratoplasty systemic immuno-

pitable host in its hinterland to achieve graft sur-

suppression with cyclosporine, myco-

vival in the long run.

phenolate mofetil or tacrolimus has to

To understand the possible targets of im-

be used to maintain clear graft survival

munosuppression and immunomodulation we

∑ As corneal transplantation is not a life-

need to take a look at the underlying immunol-

saving procedure, the side-effect profile is

ogy.

a central issue when choosing immuno-

suppressive medication

9.2

Immunology

9.1

Immunological responses against the trans-

Introduction

planted cornea remain the major cause of allo-

graft injury and loss. The innate and adaptive

Immunologic graft rejection is the single most

immune systems are variously involved in rejec-

important reason for graft failure following

tion. Several factors determine the strength and

corneal transplantation. If corneal transplanta-

nature of the immune response: (1) the nature of

tion is performed in a high-risk situation with-

the grafted cornea, i.e. whether it is a clear

out the use of systemic immunosuppression,

corneal button or a limbocorneal transplant;

corneal graft failure can be expected in over

and (2) the nature of the recipient’s graft bed

50 % of cases within the first postoperative year

(i.e. whether it is clear, vascularised or has a

[15, 33].

limbal stem cell insufficiency). Additionally,

110

Chapter 9

Current Systemic Immunosuppressive Strategies in Penetrating Keratoplasty

inflammatory responses (and graft rejection is a

ber of cells. Therefore the endothelium shows

form of inflammatory response) are physiolog-

the highest immunogenicity of a corneal graft.

ically suppressed in the anterior chamber: on

the one hand, antigens injected intraocularly

elicit deviant systemic immune responses that

9.2.2

are devoid of immunogenic inflammation (a

Major Histocompatibility Complex

phenomenon called anterior chamber associat-

ed immune deviation, ACAID). On the other

9.2.2.1

hand, the ocular microenvironment (aqueous

Direct Pathway of Allorecognition

humor, secreted by cells that surround this

chamber) suppresses intraocular expression of

Direct recognition of foreign MHC antigens by

immunogenic inflammation [51].

T cells is the primary cause of acute rejection:

These special anatomical features are re-

recipient T cells recognise donor MHC class I

sponsible for the excellent results in normal-

and class II molecules, resulting in the genera-

risk corneal transplantation when compared to

tion and clonal proliferation of helper and cyto-

solid organ transplantation or high-risk corneal

toxic T cells.

transplantation.

The nature of the host’s immune response

9.2.2.2

can be determined by its histopathology and

Indirect Pathway of Allorecognition

time course as acute or chronic rejection.

This occurs when the MHC molecules of the

donor tissues are taken up and processed by

9.2.1

antigen-presenting cells, which present the for-

Acute Rejection

eign peptides to T cells. Since MHC molecules

are highly polymorphic in nature, they are

Acute rejection which may occur weeks to years

mainly responsible for allograft rejection.

after transplantation involves both humoral

Transplantation between individuals with iden-

and cell-mediated immune reactions. T cells

tical MHC molecules may also fail in the late

play a central role in acute rejection by respond-

phase because at this time the so-called minor

ing to alloantigens, predominantly major histo-

histocompatibility antigens come into play.

compatibility complex (MHC) molecules, pre-

sented on endothelial, epithelial, or stromal

cells. Both CD4+ and CD8+ T cells contribute to

9.2.3

acute rejection. CD4+ T cells mediate acute re-

Chronic Rejection

jection by secreting cytokines and inducing de-

layed-type hypersensitivity-like reactions in the

The pathogenesis of chronic rejection is not

graft. Recognition and lyses of foreign cells by

clear [3, 30]. It is likely that most of the adaptive

cytotoxic CD8+ T cells are an important mech-

and innate immune systems are involved in this

anism of acute rejection. T cells may be activat-

process. Chronic rejection cannot be prevented

ed by two distinct mechanisms: the direct and

with current immunosuppressive drugs (which

the indirect pathway.

mainly work through their interference with T

Based on the target, immune reactions

cells), so the present strategy is to limit the

against the transplanted cornea may be divided

number of acute rejection episodes. The best

into endothelial, stromal or epithelial rejection.

prospects for overcoming late graft loss due to

The most frequent and most severe form of im-

chronic rejection may reside in a new genera-

mune response is against the endothelium. The

tion of immunosuppressive agents [28]. Many

reason is that the immunogenic epithelial cells

risk factors may increase the incidence of

are replaced within approximately 1 year by the

chronic rejection: MHC incompatibility, the

host’s epithelium and the stroma mostly consists

number and severity of acute rejection episodes

of intracellular substance and only a small num-

and the recurrence of herpetic ocular disease.

9.3

Normal-Risk Versus High-Risk Transplantation

111

9.3

9.3.3

Normal-Risk Versus High-Risk

Rationale for Systemic Immunosuppression

Transplantation

The first goal of a timely limited systemic im-

Based on their risk of graft rejection, corneal

munomodulation is the prevention of acute re-

transplants can be divided into normal-risk or

jection episodes. The second goal is the interfer-

high-risk transplants.

ence with the initial graft-host interaction in a

way that graft-protective cells and cytokines are

promoted, hence enabling a clear graft survival

9.3.1

without any further medication. We have

Normal-Risk Transplantation

already shown clinically that we can reach the

first goal in most patients when using cyclo-

In a normal-risk situation (e.g. first transplant

sporine or mycophenolate mofetil systemically.

in keratokonus or Fuchs’s endothelial dystro-

Unfortunately, we still do not have convincing

phy), a 5-month course of topical steroids (e.g.

results with our therapeutic strategies when

prednisolone acetate

1 %, Inflanefran forte®)

looking at long-term graft survival.

5 times a day, reduced by one drop every

month) accompanied by systemic steroids

(prednisolone 1 mg/kg tapered within 3 weeks)

9.3.4

is sufficient to maintain a 5-year clear graft sur-

Why Is Immunomodulation

vival of up to 90 %. Up to 20 % of normal-risk

with Topical Steroids Not Sufficient

corneal transplants experience an acute rejec-

To Prevent Immunologic Graft Rejection

tion episode which can be converted in about

in High-Risk Patients?

50 % of cases with topical and systemic steroids.

The cornea is a privileged place for transplanta-

tion, for both its anatomical features (see above)

9.3.2

and the possibility of bringing medication di-

High-Risk Transplantation

rectly to the transplanted organ, thereby reduc-

ing systemic side effects. In a high-risk situation

Postoperative systemic immunosuppression is

the immunological privilege is diminished and

widely accepted as the treatment of choice

the risk of graft loss within 1 year lies over 50 %

in immunologic high-risk groups. High-risk

without the use of systemic immunosup-

corneal transplantation can be defined as fol-

pression [15, 33]. Why are topical steroids not

lows:

enough?

∑ History of previous graft rejections

The activation of the recipient’s immune sys-

∑ Deep vascularisation of the recipient cornea

tem against the transplanted cornea, i.e. the

in more than three quadrants

priming of naïve T cells, occurs in lymphoid tis-

∑ Limbal stem cell deficiency, which requires a

sues. This hypothesis is supported by experi-

corneolimbal graft

ments in which T-cell activation and therefore

∑ Severe atopic dermatitis

graft rejection did not occur when secondary

lymphoid organs were absent [18]. These exper-

In addition to topical and systemic application

imental data indicate that leukocytes partici-

of steroids as mentioned previously, systemic

pate in host T-cell priming by migrating from

immunosuppression should be applied for at

the graft to the host’s lymph node and/or

least 6 months following transplantation.

spleen, where they activate alloreactive host T

cells in the direct and indirect pathway. Such

primed T cells circulate and target MHC mole-

cules expressed by cells of the graft.

112

Chapter 9

Current Systemic Immunosuppressive Strategies in Penetrating Keratoplasty

As topical steroids do not reach the second-

ary lymphoid organs, and even systemic

9.4

steroids do not interfere sufficiently with the

Immunosuppressive Agents

clonal expansion of activated T cells, it is essen-

tial to administer systemic immunosuppres-

9.4.1

sives in order to achieve clear graft survival.

History

Summary for the Clinician

Along with the increase in the number of solid

∑

If corneal transplantation is performed in

organ transplants, our therapeutic armamen-

a high-risk situation without the use of

tarium and knowledge of immunosuppressive

systemic immunosuppression, corneal graft

drugs in corneal transplantation has been im-

failure can be expected in over 50 % of cases

proved.

within the first postoperative year

In the 1950s the selection of immunosup-

∑

Definition of high-risk corneal transplan-

pressive drugs was limited to corticosteroids

tation:

and azathioprine. In the 1960s polyclonal anti-

- History of previous graft rejections

lymphocyte (ALG) and antithymocyte (ATG)

- Deep vascularisation of the recipient

globulins supplemented the repertoire. In the

cornea in more than three quadrants

late 1970s cyclosporine A led to a real break-

- Limbal stem cell deficiency which

through in clinical solid organ transplantation

requires a corneolimbal graft

(Table 9.1). Motivated by the encouraging re-

- Severe atopic dermatitis

sults in graft survival, the research in this im-

∑

In a high-risk situation it is crucial not only

munological field then led us to a wide range of

to work with topical or systemic steroids

but to employ immunosuppressive sub-

stances systemically

Table 9.1. Immunosuppressives: history

∑

T cells play a central role in rejection by

responding to alloantigens, predominantly

1949

Cortisone was shown to alleviate

rheumatoid arthritis

MHC molecules, presented on endothelial,

1959

Cyclophosphamide was demonstrated

epithelial, or stromal cells

to suppress the formation of antibodies

∑

The activation of the recipient’s immune

and was used for bone marrow

system against the transplanted cornea,

transplantation

i.e. the priming of naïve T cells, occurs

1960s

Azathioprine was found to delay organ

in lymphoid tissues

graft rejection

∑

As topical steroids do not reach the second-

1969

Methotrexate was shown to inhibit

ary lymphoid organs, and even systemic

antibody formation and the development

steroids do not interfere sufficiently with

of delayed hypersensitivity in guinea pigs

the clonal expansion of activated T cells,

1976

T-cell-inhibiting properties of

it is essential to administer systemic

cyclosporine were demonstrated

immunosuppressives in order to achieve

1982

Development of mycophenolate mofetil

clear graft survival

1987

Tacrolimus (FK506) was shown

∑

The first goal of a timely limited systemic

to inhibit IL-2 production

immunomodulation is the prevention

and lymphocyte proliferation

of acute rejection episodes

1980s

Interest in the antibiotic sirolimus

∑

The second goal is the interference with the

was renewed when it was shown

initial graft-host interaction in a way that

to prevent allograft rejection

graft-protective cells and cytokines are pro-

1978

Leflunomide

moted, hence enabling a clear graft survival

1981

Mizoribine, deoxyspergualin, brequinar

without any further medication

1990s

IL-2 antagonists (daclizumab,

basiliximab)

9.4

Immunosuppressive Agents

113

Fig. 9.1. Immunosuppressives:

sites of action

Table 9.2. Immunosuppressives: mode of action

Mode of action

Substances

Regulators of gene expression

Glucocorticoids, vitamin D analogs, deoxyspergualin

Alkylating agents

Cyclophosphamide

Kinases and phosphatase inhibitors

Cyclosporine, tacrolimus, everolimus, rapamycin

Inhibitors of de novo purine synthesis

First generation: mercaptopurine, azathioprine,

methotrexate

Second generation: mizoribine and MMF

Inhibitors of de novo pyrimidine synthesis

Brequinar, leflunomide, malononitrilamides

potent immunosuppressive agents with highly

produced from microorganisms, e.g. cyclo-

specific sites of action (Fig. 9.1).

sporine, tacrolimus) (Table 9.2).

According to their mode of action these new

Despite the tremendous breadth of the disci-

drugs can be divided up into agents that selec-

pline of immunosuppressive molecules, only a

tively inhibit cytokine gene transcription/

small number of drugs have made it as far as be-

expression

(cyclosporine, tacrolimus), anti-

ing used for experimental or clinical corneal

proliferative agents

(mycophenolate mofetil,

transplantation. We have decided to focus on

azathioprine) and agents that interfere with

the following agents:

intracellular signal transduction

(rapamycin,

∑ Corticosteroids

everolimus). Immunosuppressives might also

∑ Cyclosporine (Sandimmun, Neoral, CSA)

be classified as biologics which are defined as

∑ Tacrolimus (Prograf, FK506)

naturally occurring or genetically engineered

∑ Mycophenolate mofetil

mammalian proteins (thymoglobulin, basilix-

(CellCept, Myfortic, MMF)

imab and daclizumab) or xenobiotics (drugs

∑ RAD (Everolimus,Certican)

114

Chapter 9

Current Systemic Immunosuppressive Strategies in Penetrating Keratoplasty

∑ Rapamycin (Sirolimus, Rapamune)

exceptional efforts undertaken by Hill and col-

∑ FTY720

leagues in South Africa [14, 15]. These initial

∑ Biological agents (basiliximab, daclizumab)

positive clinical experiences with systemic CSA

to prevent corneal allograft rejection in high

risk keratoplasty have been confirmed by others

9.4.2

[32, 33, 35].

Corticosteroids

Despite the significant improvement of out-

come in high-risk keratoplasty, the use of CSA is

Corticosteroids prevent interleukin (IL)-1 and

limited due to its considerable toxicity and the

IL-6 production by macrophages and inhibit all

need for costly drug monitoring. The toxicity

stages of T-cell activation. Adverse effects of

is mostly caused by the CSA-cyclophilin-cal-

systemic steroids include Cushing’s disease,

cineurin-calmodulin complex, which interferes

bone disease

(e.g. osteoporosis, avascular

with tubular and endothelial cell functions:

necrosis), cataract, glucose intolerance, infec-

nephro- and hepatotoxicity, and alterations in

tions, hyperlipidaemia, and growth retardation.

glucose metabolism, hypertension, and gingival

Adverse effects of topically applied steroids

hyperplasia. To avoid the systemic toxicity,

include cataract, glaucoma and in the case of

attempts have been undertaken to apply CSA in

epithelial defects - steroid ulcers.

topical formulations including the use of colla-

gen carriers [6, 16]. The encouraging results of

these mostly experimental studies in preventing

9.4.3

corneal graft rejection did not hold true clini-

Cyclosporine A (CSA, Sandimmun,

cally [29]. However, we have shown that topical

Sandimmun Optoral, Sandimmun Neoral)

CSA is efficient in the treatment of distinct

immunological disorders of the cornea (e.g.

The fermentation product from the fungi

Thygeson’s keratitis, persistent nummular infil-

Tolypocladium inflatum Gams was first isolated

trates following adenovirus infections) [34, 36].

in 1970 by Thiele and Kis. Its immunosuppres-

sive properties were discovered in 1972 by Borel.

Sandimmun Neoral is a special galenic formula-

9.4.4

tion based on microemulsion technology.

Tacrolimus (FK506, Prograf)

Cyclosporine A binds to the intracellular im-

munophilin cyclophilin

(immunophilins are

Tacrolimus has been proven clinically superior

proteins which bind to immunosuppressive

to CSA following solid organ transplantation [5,

drugs). The CSA-cyclophilin complex blocks

54]. Tacrolimus, like CSA, is a macrolide antibi-

calcineurin-calmodulin-induced phosphoryla-

otic (structurally related to erythromycin and

tion of NFAT (nuclear factor of activated T

rapamycin) derived from a fungus, Strepto-

cells), transcription factor for IL-2 and other

myces tsukubaensis [17]. Its immunosuppressive

early T-cell specific genes (Fig. 9.1) and hence is

properties were discovered by Ochai in 1985.

highly T cell specific.

In vitro studies have shown that, even in con-

Clinical efficacy and safety data have most-

centrations

40-200 times lower than CSA,

ly been acquired in solid organ transplanta-

tacrolimus possesses extremely powerful im-

tion, and it is still the gold standard in all

munosuppressive effectiveness

[45,

55]. Al-

forms of solid organ transplantation (except

though the final step in modulating the immune

liver transplantation) mainly in combination

system is the same for CSA and tacrolimus, i.e.

with steroids, azathioprine or mycophenolate

interfering with the intracytoplasmatic cal-

mofetil.

cineurin system and hence the interleukin IL-2

production, both drugs manage this in a differ-

CSA in Corneal Transplantation. The first doc-

ent manner. Tacrolimus binds to the intra-

umented clinical experiences in corneal trans-

cellular FKBP-12 (FK-binding protein-12). The

plantation date back to the mid 1980s with the

tacrolimus-FKBP-12 complex blocks calcineurin-

9.4

Immunosuppressive Agents

115

calmodulin-induced phosphorylation of the

Unlike CSA or tacrolimus, MMF does not inter-

cytoplasmic component of NFAT transcription

fere with IL-2 pathways. Mycophenolic acid

factor for IL-2 and other “early” genes. Like CSA,

reversibly inhibits the de novo formation of

tacrolimus is a highly specific inhibitor of lym-

guanosine nucleotides [1] by inhibiting the en-

phocyte activation. Its toxicities are similar to

zyme inosine monophosphate dehydrogenase

CSA (probably due to its calcineurin-mediated

(with high affinity to the isoform II, which is

interference with tubular and endothelial cells),

expressed in activated lymphocytes).As T and B

i.e. nephro-, neurotoxicity, arterial hyperten-

cells are predominantly dependent on the de

sion, diabetogenicity.

novo synthesis of guanosine nucleotides, the

purine biosynthesis of these cells is selectively

Tacrolimus in Corneal Transplantation. Up to

inhibited [24].

now there have only been limited clinical data

As MMF is not an antimetabolite and does

available about the efficacy of tacrolimus in

not lead to genetic miscoding, it is not carcino-

corneal transplantation [49]. This might par-

genic.

tially be explained by its relatively narrow safe-

ty margins. Whereas CSA might be given in a

Mycophenolate Mofetil in Corneal Transplanta-

body weight adjusted dose (a suboptimal thera-

tion. We have been able to prove the potency of

peutic approach which is practised in some cen-

this drug and its synergistic effect on CSA and

tres in the United States), tacrolimus has to be

FK506 in delaying corneal allograft rejection in

closely monitored because the risk of overim-

the rat keratoplasty model [41]. Following these

munosuppression is great. This is also the

initial positive experiences we conducted a

reason for the initially rather unjustified poor

prospective clinical trial with MMF and CSA in

reputation of this drug: initially blood levels of

high-risk keratoplasty patients. The data from

20-30 ng/ml were targeted (with corresponding

this study show a similar efficacy of MMF and

adverse events), whereas today blood levels of

CSA in preventing allograft rejection [44]. But

5-10 ng/ml are considered to be the optimal

due to the high therapeutic margin and

range.

favourable safety profile of MMF, costly drug

The potency of this drug to inhibit experi-

monitoring is not indicated. Additionally we

mental corneal allograft rejection after systemic

used this substance in immunological disorders

administration has been proven [2, 42, 43]. As

of the eye, again with favourable results [40].

with CSA, much hope is pinned on finding an

efficient topical administration to prevent sys-

temic side effects. Experimentally the efficacy of

9.4.6

topical tacrolimus has yet been proven [9, 13, 22,

Rapamycin (Sirolimus, Rapamune)

23], and clinical studies of topical tacrolimus in

atopic conjunctivitis are under way.

Sirolimus (Rapamune) is an immunosuppres-

sive agent previously known as rapamycin. It

was under development for more than 20 years

9.4.5

before it gained FDA approval in 1999. Sirolimus

Mycophenolate Mofetil

is a macrocyclic lactone produced by Strepto-

(MMF, CellCept, Myfortic)

myces hygroscopicus found in the soil of Easter

Island. Structurally, sirolimus resembles tacro-

Mycophenolate mofetil

(MMF) is the bio-

limus and binds to the same intracellular bind-

availability-enhanced morpholinoethylester of

ing protein or immunophilin known as FKBP-

mycophenolic acid (MPA), which was originally

12. However, sirolimus has a novel mechanism

isolated from Penicillium spp. MMF is rapidly

of action. Whereas tacrolimus and cyclosporine

converted to MPA, its active compound. Its safe-

block lymphokine (e.g. IL-2) gene transcrip-

ty and effectiveness in combination with CSA

tion, sirolimus acts later to block IL2-dependent

following kidney transplantation have been

T-lymphocyte proliferation and the stimulation

proven in several clinical studies [8, 11, 48, 52].

caused by cross-linkage of CD28, possibly by

116

Chapter 9

Current Systemic Immunosuppressive Strategies in Penetrating Keratoplasty

blocking activation of a kinase referred to as the

Everolimus may have a special role in solid

mammalian target of rapamycin or “mTOR”, a

organ transplantation as it has been shown to

serine-threonine kinase that is important for

reduce chronic allograft vasculopathy in such

cell cycle progression. Therefore, sirolimus is

transplants [10].

believed to act in synergy with cyclosporine (or

tacrolimus) in suppressing the immune system.

Everolimus in Corneal Transplantation. We

Rapamycin has been shown to be highly effi-

have tested this new compound in the rat mod-

cient in preventing experimental solid organ

el of corneal transplantation both as a single

[25, 53] and clinical renal transplantation [4, 26].

therapy and in combination with CSA and

It is noteworthy that rapamycin is not nephro-

MMF. It appears that the potency of everolimus

toxic, which makes this drug especially interest-

to prevent corneal allograft rejection is compa-

ing for renal transplant recipients.

rable to CSA. Additionally we have found a syn-

ergistic effect of everolimus in a double-drug

Sirolimus in Corneal Transplantation. A cou-

regimen with CSA as well as with MMF [36, 38,

ple of experimental studies have shown the effi-

39].

cacy of sirolimus in inhibiting murine corneal

There are to date no clinical data on the effi-

allograft rejection [27, 52]. We have conducted a

cacy and safety of everolimus in corneal trans-

small clinical study with sirolimus in high-risk

plantation.

corneal transplantation. We started Rapamune

on the day of transplantation at a dose of

2 mg/day. The dose was adjusted to reach plas-

9.4.8

ma levels of 4-10 ng/ml on subsequent days, try-

FTY 720

ing to keep plasma levels close to 4 ng/ml. We

have seen that the efficacy of Rapamune in pre-

The chemical

2-amino-2[2-(4-octylphenyl)

venting corneal allograft rejection is compara-

ethyl]-1, 3, propane diol is one of a class of small-

ble to that of cyclosporine and MMF. But it is

molecule immunosuppressive agents. This

worth mentioning that we have seen a high inci-

compound was chemically synthesised in an

dence of side effects in this small group of

effort to minimise the toxic in vivo properties of

patients.

a structurally related and highly potent im-

munosuppressive agent, myriocin. The mecha-

nism of action of FTY720, although not fully

9.4.7

characterised, appears to be unique among im-

RAD (Everolimus, Certican)

munosuppressants. In vivo, FTY720 induces a

significant reduction in the number of circulat-

Everolimus is an oral rapamycin derivative pro-

ing lymphocytes. It is thought to act by altering

duced by Novartis Pharma. It is chemically de-

lymphocyte trafficking/homing patterns through

rived from rapamycin which has been obtained

modulation of cell surface adhesion receptors.

by fermentation of an Actinomycetes strain. It

Although much research has yet to be done to

has been found that everolimus (40-0-[2-hy-

unravel the nature of the mechanism of action

droxyethyl])-RPM is stable in oral formulations

of FTY720, its efficacy has been sufficiently

and that its efficacy after oral dosing is at least

proven in numerous animal models, especially

equivalent to that of rapamycin [7, 47]. The

when administered in combination with cyclo-

mode of action is equivalent to that of ra-

sporine. It has been shown that FTY720 is effica-

pamycin, i.e. binding to FKBP, inhibiting TOR1

cious in a variety of transplant and autoimmune

and 2 and hence inhibiting cell-cycle progres-

models without inducing a generalised im-

sion of activated T cells.

munosuppressed state and is effective in human

Everolimus and sirolimus are also called pro-

kidney transplantation.

liferation signal inhibitors (PSI), because they

prevent proliferation of T cells.

9.5

Guidelines for Practitioners

117

FTY720 in Corneal Transplantation. We have

9.4.9.3

been able to show the efficacy of FTY720 in in-

Basiliximab and Daclizumab

hibiting murine corneal allograft rejection [20].

There are to date no data of FTY720 in clinical

Basiliximab (Simulect) and daclizumab (Zena-

corneal transplantation.

pax) are humanised monoclonal antibodies that

target the IL-2 receptor. Clinically, both agents

are very similar, and both are used for induction

9.4.9

therapy in solid organ transplantation. These

Biologic Agents

agents have a very low prevalence of adverse

effects, although hypersensitivity reactions

Reports about the use of biological agents in

have been reported with basiliximab (Simulect),

corneal transplantation are very rare [46]. To

albeit rarely.

complete this overview their mode of action is

Perioperative basiliximab has been tested in

briefly outlined.

combination with cyclosporine postoperatively

in a small clinical study with favourable results

9.4.9.1

[46]. In 2004 we started a prospectively ran-

Polyclonal Antibodies

domised clinical trial of basiliximab as mono-

(e.g. Antithymocyte Globulins)

therapy compared to cyclosporine.

Summary for the Clinician

These agents are derived by injecting animals

with human lymphoid cells, then harvesting

∑ To date the efficacy in preventing corneal

and purifying the resultant antibody. Polyclonal

graft rejection has only been proven for

antibodies induce the complement lysis of

cyclosporine, mycophenolate mofetil and

lymphocytes and uptake of lymphocytes by the

rapamycin in prospective clinical trials

reticuloendothelial system and mask the lym-

∑ The efficacy and safety of tacrolimus in

phoid cell-surface receptors. Preparations in-

high-risk corneal transplantation has been

clude horse antithymocyte globulin (Atgam)

described in a retrospective manner

and rabbit antithymocyte globulin (thymoglob-

∑ Especially in high-risk corneal transplanta-

ulin). These agents are used for induction ther-

tion as it is not a life-saving procedure it is

apy and for the treatment of acute rejection in

important to weigh the pros and cons of

solid organ transplantation.

any immunosuppressive regimen

Adverse effects include fever, chills, throm-

∑ With respect to the profile of side effects, we

bocytopenia,leukopenia,haemolysis,respirato-

prefer cyclosporine and mycophenolate

ry distress, serum sickness, and anaphylaxis.

mofetil over rapamycin and tacrolimus

9.4.9.2

Muromonab-CD3

9.5

Guidelines for Practitioners

Muromonab-CD3 is a murine monoclonal anti-

body of immunoglobulin 2A clones to the CD3

9.5.1

portion of the T-cell receptor. It blocks T-cell

Preoperative Evaluation

function and has limited reactions with other

tissues or cells. This agent is used for induction

As systemic immunosuppression might pro-

and for the therapy of acute rejection (primary

mote tumour growth or reactivation of a chron-

treatment or steroid resistant).

ic infection, patients need to be checked by their

Adverse effects include cytokine release syn-

internists to rule out neoplasms and infections

drome (i.e. fever, dyspnoea, wheezes, headache,

(blood chemistry, abdominal ultrasound, chest

hypotension) and pulmonary oedema.

X-ray) before immunosuppression is started.

Additionally due to possible drug-specific side

effects of immunosuppressive agents, renal and

118

Chapter 9

Current Systemic Immunosuppressive Strategies in Penetrating Keratoplasty

hepatic functions should be controlled. The pa-

systemic steroids, MMF is started on the day of

tients should undergo these examinations at the

operation in a dosage of 1 g twice daily. Drug-

time they are put on the waiting list for trans-

monitoring is not mandatory due to the drug’s

plantation. If any contraindications against sys-

broad safety margins. We perform blood chem-

temic immunosuppression are found, these

istry once a month as MMF might be myelosup-

conditions need to be cleared before transplan-

pressive and might lead to a rise in liver en-

tation. If the conditions cannot be cleared, the

zymes. If side effects occur, we reduce MMF to

indication for high-risk corneal transplantation

0.5 g twice daily. In the case of drug-specific side

should be reconsidered. In this situation the use

effects or graft rejection drug-monitoring is

of an optimally matched graft might be an in-

indicated to rule out inadequate dosing.

teresting alternative to systemic immunosup-

MMF is especially valuable in herpetic ocular

pression. In the case of drug-specific con-

disease when combined with acyclovir due to its

traindications, alternative drugs should be used

synergistic antiherpetic effect [20].

(e.g. in the case of renal impairment, mycophe-

In cases of:

nolate mofetil should be used instead of CSA).

∑ Arterial hypertension

∑ Diabetes mellitus

∑ Chronic renal disease

9.5.2

∑ Low compliance

How To Use Cyclosporine

in High-Risk Corneal Transplantation

MMF is favoured above CSA or tacrolimus be-

cause of its safety margins and the profile of

In addition to perioperative topical and sys-

possible side effects.After at least 6 or 12 months

temic steroids, CSA is started on the day of op-

following transplantation, MMF is tapered and

eration in a dosage of 100 mg twice daily. With-

discontinued within 1 week. In the case of drug-

in the first postoperative week, full blood trough

specific side effects we monitor blood levels.

levels of CSA (12 h after administration) need to

be checked daily, and the dose adjusted to reach

serum levels of 120-150 ng/ml. We adjust the

9.5.4

dose by using increasing or decreasing steps of

How To Use Rapamycin

25 mg. If serum levels appear to be stable, we re-

in High-Risk Corneal Transplantation

duce drug-monitoring to once a week in the first

months and afterwards to once a month. Addi-

In our pilot study we have seen that Rapamune

tionally we check for liver and kidney functions.

effectively prevents acute allograft rejection.

Depending on the risk situation we continue

But due to the rather broad range of side effects,

therapy for at least 6 or 12 months and taper

we do not recommend Rapamune in high-risk

therapy by reducing CSA in 25-mg steps daily.

keratoplasty at this time point. Rapamune

CSA is especially helpful in high-risk pa-

should especially not be given to patients with

tients who also suffer from atopic dermatitis.

metabolic disorders (i.e. hypercholesterolaemia

CSA should only be used with great caution in

and hypertriglyceridaemia) as it aggravates

patients with renal impairment, diabetes melli-

these conditions in more than 50 % of patients.

tus and arterial hypertension.

9.5.5

9.5.3

How To Use Tacrolimus

How To Use MMF

in High-Risk Corneal Transplantation

In addition to perioperative topical and sys-

The application of MMF following high-risk

temic steroids, tacrolimus is started on the day

corneal transplantation is easier than the use of

of transplantation. As with CSA, blood levels

CSA. In addition to perioperative topical and

should be controlled daily in the 1st week. Plas-

References

119

ma levels of 5 ng/ml should be aimed for. If plas-

immunosuppressive regimen. This means first-

ma levels appear to be stable, drug monitoring

ly that the immunosuppressants may be chosen

can be reduced to once a week in the first

according to the underlying diseases of the pa-

months and once a month thereafter. Therapy

tient (i.e. CSA or tacrolimus in keratoplasty in a

should be applied for at least 6-12 months de-

patient with atopic dermatitis, MMF in kerato-

pending on the clinical course and tapered out

plasty in patients with herpetic eye disease or

within 2 weeks. Probably due to its calcineurin

patients with impaired renal function). Second-

mediated interference with tubular and en-

ly the efficacy of immunosuppression may now

dothelial cells, the profile of side effects is simi-

be adjusted to the clinical situation by adding

lar to that of CSA. Tacrolimus should not be

another immunosuppressant to a baseline med-

used in patients with diabetes mellitus, arterial

ication, thereby minimising a drug-specific tox-

hypertension or renal impairment. Due to the

ic side effect. Especially in high risk corneal

drug’s narrow safety margins, special care must

transplantation since it is not a life-saving pro-

be taken in cases where compliance is not guar-

cedure, it is important to weigh the pros and

anteed. MMF should be used in these patients

cons of any immunosuppressive regimen.

instead.

References

9.5.6

Combination Therapies

Allison A, Hovi R, Watts A, Webster A (1977) The

role of de novo purine synthesis in lymphocyte

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Benelli U, Lepri A, Del-Tacca M, Nardi M (1996)

in an oculus ultimus patient or in limbal stem

FK-506 delays corneal graft rejection in a model

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of corneal xenotransplantation. J Ocul Pharmacol

munosuppressive therapy might be indicated.

Ther 12(4):425-431

Immunosuppressive potency might be en-

Birnbaum F, Reinhard T, Bohringer D, Sundmach-

hanced by combining two immunosuppres-

er R (2005) Endothelial cell loss after autologous

sants, thereby minimising a drug-specific toxic

rotational keratoplasty. Graefes Arch Clin Exp

side effect. We have demonstrated the efficacy

Ophthalmol 243(1):57-59

Brattstrom C, Tyden G, Sawe J, Herlenius G, Claes-

and safety of a double-drug regimen with cyclo-

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blind, placebo-controlled study to determine

eratoplasty [31].

safety, tolerance, and preliminary pharmacoki-

netics of ascending single doses of orally admin-

istered sirolimus

(rapamycin) in stable renal

9.6

transplant recipients. Transplant Proc

28(2):

Conclusion

985-986

Bussutil RW, Holt CD (1997) Tacrolimus (FK506)

is superior to cyclosporine in liver transplanta-

It is now 60 years since we learned about im-

tion. Transplant Proc 29:534-538

munologic graft failure thanks to the pioneer-

Chen YF, Gebhardt BM, Reidy JJ, Kaufman HE

ing work of Medawar and Maumenee in the

(1990) Cyclosporine-containing collagen shields

1940s. Since that time impressive developments

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have been undertaken in the field of pharma-

thalmol 109(2):132-137

ceutical immunosuppression. CSA has for many

Crowe A, Lemaire M (1998) In vitro and in situ ab-

sorption of SDZ-RAD using a human intestinal

years been the gold standard in organ and high-

cell line

(Caco-2) and a single pass perfusion

risk corneal transplantation, but it is now ac-

model in rats: comparison with rapamycin.

companied by mycophenolate mofetil and

Pharm Res 15(11):1666-1672

tacrolimus.

Deierhoi M, Sollinger H, Diethelm A et al. (1993)

The new compounds now give us the possi-

One-year follow-up results of a phase 1 trial of

bility not just to maximise immunosuppressive

mycophenolate mofetil (RS-61443) in cadaveric

potency but to apply a more patient-oriented

renal transplantation. Transplant Proc 25:693f

120

Chapter 9

Current Systemic Immunosuppressive Strategies in Penetrating Keratoplasty

Dickey JB, Cassidy EM, Bouchard CS (1993) Peri-

22.

Mills RA, Jones DB,Winkler CR,Wallace GW,Wil-

ocular FK-506 delays allograft rejection in rat

helmus KR (1995) Topical FK-506 prevents exper-

penetrating keratoplasty. Cornea 12(3):204-207

imental corneal allograft rejection. Cornea 14(2):

10.

Eisen HJ, Tuzcu EM, Dorent R et al. (2003)

157-160

Everolimus for the prevention of allograft rejec-

23.

Minamoto A, Sakata H, Okada K, Fujihara M

tion and vasculopathy in cardiac-transplant re-

(1995) Suppression of corneal graft rejection by

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subconjunctival injection of FK-506 in a rat mod-

11.

European Mycophenolate Mofetil Cooperative

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39(1):12-19

mycophenolate mofetil combined with cyclo-

24.

Morris R, Hoyt E, Murphy P (1990) Mycophenolic

sporin and corticosteroids for the prevention of

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acute rejection. Lancet 345:1321-1325

immunosuppressant that prevents and halts

12.

Mayer K, Birnbaum F, Reinhard T, Reis A, Braun-

heart allograft rejection by selective inhibition of

stein S, Claas F, Sundmacher R (2004) FTY720

T- and B-cell purine synthesis. Transplant Proc

prolongs clear corneal allograft survival with a

22:1659

differential effect on different lymphocyte popu-

25.

Morris RE, Huang X, Gregory CR, Billingham ME,

lations. Br J Ophthalmol 88(7):915-919

Rowan R, Shorthouse R, Berry GJ (1995) Studies

13.

Hikita N, Lopez JS, Chan C, Mochizuki M,

in experimental models of chronic rejection: use

Nussenblatt RB, de Smet MD (1997) Use of topical

of rapamycin (sirolimus) and isoxazole deriva-

FK506 in a corneal graft rejection model in Lewis

tives (leflunomide and its analogue) for the sup-

rats. Invest Ophthalmol Vis Sci 38:901-909

pression of graft vascular disease and obliterative

14.

Hill JC, Maske R (1988) An animal model for

bronchiolitis. Transplant Proc 27(3):2068-2069

corneal graft rejection in high-risk keratoplasty.

26.

Murgia MG, Jordan S, Kahan BD (1996) The side

Transplantation 46(1):26-30

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15.

Hill JC (1995) Systemic cyclosporine in high-risk

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keratoplasty: long-term results. Eye 9(4):422-428

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16.

Hoffmann F, Wiederholt M (1986) Topical cyclo-

27.

Olsen TW, Benegas NM, Joplin AC, Evangelista T,

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Mindrup EA, Holland EJ (1994) Rapamycin in-

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17.

Kino T, Hatayama H, Hashimoto M et al. (1987)

larization. Arch Ophthalmol 112(11):1471-1475

FK-506, a novel immunosuppressant isolated

28.

Otto Ch, Ulrichs K (2004) The immunology of

from Streptomyces: 1. Fermentation, isolation, and

allograft rejection. Tx Med 16:158-171

physio-chemical and biological characteristics.

29.

Perry HD, Donnenfeld ED,Acheampong A, Kanel-

J Antibiot 40:1249-1255

lopoulos AJ, Sforza PD, D’Aversa G, Wallerstein A,

18.

Lakkis FG, Arakelov A, Koniecny BT, Inoue Y

Stern M

(1998) Topical cyclosporine A in

(2000) Immunologic “ignorance” of vascularized

the management of postkeratoplasty glaucoma

organ transplants in the absence of secondary

and corticosteroid-induced ocular hypertension

lymphoid tissue. Nat Med 6:686-688

(CIOH) and the penetration of topical 0.5 % cy-

19.

Mayer K, Reinhard T, Reis A, Voiculescu A, Sund-

closporine A into the cornea and anterior cham-

macher R (2003) Synergistic antiherpetic effect of

ber.CLAO J 24(3):159-165

acyclovir and mycophenolate mofetil following

30.

Reinhard T, Bohringer D, Enzmann J, Kogler G,

keratoplasty in patients with herpetic eye disease:

Wernet P, Bohringer S, Sundmacher R (2004)

first results of a randomised pilot study. Graefes

HLA class I/II matching and chronic endothelial

Arch Clin Exp Ophthalmol 241(12):1051-1054

cell loss in penetrating normal risk keratoplasty.

20.

Mayer K, Reinhard T, Reis A, Voiculescu A, Sund-

Acta Ophthalmol Scand 82(1):13-18

macher R (2003) Synergistic antiherpetic effect of

31.

Reinhard T, Kontopoulos T, Wernet P, Enzmann J,

acyclovir and mycophenolate mofetil following

Sundmacher R (2004) Long-term results of ho-

keratoplasty in patients with herpetic eye disease:

mologous penetrating limbokeratoplasty in total

first results of a randomised pilot study. Graefes

limbal stem cell insufficiency after chemical/

Arch Clin Exp Ophthalmol 241(12):1051-1054

thermal burns. Ophthalmologe 101(7);682-687

21.

Mayer K, Birnbaum F, Reinhard T, Reis A, Braun-

32.

Reinhard T, Moller M, Sundmacher R (1999) Pen-

stein S, Claas F, Sundmacher R (2004) FTY720

etrating keratoplasty in patients with atopic der-

prolongs clear corneal allograft survival with a

matitis with and without systemic cyclosporin A.

differential effect on different lymphocyte popu-

Cornea 18(6):645-651

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43.

Reis A, Reinhard T, Sundmacher R, Godehard E,

P (1997) [Preventive systemic cyclosporin A after

Braunstein C (1998) FK506 and mycophenolate

keratoplasty at increased risk for immune reac-

mofetil: two novel immunosuppressants in murine

tions as the only elevated risk factor.] Systemis-

corneal transplantation. Transpl Proc 30(8)

che Cyclosporin-A-Prophylaxe nach Keratoplas-

44.

Reis A, Reinhard T, Voiculescu A, Kutkuhn B,

tiken mit erhohtem Risiko für Immunreaktionen

Godehardt E,Spelsberg H,Althaus C,Sundmach-

als einzigem erhohten Risikofaktor. Ophthal-

er R (1999) Mycophenolatemofetil versus cyclo-

mologe 94(7):496-500

sporin A in high-risk keratoplasty patients: a

34.

Reinhard T, Sundmacher R (1996) Local cyclo-

prospectively randomized clinical trial. Br J Oph-

sporin A therapy in Thygeson superficial punc-

thalmol 83:1268-1271

tate keratitis - a pilot study. Klin Monatsbl Au-

45.

Sawada S, Suzuki G, Kawase Y, Takaku F (1987)

genheilkd 209(4):224-227

Novel immunosuppressive agent, FK-506: in vitro

35.

Reinhard T, Sundmacher R (1992) Perforating

effects on the cloned T cell activation. J Immunol

keratoplasty in endogenous eczema. An indica-

40:1249-1255

tion for systemic cyclosporin A - a retrospective

46.

Schmitz K, Hitzer S, Behrens-Baumann W (2002)

study of 18 patients. Klin Monatsbl Augenheilkd

Immune suppression by combination therapy

201(3):159-163

with basiliximab and cyclosporin in high risk

36.

Reinhard T, Sundmacher R (1999) Topical cy-

keratoplasty. A pilot study. Ophthalmologe 99(1):

closporin A in Thygeson’s superficial punctate

38-45

keratitis. Graefes Arch Clin Exp Ophthalmol

47.

Schuurman HJ, Schuler W, Ringers J, Jonker M

237(2):109-112

(1998) The macrolide SDZ RAD is efficacious in a

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immunosuppressive macrolide in murine corneal

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Simmons WD, Rayhill SC, Sollinger HW (1997)

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Spelsberg H, Braunstein C, Sundmacher R (2000)

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Streilein JW, Okamoto S, Sano Y, Taylor AW

Coadministration of the new macrolide immuno-

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The Tricontinental Mycophenolate Mofetil Renal

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Transplantation Study Group (1996) A blinded,

40. Reis A, Reinhard T, Sundmacher R, Althaus C,

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Trephination in Penetrating Keratoplasty

Berthold Seitz, Achim Langenbucher, Gottfried O.H. Naumann

Core Messages

10.1

∑ Donor and host trephination should be

Introduction

performed with the same system from

the epithelial side

Zirm in 1905 was the first surgeon to perform a

∑ A horizontal position of the limbal plane

successful homologous penetrating keratoplas-

is essential

ty (PKP) in a human patient [84]. The operation

∑ The graft size should be adjusted individu-

became more successful with the development

ally (“as large as possible, and as small as

of more delicate instruments, use of the operat-

necessary”)

ing microscope, and the availability of antibi-

∑ Limbal centration is to be preferred over

otics, antivirals and corticosteroids. Today, still

pupil centration (especially in keratoconus!)

unsolved problems include:

(1) high/irregu-

∑ Avoid excessive graft over- or undersize

lar astigmatism, (2) trephination of unstable

∑ Intraoperative adjustment is required of

cornea, (3) surface pathologies, (4) immunolog-

double running suture

ic graft rejection, (5) secondary glaucomas, (6)

∑ Nonmechanical excimer laser trephination

chronic endothelial cell loss of the transplant,

results in:

(7) recurrences of the disease, and (8) a lack of

– Lower astigmatism

donor tissue.

– Higher regularity of topography

With the improved understanding and man-

– Better visual acuity - especially

agement of immunologic problems during past

in young patients with keratoconus

few decades, the microsurgeon’s main attention

∑ In unstable corneas (e.g., after RK, iatro-

in corneal transplantation has shifted from pre-

genic keratectasia after LASIK, descemeto-

serving a “clear graft” towards achieving a good

cele, perforated ulcer), laser application

refractive outcome. Thus, PKP today is no

makes trephination feasible

longer just a “curative” but has also become a

∑ New nut-and-bolt type variants for

sort of “refractive” procedure. Today, a crystal

potentially self-sealing donor/host

clear corneal graft after PKP with high and/or

appositions are on the horizon (“no-stitch

irregular astigmatism - especially if in associa-

keratoplasty”)

tion with high anisometropia - can no longer be

∑ Femtosecond laser application may be the

considered “successful” in normal-risk kerato-

“excitement of tomorrow” in microsurgery

plasties. Deluded by advertisements of refrac-

of the cornea

tive surgery, patients expect an optimal visual

acuity preferably without spectacles. Many pa-

tients consider the necessity of wearing contact

lens as representing a partial failure of the inter-

vention. Especially older PKP patients cannot

cope with contact lenses manually and/or men-

tally. Additional “dysfunctional tear syndrome”

and blepharitis further promote contact lens

124

Chapter 10

Trephination in Penetrating Keratoplasty

intolerance in this age group. Persisting corneal

Table 10.1. Assessment of astigmatism and visual

hypesthesia after PKP for many years can delay

acuity after keratoplasty

(SRI, surface regularity

index; SAI, surface asymmetry index; PVA, potential

recognition of contact lens induced damage to

visual acuity)

the cornea.

It has been debated whether cutting or sutur-

Uncorrected visual acuity

ing is more important for the regularity of the

Keratometry

transplant curvature. We have always stressed

a) Absolute values

that: (1) early postoperative astigmatism with

b) Angle of steep and flat meridian

sutures in place should be differentiated from

separately (Dπ90°)

(2) late persisting postoperative astigmatism

c) Classification of irregularity [59, 62]

without sutures [59].

Topography analysis

a) Meridians

Summary for the Clinician

b) Hemimeridians

Two major types of post-PKP astigmatism

c) Irregularity (SRI, SAI)

need to be distinguished:

d) Semiquantitative classification [29]

1. Early postoperatively with sutures in place

Objective refractometry/retinoscopy

predominantly depending on:

Subjective refractometry

- Symmetry of suture positions

and spectacle-corrected visual acuity

- Depth of suture track in graft

Pinhole

and recipient

Diagnostic contact lens

- Homogeneity of suture tension

- Microsurgeon’s “hand writing”

2. Late postoperatively persisting without

sutures predominantly depending on:

- Cut quality

- Wound configuration

(horizontal/vertical)

- Symmetry of graft placement

- Wound healing

10.2

Astigmatism and Keratoplasty

10.2.1

Definition of Post-keratoplasty

Fig. 10.1. Semiquantitative classification of regular-

ity of keratometry mires (ophthalmometer, type H,

Astigmatism

190071, Zeiss, Jena, Germany) (0, regular; 1, mildly

irregular; 2, severely irregular; 3, not measurable) [59,

The cornea contributes about two-thirds of the

62, 83]

refractive power of a human eye. Surgical proce-

dures on the cornea may therefore influence the

state of refraction considerably. Corneal astig-

matism is an optical aberration, resulting from

hemimeridians. In addition, the refractive pow-

unequal refraction of entering light in different

er of corresponding hemimeridians may differ.

meridians of the corneal surface. Astigmatism

Especially with sutures in place, patients accept

after PKP is often irregular, i.e., two or more

much less subjective cylinder than indicated by

meridians are separated from each other by

objective measures such as keratometry or to-

an angle not equal to 90°. Two or more steep

pography analysis [20]. In cases of highly irreg-

hemimeridians are not located opposite to each

ular astigmatism, good visual acuity can only be

other. The same may be true for the flat

achieved by hard contact lenses (Table 10.1).

10.2

Astigmatism and Keratoplasty

125

Fig. 10.2. Semiquantitative

classification of corneal topo-

graphy after PKP [29]: 1, ortho-

gonal symmetric (i.e., difference

of maximal powers of opposing

hemimeridians is less than

2 diopters and deviation of axis

of opposing hemimeridians is

less than 20°); 2, orthogonal

non-symmetric; 3, non-ortho-

gonal symmetric; 4, non-

orthogonal non-symmetric;

5, keratoconus-like (a steep

sector is opposing a flat sector

at the apex, difference between

steep and flat hemimeridian

at least 2 diopters); 6, polyaxi-

gonal (at least three steep/flat

sectors can be recognized, at

least 2 diopters of power differ-

ence between steep and flat

hemimeridians); 7, irregular

After PKP we recommend documenting the

∑ Astigmatism with “all-sutures-out”

keratometric refractive power separately in the

and vector-corrected astigmatism

steep and in the flat meridian with individual

axis notation and assessment of the degree of

“keratometric irregularity” (Fig. 10.1). Instead

10.2.2

of “42.0+4.5/0°,” we suggest writing “42.0/0°

Reasons for Astigmatism After Keratoplasty

(irreg. 1); 46.5/70° (irreg. 2)” [62].

(Table 10.2)

Besides keratometry, topography analysis is

indispensable for mapping the corneal power

Each of the multiple steps from donor selection,

over the entire graft. Refractive powers and in-

intraoperative trephination and suturing tech-

dividual axes of the four hemimeridians are

nique to type and quality of postoperative care

complemented by system specific indices, e.g.,

can determine not only the clarity of the graft

SRI (surface regularity index) and SAI (surface

but also its final refractive result.

asymmetry index) of the TMS-1 topography

Besides intrinsic factors of donor and recipi-

system. In addition, we suggest a semiquantita-

ent, the short-term astigmatism with sutures in

tive classification of post-keratoplasty topogra-

place seems to depend more on the symmetry of

phy in seven groups (Fig. 10.2).

the sutures including methods of intra- and

postoperative suture adjustments. After suture

Summary for the Clinician

removal corneal curvature typically becomes

Studies intending to compare the corneal

more regular [35, 62], but the amount of net

curvatures after different trephination or

astigmatism may increase considerably [36, 38].

suturing techniques for PKP should include

Thus, it has been concluded that factors di-

the following:

rectly or indirectly related to the quality of the

∑ Subjective cylinder and keratometric/

wound geometry have a predominant influence

topographic astigmatism

on the long-term residual astigmatism after

∑ Portion of irregular/not measurable

suture removal [59].

astigmatism

126

Chapter 10

Trephination in Penetrating Keratoplasty

Table 10.2. Potential causative factors of high and/or irregular astigmatism after keratoplasty [59]

Preoperative factors

a) Age of donor (infant!)

b) Size of recipient cornea

i) Keratoconus >Fuchs’ dystrophy [60]

ii) Microcornea

c) Topography of donor

d) Topography of recipient

e) Disharmony between donor and recipient topography

f) Pathologic properties of recipient

i) Peripheral thinning or ectasia

ii) Focal edema/focal scar

iii) Defects in Bowman’s layer

iv) Vascularization

v) Preceding keratoplasty (especially decentered)

g) Aphakia

Intraoperative factors

a) Decentration of donor excision and/or recipient bed

“Vertical tilt” due to discrepancies of wound configuration [42]

i) Application of different trephine systems for donor and recipient

ii) Trephine tilt (i.e., not parallel to optical axis)

iii) Limbal plane not horizontal

iv)

“Shifting” of trephine during cutting

v) Too high/low intraocular pressure

“Horizontal torsion” [42]

i) Asymmetric placement of second cardinal suture (Dπ180°)

ii) Mismatch of donor and recipient due to form incongruence

iii) Focal overlap or dehiscence of donor button in recipient bed

d) Excessive over-/undersize of donor

e) Distortion and squeezing of cornea (e.g., due to dull trephine)

f) Traumatizing the cornea with instruments

g) Suture-related factors

i) Suture material

ii) Suture technique (interrupted, single running, double running, combinations)

iii) Length of stitch

iv) Depth of stitch

v) Angle of stitch towards graft-host apposition

vi) Suture tension

vii) “Depth disparity”

h) Simultaneous intraocular surgery (e.g., triple procedure, IOL exchange)

i) Fixation rings and lid specula

j) Surgeon’s experience

3. Postoperative factors

a) Suture-related factors

“Cheese wiring” of sutures

ii) Suture loosening

iii) Suture adjustment/selective suture removal

iv) Time point of suture removal

b) Wound healing processes

i) Wound dehiscence

ii) Retrocorneal membrane

iii) Incarceration of overlapping tissue

iv) Focal vascularization

c) Medication (e.g., corticosteroids)

d) Postoperative trauma

10.2

Astigmatism and Keratoplasty

127

10.2.2.1

Preoperative Determinants

Infant corneas have high refractive power

(>50 diopters) and tend to steepen further after

transplantation due to the biomechanical insta-

bility of the tissue. Thus, Pfister and Breaud sug-

gested using infant corneas to compensate for

aphakia. However, the refractive outcome var-

ied considerably and was not predictable [49].

Thus, we do not recommend the use of infant

donor corneas for grafting.

Today, donor topography is still rarely per-

formed. The higher the immanent preoperative

astigmatism of donor and recipient, the more

Fig. 10.3. Main reasons for high post-keratoplasty

probable it is that dysharmony between donor

astigmatism: top decentration of donor and/or recip-

and recipient topography results in high astig-

ient trephination; middle “vertical tilt” due to incon-

matism after suture removal [10, 15, 56]. Espe-

gruent cut angles; bottom “horizontal torsion”due to

cially high congenital astigmatism, keratoconus

asymmetric suturing (modified from [42])

and previous corneal refractive surgery must be

ruled out in potential donors.

10.2.2.2

Intraoperative Determinants (Fig. 10.3)

astigmatism with an 8-mm-diameter graft [22].

Especially tilted hand-held trephines and ne-

Asymmetrically placed fixation rings

(e.g.,

glecting the horizontal position of the limbal

Flieringa or McNeill-Goldmann) may induce an

plane are reasons for the “vertical tilt” phenom-

astigmatism of up to 10 diopters [45]. Thus,

enon. In addition, application of different

post-PKP astigmatism is typically higher in

trephine systems and different trephination di-

aphakic than in phakic or pseudophakic PKP

rections (e.g., punching the donor from the

[48]. Even simple lid specula may be responsible

endothelial side) in donor and host are crucial

for 3 diopters of with-the-rule astigmatism [45].

factors.

Decentration. Besides a higher incidence of

“Horizontal Torsion.” One of the major predis-

immunologic graft reactions due to proximity

positions for regular all-suture-out curvature

to the limbal vessels, decentration of host

after PKP is the 360° symmetric apposition of

trephination

(>1 mm) may result in higher

the donor button in the recipient bed. Especial-

astigmatism. The flat axis of astigmatism points

ly the correct positioning of the second cardinal

towards the direction of decentration [30, 75].

suture opposite to the first one is crucial. Asym-

Due to the thickness gradient from the center to

metric placement of the second cardinal suture

the periphery, donor decentration may also

results in a tissue deficit on one side which

have a minor impact on post-PKP astigmatism

needs to be compensated by forced suture adap-

[61].

tation. In the case of long shallow suture bites, a

regional flattening may result. In the case of

“Vertical Tilt.” The amount of persisting post-

short and deep suture bites, a central steepening

PKP astigmatism after suture removal depends

may result, in analogy to sutured wedge resec-

significantly on the incongruences (“mismatch-

tions. On the other side a tissue surplus may re-

es”) of shape and cut angles of donor and recip-

sult in peripheral donor tissue compression

ient wounds [50, 74, 75]. Theoretically, a trephine

with peripheral steepening and consecutive

tilt of

5° (10°) can induce 1.6 (5.9) diopters of

central flattening [74].

128

Chapter 10

Trephination in Penetrating Keratoplasty

An analogous situation arises when the re-

10.2.2.3

cipient bed is cut asymmetrically elliptical

Postoperative Determinants

[34, 46, 78]. This may result from asymmetric

bulging of the unstable cornea into the trephine

Postoperative suture adjustment or selective

opening or even by using an obturator in the

removal of single sutures may have a favorable

case of keratoconus [21]. Mechanical trephines,

impact on the early post-PKP astigmatism.

such as hand-held or motor trephines, may re-

However, changes of corneal curvature are un-

sult in oval-shaped host beds even if a circular

predictable after suture removal [36, 38]. At this

round excision was intended [9].

time there is still no reliable indicator available

Likewise, in donor trephination a trephine

to the microsurgeon instructing him about the

tilt of

20° may induce a difference of about

amount and direction of impending astigma-

0.5 mm between the maximal and minimal di-

tism changes of the graft after suture removal.

ameter, resulting in an elliptical donor button

There is some evidence that a high coincidence

[45]. Suturing of such an elliptical donor button

of the axes of refractive, keratometric and topo-

in a round bed will result in a peripheral steep-

graphic astigmatism with the suture in place

ening in the major axis due to tissue compres-

speaks in favor of decreasing astigmatism to be

sion and - consequently - a central flattening in

expected after suture removal [54]. Thus, in the

this (hemi-)meridian [8]. A wound disparity of

case of intact sutures, lack of vascularization, a

0.1 mm is supposed to create an astigmatism of

low amount of astigmatism, and high topo-

about 1 diopter [45, 74].

graphic regularity resulting in good spectacle-

Undoubtedly, the technique for adequate

corrected visual acuity, microsurgeons will tend

graft-host adaptation by means of four to eight

to leave the suture in place for a longer period of

cardinal sutures is determined - at least in part

time under regular controls and adequate coun-

- by the experience of the microsurgeon. The

seling of a compliant patient. However, it must

same holds true for the correct performance, in-

be considered an illusion that keeping the su-

terpretation and consequences of intraopera-

tures in place for a longer time would help to

tive keratoscopy. However, even if adequate

preserve a favorable topography after final su-

suture distribution and tension as well as intra-/

ture removal [11, 14, 36, 38, 70]. Especially step

postoperative suture adjustments compensate

formations after suture removal - often after

for the fundamental intraoperative determi-

inadequate trauma

- will result in a flat

nants of post-PKP astigmatism in the early

hemimeridian and irregular high astigmatism.

stage, suture removal - even after years - may

For this reason, such steps at the graft-host

result in major changes of topography and a

junction need immediate surgical repair to pre-

dramatic increase in astigmatism [36, 38].

serve a good long-term refractive result even if

the anterior chamber is not opened [18].

Summary for the Clinician

Major intraoperative determinants for high/

irregular astigmatism after suture removal

The pathomechanism of astigmatism increase

include [42]:

after suture removal may be as follows:

∑ Decentration (donor and/or recipient

∑ A low quality of trephination wound

trephination)

and geometric incongruences (horizontal

∑

“Vertical tilt” (incongruent cut angles

and vertical) require a higher suture

between donor and host)

tension to guarantee:

∑

“Horizontal torsion” (horizontal discrepan-

- Watertight wound closure

cy of donor and host shape or asymmetric

- A pseudo-optimal topography early

suturing - second cardinal suture!)

postoperatively

∑ Asymmetric regional forces between donor

and host may cause inhomogeneous wound

healing

10.2

Astigmatism and Keratoplasty

129

∑ Removal of sutures liberates forces due to:

An analogous approach was followed by in-

(1) geometric incongruences and (2) inho-

troducing an inverse mushroom-shaped trephi-

mogeneous wound healing

nation with the larger diameter of the graft at

∑ Thus: horizontal, vertical and topographic

the level of Descemet’s membrane [7, 67].

discrepancies between donor and host

In order to leave the architecture of the cen-

intraoperatively are responsible for an

tral cornea untouched, endothelial cell trans-

increase in astigmatism after suture

plantation has been investigated and posterior

removal

lamellar keratoplasty (PLKP) has been intro-

duced into clinical routine by Melles [37] in Eu-

rope in 1998 and later modified by Terry in the

10.2.3

United States [71] in cases of sole endothelial

Prevention/Prophylaxis

failure.

of Astigmatism After Keratoplasty

10.2.3.2

The large number of treatment options for

Ten Precautions During Surgery

astigmatism after PKP leads to the conclusion

that none of the methods is really convincing.

Donor topography should be attempted for

Therefore, prophylaxis of high and/or irregular

exclusion of previous refractive surgery,

astigmatism is preferred over treatment [59].

keratoconus/high astigmatism, and “harmo-

nization” of donor and recipient topography

10.2.3.1

[16, 56, 59].

Alternatives “Without Sutures”

Donor and recipient trephination should be

performed from the epithelial side with the

Alternatives

“without sutures” include pho-

same system, which - from our point of view

totherapeutic keratectomy (PTK) in the case of

- predisposes to congruent cut surfaces and

superficial corneal diseases. PTK yields good

angles in donor and recipient. For this pur-

results especially with recurrences of corneal

pose an artificial anterior chamber is used

dystrophies after PKP. In order to avoid sutures

for donor trephination although the whole

involving Bowman’s layer, potentially self-seal-

globe would yield even better results [27].

ing nut-bolt variants of donor-recipient apposi-

Orientation structures in donor and host fa-

tion have been investigated. One approach is di-

cilitate the correct placement of the first four

vergent cut angles that may be created using

cardinal sutures to avoid horizontal torsion

lasers [57]. The increased contact area reduces

[2].

the probability of wound dehiscence, the small-

A measurable improvement seems possible

er diameter at the level of Bowman’s layer in-

using the Krumeich guided trephine system

creases the distance from the limbal vessels with

(GTS) [4], the second generation Hanna

favorable effects concerning immunologic graft

trephine [81] and our technique of nonme-

reactions, and the larger diameter at the level of

chanical trephination with the excimer laser

Descemet’s membrane increases the amount of

[58, 66].

transplanted endothelial cells with favorable

Horizontal positioning of head and limbal

effects in Fuchs’ dystrophy and aphakic/

plane is indispensable for state-of-the-art

pseudophakic bullous keratopathy. It has been

PKP surgery in order to avoid decentration,

shown that the stability of the graft in the recip-

vertical tilt and horizontal torsion [59].

ient bed increases with increasing divergence of

Graft size should be adjusted individually

the cut angles [57]. Additional application of tis-

(“as large as possible, as small as necessary”)

sue glue, a temporary therapeutic contact lens

[60, 62].

or an intrastromal suture may further increase

the stability of the graft-host junction.

130

Chapter 10

Trephination in Penetrating Keratoplasty

7. Limbal centration should be preferred over

Table 10.3. Principal indications for keratoplasty

pupil centration (especially in keratoconus -

(modified from [40])

“optical displacement of pupil”) [31].

8. Excessive graft over- or undersize should be

1. Optical

a) Opacities

avoided to prevent stretching or compres-

b) Pathologic curvature

sion of peripheral donor tissue [19, 47, 82].

2. Curative

9. As long as Bowman’s layer is intact, a double

a) Deep keratitis (e.g., herpetic keratitis with

running cross-stitch suture

(according to

granulomatous reaction to Descemet’s

Hoffmann [17]) is preferred since it results in

membrane or Acanthamoeba keratitis)

greater topographic regularity, earlier visual

b) Endothelial diseases

rehabilitation and less loosening of sutures,

(primary or secondary)

with suture replacement only rarely required.

c) Perforated corneal ulcer

10.Intraoperative keratoscopy should be ap-

3. Tectonic

plied after removal of lid specula and fixa-

a) Traumatic corneal defects

tion sutures. Unstable donor epithelium

b) Infectious corneal defects

c) Postoperative fistula after cataract

would be better removed to allow for repro-

extraction or antiglaucomatous surgery

ducible results. Adjustment of double run-

d) After “block excision” [44]

ning sutures or replacement of single sutures

i) Uveal tumors

may be indicated [3].

ii) Localized epithelial downgrowth

(cysts)

Summary for the Clinician

e) Reconstruction of the anterior segment

Requirements for “the optimal trephination”

include:

∑ Full visual control

∑ No contact

material, the vertical shape of the graft, the hor-

∑ Optimal donor and host centration

izontal shape of the graft and the location of the

∑ Identical shape of donor and host

graft within the host (Table 10.4) [40].

(typically circular)

A few general technical details concerning

∑ Congruent cut angles

PKP need to be mentioned [40, 42]:

∑

360° symmetric donor host alignment

1. General anesthesia has advantages over local

∑ No necessity to complete trephination

anesthesia. The arterial blood pressure

by scissors

should be kept low as the eye is opened

∑ No damage to intraocular tissues

(“controlled arterial hypotension”).

∑ Future: self-sealing donor/host apposition

2. To protect the crystalline lens in phakic ker-

atoplasty, usually the pupil is constricted.

3. Before recipient trephination, a stab-like

paracentesis at the limbus is performed.

10.3

4. The limbal plane must be horizontal during

Trephination Techniques

trephination.

5. An iridotomy prevents pupillary block and

The principal indications for keratoplasty in-

acute angle closure glaucoma (so-called Ur-

clude optical, curative and tectonic factors

rets-Zavalia syndrome in the case of dilated

(Table 10.3). Overlaps between the different cat-

pupil with iris sphincter necrosis [43]).

egories may occur. But corneal transplants may

6. The second cardinal suture is crucial for graft

also be classified according to the type of donor

alignment.

10.3

Trephination Techniques

131

Table 10.4. Terminology of various types of keratoplasty (modified from [40])

Donor cornea

Vertical shape

Horizontal

Location

of graft

shape of graft

within the host

Autologous (autograft)

Lamellar

Circular

Central

(anterior vs. posterior)

Homologous (allograft)

Elliptical

Eccentric

Penetrating

Heterologous (xenograft)

Semilunar

Marginal

Mushroom

Alloplastic (keratoprosthesis)

Rectangular

Inverse mushroom [67]

Triangular

Ring-shaped

Since the development of “artificial anterior

10.3.1

chambers” [23], microsurgeons have had the

Principal Considerations

opportunity to perform donor trephination

from the epithelial side, which is the same di-

10.3.1.1

rection as in the host. If pressure in the arti-

Donor Trephination

ficial anterior chamber is kept normal (e.g.,

22 mmHg), the advantages with respect to

From a 16-mm corneoscleral button as provided

cut angles are obvious [55]. However, fixing

by the Eye Bank, the transplant can be created in

the corneoscleral button in an artificial ante-

two principal ways:

rior chamber may induce a considerable

1. The original method used is for the donor

amount of astigmatism. This problem can be

button to be punched from the endothelial

overcome by using an artificial anterior

side against a firm surface (such as a paraffin

chamber with a larger central opening, leav-

or Teflon block) using special trephines

ing the limbus untouched during fixation

(Lochpfeifentrepan) [6, 80]. Care must be

for trephination from the epithelial side. In

taken to ensure a proper alignment when

this setting the corneoscleral limbus seems

cutting since a beveled cut will result if the

to have a protective effect concerning the

blade is not perpendicular to the cutting

central corneal topography of the fixated

block. This risk may be decreased by the use

cornea [27].

of “guided donor trephine” systems (e.g.,

Summary for the Clinician

“guillotines”) (Fig. 10.4).

On histological evaluation, the cut surfaces

∑

Trephination of the donor button should

without consideration of the cut angles seem

preferably be performed from the epithelial

to be almost “perfect.” However, deviation of

side using an artificial anterior chamber

the cut direction outwards results in conver-

with a large central opening

gent cut angles due to a smaller diameter at

∑

Punching the donor from the endothelial

the level of Descemet’s membrane and a larg-

side results in an undercut at the level of

er diameter at the level of Bowman’s layer

Descemet’s membrane with convergent cut

(“undercut”) (Fig. 10.4 D) [76].

angles

10.3

Trephination Techniques

133

∑ The higher the intraocular pressure

(iatrogenic!) the more divergent are the

cut angles to be expected [55]

10.3.1.3

Graft Size and “Oversize”

Graft Size. In a quantitative study we found

that the corneal diameter of keratoconus pa-

tients was larger than that of Fuchs’ patients

(mean horizontal diameter of 11.8 mm in kera-

Fig. 10.5. Combination of donor trephined from the

toconus patients and 11.3 mm in Fuchs’ patients)

endothelial side (convergent cut angle) and mechani-

[60]. In general, a good optical performance

cally trephined recipient (divergent cut angle) results

requires a larger graft, whereas a low rate of

in a triangular-shaped tissue deficit at the level of

immunologic graft reactions tends to be seen

Descemet’s membrane which has to be compensated

with smaller grafts. Therefore, the graft should

by suture tension resulting in central flattening and

vertical tilt

be “as large as possible, but as small as neces-

sary.” For many eyes with keratoconus an

8.0-mm diameter and in many eyes with Fuchs’

Investigations by Van Rij and Waring

dystrophy a 7.5-mm diameter prove to be good

demonstrated that in recipient trephination all

options as a prerequisite for obtaining tissue

trephine systems result in an opening larger

from the Eye Bank. Today, graft diameters of

than the trephine size. In addition, the diameter

5.5-7.0 mm are only rarely required and justi-

is larger at the level of Descemet’s membrane,

fied.

resulting in divergent cut angles [76]. This can

It has been supposed that smaller grafts

be explained by the “ballooning” of the cornea

might be associated with a higher post-kerato-

to be excised into the trephine opening due to

plasty astigmatism. In a recent study we found

the pressure executed. The higher the intraocu-

[62]:

lar pressure, the more divergent the angles to be

1. A flatter curvature with smaller grafts

expected [55]. This phenomenon of “balloon-

2. A higher topographic irregularity with

ing” is one of the major drawbacks of a mechan-

smaller grafts

ical trephine and can be prohibited - at least in

3. A higher proportion of unmeasurable ker-

part - by the use of an “obturator.” However,

atometry mires with smaller grafts

Kaufman stresses that the use of an obturator in

4. A tendency towards regularization of topo-

keratoconus may result in other than round

graphy after suture removal

host openings such as pear-shaped holes [21].

5. No difference concerning the amount of net

The combination of a donor punched from

astigmatism between different graft sizes

the endothelial side with convergent cut angles

either with or without sutures

and a host opening with divergent cut angles

will result in a triangular-shaped tissue defect at

The major reason for the flatter and more irreg-

the level of Descemet’s membrane that has to be

ular graft with smaller diameters seems to be

compensated for with increased suture tension

the closer position of the proximal suture ends

and - consequently - vertical tilt (Fig. 10.5).

in relation to the optical center of the graft. This

will be pronounced in particular with wider su-

Summary for the Clinician

ture bites. After suture removal the potentially

∑ Horizontal positioning of limbal plane is

topography disturbing circular scar at the graft-

indispensable

host junction is located closer to the line of sight

∑ Flieringa ring is only necessary in aphakic

with smaller grafts. This may explain that over-

eyes

all the regularity of graft topography increases

134

Chapter 10

Trephination in Penetrating Keratoplasty

with suture removal but that major differences

side) may result in significantly increased

between various graft sizes do persist.

corneal astigmatism [47]. In keratoconus, same

Larger sizes may be considered for eccentric

size donors were found to reduce resulting

tectonic corneoscleral grafts

(e.g., after the

myopia. We do not recommend undersizing of a

block excision of tumors of the anterior uvea or

graft!

cystic epithelial downgrowth [44]) and in buph-

In contrast, with guided trephines and laser

thalmos [73]. But we do not recommend graft

trephination (donor from the epithelial side),

sizes over 8.5 mm in buphthalmos for immuno-

attempted diameters are indeed achieved with

logic reasons [52].

congruent cut angles. Thus, donor oversize is

Recent studies indicate that the rate of

not necessary.

chronic endothelial cell loss after PKP depends

Summary for the Clinician

on the initial diagnosis [32, 53]. Endothelial mi-

gration from donor to recipient in pseudopha-

∑

Typically, keratoconus corneas are larger

kic bullous keratopathy along a density gradient

than Fuchs’ dystrophy corneas

is thought to be the reason for this phenome-

∑

Graft size has to be judged by the micro-

non. Therefore, eyes with bullous keratopathy

surgeon individually in every single case

may require a larger graft not just to improve

before recipient trephination to achieve the

the optical performance but rather to transplant

best compromise between immunologic

as many endothelial cells as possible. Neverthe-

purposes and optical quality

less, graft size has to be judged by the surgeon in-

∑

Donor trephination from the endothelial

dividually in every single case before recipient

side results in a smaller donor button than

trephination to achieve the best compromise

trephine size and convergent cut angles

between immunologic purposes and optical

(“undercut”)

quality [59, 60].A slit lamp with a measuring de-

∑

Recipient trephination results in larger

vice

(scale), e.g., a Haag-Streit slit lamp, or

openings than trephine size and divergent

calipers for intraoperative application may be

cut angles

helpful. Prior removal of vascularized pannus

∑

This discrepancy makes a donor “oversize”

(in contrast to vascularized stromal scars) may

of ≥0.25 mm necessary

render a larger “individual optimal graft size”

∑

Same size grafts are feasible if the donor is

possible for transplantation of more endothelial

created by means of an artificial anterior

cells and better graft topography.

chamber from the epithelial side

∑

Undersizing the graft for simultaneous

Graft “Oversize.” In mechanical trephination,

correction of myopia in keratoconus is

the diameter of the recipient bed tends to be

not recommended (watertight wound!

larger and the diameter of the donor button,

irregular astigmatism!)

punched from the endothelial side, tends to be

smaller than the trephine diameter, which may

10.3.1.4

affect the resulting spherical equivalent [76].

Pupil Versus Limbal Centration

Thus, “oversizing” the donor button by 0.25-

0.50 mm is commonly done to compensate for

Centration is crucial with respect to immuno-

refractive effects and to reduce crowding of the

logic graft reaction and post-PKP astigmatism.

chamber angle and therefore postoperative

Typically a compromise between limbal and

“glaucoma” [47]. An oversize of 0.25 mm com-

pupil centration is attempted in the case of non-

pared to one of 0 mm or 0.5 mm may account

traumatized pupils. However, limbal centration

for a difference in keratometric readings of

is preferred especially in keratoconus, scars

1.5 diopters after suture removal. Javadi et al.

after trauma or irregular astigmatism of other

found no difference in astigmatism in compar-

origins. In such eyes the center of the visible

ing 0.25 mm and 0.50 mm graft oversize [19].

(“entrance”) pupil may be dislocated from that

However, Perl et al. stressed that oversizing the

of the real anatomic pupil [31].

graft by 0.5 mm (punched from the endothelial

10.3

Trephination Techniques

135

for the amount of relative change in curvature

after suture removal. Therefore, “harmoniza-

tion” of donor and recipient topography should

allow for minimization of the residual astigma-

tism for a given pair of donor and recipient [56].

The use of an artificial anterior chamber en-

ables donor topography analysis and allows the

“contour line” of the trephination edges in both

donor and recipient to be calculated. A comput-

erized simulation of graft rotation in the recipi-

ent bed may help to find an angle of graft rota-

tion at which topographical misalignment is

minimal.

Grütters et al. have proposed “astigmatism-

Fig. 10.6. An eight-line radial keratotomy marker

oriented perforating keratoplasty”, i.e., match-

(colored with methylene blue) may be used to facili-

ing the flat axis of the donor with the steep axis

tate limbal centration

of the host cornea [16].

Summary for the Clinician

An eight-line radial keratotomy marker may

Consideration of donor topography may:

be used to ensure centration (Fig. 10.6). An

∑ Eliminate the use of donors with abnormal

additional central dot-like mark may be helpful

or unusual curvatures (such as high astig-

for certain trephine systems (e.g., Hessburg-

matism, keratoconus, previous refractive

Baron).

surgery)

If the broadening of the superior limbus due

∑ Allow for “harmonization” of donor and

to a vascularized pannus is neglected intraoper-

recipient topography

atively, an inferior decentration may be recog-

nized on the next day at the slit-lamp.

10.3.1.6

The Vascularized Cornea

Summary for the Clinician

∑ In doubt, limbal centration is preferred over

Excessive bleeding after trephination of vascu-

pupil centration

larized corneas with blood clots left in the ante-

rior chamber may result in increased risk of

10.3.1.5

immunologic graft reaction and peripheral

“Harmonization” of Donor

anterior synechiae due to contraction. Thus, the

and Patient Corneal Topography

following precautions should be taken:

Before trephination the microsurgeon

Keratometric readings of the donor cornea are

should differentiate between vascularized pan-

still usually neglected. However, it might be bet-

nus tissue (“plus”) and vascularized scars (“mi-

ter to consider them to improve predictability of

nus”). Vascularized fibrous tissue between the

the final refractive outcome after PKP [10, 16, 56].

epithelium and Bowman’s layer or the superfi-

This may help to avoid transplantation of corneas

cial stroma in the case of defective Bowman’s

with unusual or abnormal curvatures. In addi-

layer can be removed easily with a hockey knife.

tion, it may allow a more accurate selection of

Typically, bleeding stops after a few minutes

intraocular lens power in triple procedures.

without additional measures. In contrast, dis-

The vertical difference at the graft-host junc-

tinct “feeder vessels” of vascularized scars may

tion due to the different curvatures of donor

be incised with a pointed scalpel at the limbus.

and recipient must be compensated intraopera-

Pillai et al. have proposed sophisticated kauteri-

tively by suture tension to avoid a step forma-

zation techniques for coagulation of afferent

tion. The resulting forces may be co-responsible

and efferent vessels [51]. In the case of diffusely

136

Chapter 10

Trephination in Penetrating Keratoplasty

capillarized scars, ice-cold balanced salt solu-

Due to inhomogeneous corneal thickness, an

tion (BSS) or topical alpha-mimetic vasocon-

early perforation at the site of the thinned

stringent drops (such as naphazoline nitrate)

cornea is to be expected. This has to be taken

may help to reduce bleeding during trephination.

into account with conventional trephines to

avoid inadvertent injury of the iris or even the

Summary for the Clinician

lens.

∑ Removal of vascularized pannus tissue may

Peripheral thinning of the host cornea, e.g.,

help to increase the “individually optimal

with keratotorus (= pellucid marginal degener-

graft size”

ation) or Fuchs-Terrien marginal degeneration,

∑ Incision or kauterization of distinct “feeder

is very rare but difficult to treat. Treatment op-

vessels” of scars at the limbus may reduce

tions include an eccentric semilunar lamellar/

bleeding during trephination

penetrating graft or an overdimensioned

preferably elliptical eccentric through-and-

10.3.1.7

through graft.

Keratoconus and Disabling High

Astigmatism of a Graft

Disabling High Astigmatism of a Graft. Eyes

with high disabling astigmatism after PKP are

Keratoconus. In keratoconus, a central round

often - but not always - associated with small

PKP is indicated as soon as hard contact lenses

and/or decentered grafts. The re-graft should be

are no longer tolerated. Excessively steep

well centered and large enough to cut out the

corneas before surgery do not have less favor-

previous graft entirely. However, in some cases

able outcome than less deformed corneas after

the previous graft-host junction cannot be

PKP using the excimer laser for nonmechanical

excised in toto (cf. Sect. 10.3.1.3,“Graft Size” and

trephination [83].

“Oversize”), leaving a “wedge” of the first donor

Keratoconus eyes have larger corneas than

tissue in situ.

normal eyes and other dystrophies allowing for

After second suture removal, astigmatism

larger graft diameters (typically 8.0 mm) [60].

may increase again and may no longer be signif-

A larger graft diameter in keratoconus patients

icantly different in comparison to the preopera-

may help to preserve a sufficiently thick cornea

tive values [70].

at the trephination margin in the patient since

Our own results suggest a potentially impor-

the “cone” can be excised almost completely.

tant role of the remaining second running su-

Kauterization of the cone has been suggested to

ture in keeping corneal astigmatism values low

avoid divergent cut angles, but its effect may not

and topographic regularity high after repeat

be reproducible. Thus, we do not advocate kau-

PKP in patients with high and/or irregular post-

terization of the cone. Kaufman has suggested

keratoplasty astigmatism. After removal of the

not using obturators in the case of keratoconus

last suture, the curvature may change in an un-

to prevent unintended creation of elliptical or

predictable and often unfavorable manner. The

pear-shaped openings [21].

presumed original instability of the host rim,

We do not advocate centering the trephina-

which on final suture removal may be trans-

tion on the cone, thereby typically decentering

ferred to the center of the graft (“memory ef-

the trephination with respect to the limbus. In

fect”), is probably responsible for the increase in

addition, pupil centration may be misleading

astigmatism and the increase in irregularity of

due to “optical displacement” of the visible pupil

the corneal surface. In addition, the host rim in-

because of irregular refraction of incoming rays

stability may be exacerbated by incomplete ex-

of light by the irregularly curved corneal sur-

cision of the previous graft-host junction in se-

face in keratoconus [31]. We do not advocate un-

verely decentered first grafts. However, the exact

dersizing of the donor to reduce myopia, since

role of any such residual tissue has yet to be

irregular astigmatism is to be expected.

clarified.

10.3

Trephination Techniques

137

The long-term value of so-called “intra-

corneal rings” inside the graft-host junction

with respect to stabilization of the topography

in such eyes has yet to be determined [13, 24].

Summary for the Clinician

∑ With keratoconus a large excision should be

centered at the limbus (not the “cone”) and

non-contact laser trephination is preferred

to prevent “other-than-round” recipient

openings

∑ Where repeat PKP is performed in eyes

with high and/or irregular astigmatism in

clear grafts, visual rehabilitation may be

limited by an increase in astigmatism and

topographic surface irregularity after re-

moval of the last running suture

∑ In such eyes it may be advantageous to

postpone final suture removal for as long

as possible

10.3.1.8

Fig. 10.7.

A Descemetocele after ipsilateral autolo-

The Unstable Cornea

gous keratoplasty for localized central herpetic scar;

B eccentric elliptical triple procedure à chaud

Unstable corneas include:

(7.0¥8.0 mm/7.1/8.1 mm, excimer laser trephination)

1. Corneal perforations or descemtoceles typi-

cally arising from ulcerative necrotizing

stromal keratitis of herpetic or bacterial ori-

gin

sertion of viscoelastic agent via paracentesis

2. Eyes after unfavorable keratorefractive sur-

(“valve”). A larger than usual graft oversize

gery such as after radial keratotomy and ia-

(e.g., 0.5 mm) is recommended to avoid periph-

trogenic keratectasia after laser in-situ ker-

eral synechiae in eccentric or even peripheral

atomileusis (LASIK)

grafts.

In the case of excisions involving the limbus,

In the “open eye” situation mechanical tre-

the scleral spur has to be preserved during

phines may lead to compression and distortion

(partly lamellar) trephination. In the case of pe-

of the cornea although a high-viscosity vis-

ripheral small perforations, an eccentric mini-

coelastic agent is used to stabilize the anterior

keratoplasty may have immunologic advan-

chamber. Especially with large perforations the

tages. Wide limbus-parallel perforations

trephine can only be used to mark the excision,

typical of rheumatoid origin - may best be

the keratotomy has to be deepened with a dia-

treated with a crescent graft. For this partly

mond knife and the excision is completed with

“freehand” procedure, an outer segmental

scissors. Nonmechanical laser trephination has

trephination with a smaller diameter

(e.g.,

been advocated since it may allow non-contact

10 mm) is combined with an inner segmental

round and elliptical trephinations

(Fig. 10.7)

trephination with a larger diameter

(e.g.,

[26]. One suggestion has been to insert a

16 mm). Adequate preparation of the slightly

trimmed part of a soft contact lens via large

oversized graft is best achieved from an intact

paracentesis, unrolling it inside the anterior

donor globe but is quite difficult using a cor-

chamber and thus achieving a stable eye for

neoscleral button from the Eye Bank (protec-

trephination after pressurizing the globe by in-

tion of endothelium!).

138

Chapter 10

Trephination in Penetrating Keratoplasty

After excessive radial keratotomies resulting

in irregular astigmatism and glare/halos due to

scars in the optical field, deep epithelial plugs

are typically present inside the original radial

cuts for years. Instability leads to opening of

these plugs during mechanical trephination.

Certain types of circular sutures have been pro-

posed before trephination. However, non-con-

tact laser trephination seems to be the method

of choice for such eyes. In analogy, iatrogenic

keratectasia after LASIK is prone to opening of

the lamellar interface between the stromal bed

and flap during conventional contact trephina-

tion. This may result in oval host wounds and

different sizes of the excised button at the flap

Fig. 10.8. Well centered (1) trephination, (2) capsu-

and bed levels [64]. Again, non-contact laser

lorhexis, and (3) posterior chamber lens inside the

trephination seems to be the method of choice

capsular bag after triple procedure in Fuchs’ dystro-

for such eyes, the incidence of which is sup-

phy (7.5/7.6 mm, excimer laser trephination with eight

posed to increase over the next few decades.

“orientation teeth/notches”)

Summary for the Clinician

∑ In the “open eye” situation conventional

should be centered along the optical axis

trephines typically only mark the host exci-

(Fig. 10.8). If possible, performing the capsu-

sion which has to be completed freehand

lorhexis under controlled intraocular pressure

with diamond knife and scissors

conditions prior to trephination may help to

∑ With unstable corneas non-contact non-

minimize the risk of capsular ruptures. In the

mechanical laser trephination has major

case of excessive corneal clouding, a capsu-

advantages over conventional mechanical

lorhexis forceps is used via the “open sky” ap-

trephination

proach. Delivery of the nucleus is achieved via

the “open sky” approach by means of manual

10.3.1.9

irrigation, and removal of the lens cortex by

The Triple Procedure

automated irrigation-aspiration.

The major advantage of the triple procedure

Since the introduction of the triple procedure

is the faster visual rehabilitation achieved and

[= simultaneous penetrating keratoplasty

less effort required for the mostly elderly pa-

(PKP), extracapsular cataract extraction and

tients. In contrast, sequential cataract surgery

implantation of a posterior chamber intraocu-

has the potential for a simultaneous reduction

lar lens (PCIOL)] in the mid-1970s, there has

of corneal astigmatism (appropriate location of

been an ongoing discussion among corneal mi-

the incision, simultaneous refractive kerato-

crosurgeons concerning the best approach (si-

tomies or implantation of a toric PCIOL). Dis-

multaneous or sequential) for combined corneal

advantages may include the loss of graft en-

disease and cataract [65]. For the refractive re-

dothelial cells and the theoretically increased

sults after the triple procedure, some intraoper-

risk of immunologic allograft reactions. After

ative details are crucial: trephination of recipi-

the triple procedure, major deviations from tar-

ent and donor from the epithelial side without

get refraction have been reported. However, in-

major oversize (guided trephine system or non-

dividual multiple regression analysis may help

mechanical excimer laser trephination) should

to minimize this problem with appropriate

preserve the preoperative corneal curvature.

methods of trephination [77]. Since suture re-

Graft and the PCIOL placed in the bag after

moval after PKP may result in major individual

large continuous curvilinear capsulorhexis

changes of the corneal curvature, IOL power

10.3

Trephination Techniques

139

calculation for the sequential approach requires

all sutures to be removed at the time of cataract

surgery. However, even after complete suture re-

moval the abnormal proportions between ante-

rior and posterior curvatures and/or the irregu-

lar topographies after PKP may be responsible

for marked IOL power miscalculations in the in-

dividual eye [65].

Summary for the Clinician

∑ The postulated better prediction of refrac-

tion after sequential keratoplasty and

cataract surgery is opposed by a markedly

Fig. 10.9. Typical double running 10-0 nylon cross-

delayed visual rehabilitation

stitch suture with 8 bites each (according to Hoff-

∑ We consider the triple procedure including

mann [17]) in keratoconus (8.0/8.1 mm, excimer laser

cataract extraction via “open sky” in gener-

trephination with eight “orientation teeth/notches”)

al anesthesia as the method of choice for

combined corneal and lens opacities

visual rehabilitation with these sutures in place

10.3.1.10

in contrast to single sutures is due to a more reg-

Impact of Trephination on Suturing

ular corneal topography avoiding cornea plana.

Summary for the Clinician

The trephination modality may have a major

impact on the correct placement of the first four

∑ The better the trephination the easier

or eight cardinal sutures. The predominant pur-

watertight wound closure is achieved

pose of the cardinal sutures is: (1) symmetric

∑ Inadequately high suture tension to achieve

horizontal distribution of donor tissue in the re-

watertight wound closure may deteriorate

cipient bed, (2) good adaptation of graft and

the regularity of the topography after PKP

host on Bowman’s level (external steps are to be

and delay visual recovery

avoided, internal steps may be tolerated in the

case of thin recipient corneas such as in pellucid

marginal degeneration or herpetic scars), and

10.3.2

(3) stabilization of the anterior chamber for

Conventional Mechanical Trephines

further homogeneous suturing.

(Table 10.5)

Unintentionally other than round host open-

ing may create a challenge even for the experi-

In 1886 Arthur von Hippel was the first to

enced PKP surgeon concerning the correct

use a mechanical clock-watch driven trephine

placement of the second cardinal suture. After

(Fig. 10.10) for transplantation of a lamellar

removal of the cardinal sutures the quality of

corneal graft from a rabbit to a human [79]. The

the trephination and graft positioning are major

same trephine was used by Eduard Zirm for his

determinants for watertight wound closure. The

first successful PKP in a patient in 1905 [84].

better the trephination, the smaller the final su-

Conventional mechanical trephination is

ture tension required for watertight wound clo-

associated with deformation of corneal tissue

sure after removal of the cardinal sutures. The

including a distortion of the cut margin with

smaller the final suture tension, the better the

rough-cut edges as a consequence of axial and

visual acuity as long as the sutures are in place.

radial forces induced by the trephine. The cut

Generally, in cases where Bowman’s layer is

angle deviates from the perpendicular and it

intact, a 16-bite double-running diagonal cross-

may be different in donor and recipient, espe-

stitch suture (10-0 nylon) according to Hoff-

cially if the donor trephination is undertaken

mann (Fig. 10.9) is preferred. The more rapid

from the endothelial side. The fitting of the

140

Chapter 10

Trephination in Penetrating Keratoplasty

Table 10.5. Characteristics of mechanical trephines

Type

Geuder

Moria

GTS

Hessburg-

Asmotom

Micro-Keratron

(Hanna)

(Krumeich)

Barron

(Gliem &

(discontinued)

Franke)

Motorized cutter

Yes

Vacuum fixation

Yes

Double

for recipient

(limbus)

(cornea)

Cutter feed

Yes

Depth adjustment

Yes

Limited

Yes

Auto-retract

Yes

Anterior chamber

Yes

Possible

maintainer required

for donor

Automation

Yes

Table 10.6. Trephines used in Germany in the year 2002 for 4583 penetrating keratoplasties (German Kerato-

plasty Registry Erlangen) (122 institutions contributed) [5]

GTS

Manual

Barron

Motor trephine

Asmotom

Excimer laser

Unknown

Donor

1555

1040

716

415

393

313

151

33.9

22.7

15.6

9.1

8.6

6.8

3.3

Recipient

1570

818

745

640

346

313

151

34.3

17.8

16.3

13.9

7.6

6.8

3.3

Fig. 10.10 A, B. Mechanical trephines. A Arthur von Hippel’s clock-watch driven trephine. B “Modern”

mechanical trephines (motor trephine, Lochpfeiffentrepan, hand-held trephine [39])

10.3

Trephination Techniques

141

donor tissue into the malleable recipient cornea

10.3.2.2

is extremely difficult to achieve in a perfectly

Motor Trephines

symmetric fashion. After suturing the incon-

(Mikro-Keratron, Asmotom)

gruent cut edges in order to achieve watertight

wound closure, wound healing may cause

Mikro-Keratron. The Geuder Micro-Keratron

marked distortion of the surface topography

trephine is a non-automated motor-driven

after suture removal due to this “vertical tilt.” In

trephine system for PKP. The depth of the cut is

addition, asymmetric cardinal suture place-

not preadjustable, so that this trephine system

ment may result in unequal donor tissue distri-

has no impact on lamellar keratoplasty. Rota-

bution in the host wound, particularly if the sec-

tion

(variable speed) may be started and

ond cardinal suture is not placed exactly

stopped by pressing down and releasing a foot

opposite to the first (“horizontal torsion”) [42].

pedal. Different blades mounted on the unit al-

A questionnaire was sent to all German ker-

low for a wide range of trephination diameters.

atoplasty surgeons in 2002 asking for their pre-

To trephine the donor cornea from the epithe-

ferred technique of trephination. As outlined

lial side, the tissue has to be mounted into an

in Table 10.6 for recipient trephination, most

artificial anterior chamber maintainer. Motor

surgeons use the GTS (34.3 %), the hand-held

trephine rotation may lead to “shifting” of the

trephine

(17.8 %) or the Hessburg-Barron

trephine within the corneal stroma.

trephine

(16.3 %). Motor trephines are used

more rarely and the laser trephination has still

Asmotom. The Asmotom ATS is an automated

not entered many operating theaters because it

trephine system for PKP. The trephination of

is bulky and expensive. As many as 12 % of all

patient and donor eyes as well as corneoscleral

procedures were performed with different

disks is performed with separate instrumenta-

trephine systems for donor and recipient [5]!

tion sets. For non-perforating cuts the cutting

depth is preadjustable with offset rings for the

10.3.2.1

patient. The cutter sets provided by the distrib-

Freestanding Blade/Hand-Held Trephines

utors include five different diameters

(6.0-

8.2 mm). The ATS uses an innovative double fix-

Hand-held trephines are available in a wide

ation design.Vacuum is applied to both the cen-

range of diameters from very small

(e.g.,

tral and the peripheral section of the cornea.

1.5 mm) to very large (e.g., 16.0 mm). Hand-held

The trephine rotates between the two concen-

trephines may be dull with reduced visual con-

tric areas of fixation, using an automatic feed.

trol under the operating microscope despite re-

Once the pre-set depth is reached, the cutter re-

cent improvements [39]. Thus, centration may

tracts back into its initial position, holding on to

be a problem. Typically, the donor is punched

the separated central portion, until vacuum is

from the endothelial side

(Lochpfeiffentre-

released. The ATS marker facilitates the center-

pan). Francheschetti-type freestanding blades

ing of the trephination cut to the cornea. The

(Fig. 10.11) seem to create more reproducible

system does not require an artificial chamber

cuts than other hand-held trephines [72, 76].

maintainer for graft trephination.

Fig. 10.11. Francheschetti-type freestanding blades

are available in a wide range of diameters

142

Chapter 10

Trephination in Penetrating Keratoplasty

periphery of the blade-containing part and four

corresponding peripheral holes in the suction-

containing part (Fig. 10.12 B). In addition, four

small holes inside the cut area which are colored

before the corneoscleral button is placed inside

give a reference with respect to the first four car-

dinal sutures. The donor is typically oversized

by 0.25 mm [12].

Recently, a single-use artificial anterior

chamber has been available, to create donor

trephination from the epithelial side using the

recipient trephine for donor trephination first.

10.3.2.4

Guided Trephines (GTS, Hanna)

The guided trephines result in the best cut qual-

ities possible with mechanical trephines [72,

76]. These new generation suction trephines

such as the Hanna trephine

[80] and the

Krumeich trephine (“guided trephine system,”

GTS) [4, 23] are preferred over the Hessburg-

Barron trephine because they stabilize the globe

Fig. 10.12 A, B.

Hessburg-Barron suction trephine.

by suction at the limbus - not the peripheral

A Recipient trephine with cross-hairs for centration;

cornea. Thus - at least theoretically - the cut

B Donor trephination is performed from the endo-

thelial side

angles should be parallel to the optical axis, the

dimensions for donor and recipient should be

equal and, therefore, no graft oversize is re-

10.3.2.3

quired [50]. Overall, handling of both trephines

Suction Trephines (Hessburg-Barron)

requires a special introduction to the micro-

surgeon and the staff before application in

The classical Hessburg-Barron trephine (HBT)

patients.

has been on the market for over 25 years. The

HBT vacuum trephine is an easy to handle sin-

GTS (Fig. 10.13). The Krumeich guided trephine

gle-use product. The suction is applied to the

system (GTS) is designed for PKP, lamellar ker-

peripheral cornea. The depth of the lamellar

atoplasty, and circular keratotomy. The GTS can

trephination can be predicted to a certain de-

be used with and without an obturator prevent-

gree. One full rotation is presumed to achieve

ing ballooning of the excised tissue into the

250 mm of corneal depth. Perforation is typical-

trephine opening.

ly limited to one-third to one-half of the cir-

Advantages of the GTS include: (1) trephina-

cumference of the excision. The recipient

tion of donor and recipient from the epithelial

trephine has cross-hairs for centration. No ob-

side using an artificial anterior chamber, (2)

turator is applied (Fig. 10.12 A). The Hessburg-

pre-defined depth of trephination, e.g., for

Barron trephine leads to divergent cut angles

lamellar procedures, and (3) in experienced

and a larger diameter of the hole at the level of

hands through-and-through trephination with-

Descemet’s membrane [72, 76].

out the necessity of cut completion with scissors

In the classic version the donor is punched

can be achieved.

from the endothelial side with the aid of a suc-

Potential disadvantages of the GTS include:

tion device for fixating the donor epithelial side

(1) it is difficult to apply in patients with narrow

down. Tilt is avoided by four metal rods in the

lid fissure or deeply set eyes with prominent or-

10.3

Trephination Techniques

143

Fig. 10.14. The Hanna (Moria) trephine system. In

patients this trephine attaches firmly to the eye

through suction applied to the limbal conjunctiva.

The Hanna trephine in combination with the artifi-

cial anterior chamber allows the surgeon to trephine

both the recipient and the donor cornea from the

Fig. 10.13. The Krumeich guided trephine system

epithelial side

(GTS) is designed for PKP, lamellar keratoplasty, and

circular keratotomy. In patients, the GTS can be used

with and without an obturator preventing ballooning

both the recipient and the donor cornea from

of the excised tissue into the trephine opening

the epithelial side, thus reducing shape disparity.

In the original version the donor trephination

was performed from the endothelial side [81].

bital bones (which is not an uncommon issue in

Summary for the Clinician

keratoconus), preexisting filtering blebs or con-

junctival chemosis, (2) centration is difficult

∑ If conventional trephines are used it is rec-

due to the limited view, (3) injury if the iris and

ommended to use at least the same system

lens are not securely prohibited, and (4) eccen-

with trephination of the donor from the

tric mini-keratoplasty with a small diameter

epithelial side using an artificial anterior

(e.g., 4 mm) cannot be accomplished.

chamber for placement of the corneoscleral

button from the Eye Bank

Hanna Trephine (Fig. 10.14). The Hanna (Mo-

∑ The trephine should be as sharp as possible

ria) trephine system is one of the most advanced

trephines which is designed to create a proper

donor/recipient match. The Hanna trephine at-

10.3.3

taches firmly to the eye through suction applied

Nonmechanical Laser Trephination

to the limbal conjunctiva. Uniform support over

the whole cornea during trephination prevents

Hypothesizing that the properties of the wound

corneal vaulting. From a fully retracted position,

bed are much more important for the final “all-

the blade rotates while descending to a preset

suture-out” astigmatism and the final optical

depth, after which the blade rotates without fur-

performance of the graft than various types of

ther descent, cutting the displaced tissue

suture techniques or methods of suture adjust-

and creating a uniform incision. The Hanna

ment, we have developed and optimized the

trephine in combination with the artificial ante-

technique of nonmechanical corneal trephina-

rior chamber allows the surgeon to trephine

tion since 1986.

144

Chapter 10

Trephination in Penetrating Keratoplasty

Fig. 10.15. Principle of excimer laser trephination in donor and recipient (schematic drawing, sagittal view)

10.3.3.1

Table 10.7. Indications for 1656 consecutive non-

The 193-nm Excimer Laser

mechanical excimer laser keratoplasties (06/1989 to

04/2005 in Erlangen)

Since 1989 more than 1650 human eyes have

Keratoconus

607

(36.7 %)

been treated successfully with the Meditec

MEL60 excimer laser (Fig. 10.15). Keratoconus

Fuchs’ dystrophy

323

(19.5 %)

has been by far the leading indication (around

Bullous keratopathy

275

(16.6 %)

37 %) for PKP with this non-contact technique

Avascular scars

181

(10.9 %)

(Table 10.7). For donor trephination from the

Graft failure

(4.6 %)

epithelial side an artificial anterior chamber is

Corneal ulcer

(3.9 %)

used [41, 42, 58, 66].

Stromal dystrophies

(2.9 %)

Disabling astigmatism

(2.4 %)

Technique (Fig. 10.16). Before starting trephi-

Others

(2.5 %)

nation, the limbus is centered on the perpendi-

cular HeNe aiming beam in donor and patient

to ensure a reproducible position of the eye rel-

ative to the laser and symmetric cut angles over

may be reduced by employing eight orientation

the entire circumference without tilt. The hori-

teeth at the donor trephination margin and

zontal positioning of the limbal plane can be

eight corresponding notches in the recipient

controlled using the focusing device of the laser

bed (a technique which allows the use of eight

at 3, 6, 9, and 12 o’clock at the limbus before

symmetric cardinal sutures) [2].

focusing the laser at the trephination edge (“tri-

For donor trephination from the epithelial

angulation”). “Horizontal torsion” of the graft side using the 193-nm excimer laser MEL60

10.3

Trephination Techniques

145

Fig. 10.16 A-D. Nonmechanical trephination using

C laser arm and joystick for recipient trephination;

the 193-nm excimer laser in combination with metal

D metal recipient mask with eight “orientation notch-

masks with “orientation teeth/notches.” A Curved

es” on top of patient’s cornea. A 1.5¥1.5-mm laser spot

donor mask on top of corneoscleral button fixed in a

is guided along the inner edge of the mask, half of the

modified Krumeich artificial anterior chamber;

beam on the mask and half of it on the cornea

B metal donor mask with eight “orientation teeth”;

(Carl Zeiss Meditec, Jena, Germany), a circular

trephination, centration relative to the limbus is

round metal aperture mask

(diameter

5.6-

achieved by lining up the eight notches with the

8.6 mm, central opening 3.0 mm for centration,

eight lines of a radial keratotomy marker under

thickness 0.5 mm, weight 0.2 g, eight orientation

microscopic control (Fig. 10.6).

teeth

0.15¥0.3 mm) is positioned on a cor-

neoscleral button (16 mm diameter) fixed in

Advantages (Table 10.8). The main advantage

an artificial anterior chamber (Polytech, Ross-

of this novel laser cutting technique performed

dorf, Germany) under microscopic control

from the epithelial side in donor and recipient is

(Fig. 10.16 A, B). The pressure within the artifi-

the avoidance of mechanical distortion during

cial anterior chamber is adjusted to 22 mmHg.

trephination, resulting in smooth cut edges

An automated rotation device for the artificial

(Fig. 10.17 A) which are congruent in donor and

anterior chamber is used.

patient, potentially reducing “vertical tilt” [33].

For recipient trephination exclusively per-

Such cut edges in combination with “orientation

formed with the manually guided excimer laser,

teeth” (Fig. 10.17 B) at the graft margin [2] and

a corresponding metal mask is used (diameter

corresponding notches at the recipient margin

12.9 mm, central opening 5.5-8.5 mm), thickness

for symmetric positioning of the eight cardinal

0.5 mm, weight 0.4 g, eight orientation notches

sutures minimize

“horizontal torsion,” thus

0.15¥0.3 mm (Fig. 10.16 C, D). Before starting the potentially improving the optical performance

146

Chapter 10

Trephination in Penetrating Keratoplasty

Table 10.8. Advantages of nonmechanical trephination with the 193-nm excimer laser along metal masks

with “orientation teeth/notches” [41, 42, 58, 66]

1. No trauma to intraocular tissues

2. Avoid deformation and compression of tissue during trephination

3. Reduction of horizontal torsion (“Erlangen orientation teeth/notches”)

4. Reduction of vertical tilt (congruent cut edges)

5. Reduction of host and donor decentration

6. Feasibility of “harmonization” of donor and host topography

7. Reduction of anterior chamber inflammation early after PKP

8. Reduction of astigmatism after suture removal

9. Higher regularity of corneal topography

10. Significantly better visual acuity with spectacle correction

11. Feasibility of trephination with unstable cornea (e.g.,“open eye”, descemetocele, after radial

keratotomy, iatrogenic keratectasia after LASIK)

12. Arbitrary shape (e.g., elliptical) [28]

after transplantation [42]. Furthermore, recipi-

ent and donor decentration may be reduced [30,

61]. The use of metal masks allows for arbitrary

shapes of the trephination [28].

These favorable impacts on major intra-

operative determinants of post-keratoplasty

astigmatism

(cf. Table 10.2) result in lower

keratometric astigmatism, higher topographic

regularity and better visual acuity after suture

removal. After sequential removal of a double

running suture, keratometric astigmatism in-

creased in

80 % of eyes with conventional

trephination, but further decreased in 52 % of

eyes with laser trephination [58]. In addition to

less blood-aqueous barrier breakdown during

the early postoperative time course after PK

[26], laser trephination induces neither cataract

formation nor higher endothelial cell loss of the

graft. Likewise, the rates of immunologic graft

rejection and secondary ocular hypertension

are comparable using either technique. In addi-

tion, trephination of an unstable cornea, such as

in (pre-)perforated corneal ulcers or after RK or

Fig. 10.17 A, B. Donor trephination immediately

LASIK, is facilitated [64].

before perforation. A Histologic view with smooth

almost perpendicular cut edge; B macroscopic view

with smooth cut surfaces and “orientation teeth”

Practical Considerations for the Microsurgeon

[66]. The longer trephination time of around

6 min for the donor and around 4 min for the re-

cipient are by far compensated for by practical

advantages for the microsurgeon during the

subsequent course of surgery: (1) injuries of in-

10.3

Trephination Techniques

147

tures with adverse effects on graft topography

required at the end of surgery. (8) In addition,

the so-called “barrel-top formation” at the prox-

imal suture endings inducing a relative cornea

plana and delaying optical rehabilitation can be

avoided. (9) After removal of lid speculum and

fixation sutures, the use of a Placido’s disk often

enables an almost round projection image to be

achieved during intraoperative suture adjust-

ment.

Summary for the Clinician

∑ Nonmechanical trephination using the

193-nm excimer laser along metal masks

has improved functional outcome after PKP

with all-sutures-out

∑ The application of excimer lasers allows

controlled trephination of unstable corneas

such as perforated ulcers or iatrogenic

keratectasia after LASIK

10.3.3.2

The 2.94-µm Erbium:YAG Laser

Fig. 10.18.

Correct

position

second

cardinal

suture

(arrow) is facilitated by orientation tooth

The erbium:YAG laser was investigated to im-

(donor) and corresponding notch (host)

prove handling, reduce acquisition and mainte-

nance costs, and provide solid state laser safety

but keep the morphological advantages of the

traocular structures are impossible with the

excimer laser trephination [1]. However, shrink-

laser - even in beginner’s hands - since the ab-

age effects due to thermal damage of the cut

lation stops as soon as aqueous humor fills the

edges especially in the free-running but even

trephination groove after focal perforation. (2)

with Q-switched laser pulses are major draw-

The need for completion of the cut by scissors is

backs of this infrared laser [69]. The induced

reduced to a minimum. (3) The localization

thermal damage of the Q-switched mode er-

of the first eight cardinal sutures is unequivo-

bium:YAG laser has been detected to be around

cally given by the “orientation teeth/notches”

2-15 mm, in comparison to only 200 nm using

(Fig. 10.18). (4) Crescent-shaped tissue deficits

the excimer laser [54, 68].

at the graft-host junction (e.g., at other than

Summary for the Clinician

round recipient openings in keratoconus) are

avoided, thus achieving a latent watertight

∑ The erbium:YAG laser will probably

wound closure often as soon as after four cardi-

not substitute the excimer laser for non-

nal sutures. (5) During further suturing the an-

mechanical trephination in the near future

terior chamber tends to remain stable. (6) The

without a loss of advantages

final double running suture needs very little

tension to keep a watertight wound after re-

moval of the eight cardinal sutures. (7) There-

fore, only very rarely are additional single su-

148

Chapter 10

Trephination in Penetrating Keratoplasty

10.3.3.3

Summary for the Clinician

The Femtosecond Laser

∑ Femtosecond laser application is the

In contrast to the excimer laser, which allows

“excitement of tomorrow” in microsurgery

only surface ablation, the femtosecond

of the cornea

10^-15 s) laser allows the cornea to be cut within

∑ New nut-and-bolt type variants for poten-

the stroma, enabling truly three-dimensional

tially self-sealing donor/host appositions

cuts without opening the eye and without ther-

are on the horizon, offering a promising

mal damage. No masks but an ultra-fast eye

approach towards minimally invasive

tracking system is required. There is no signifi-

“no-stitch keratoplasty”

cant tissue loss to be compensated. For PKP es-

pecially in keratconus a non-contact approach

of laser application is favored to avoid deforma-

10.4

tion.

Concluding Remarks

Self-sealing keratoplasty wounds would be a

major step towards rapid visual rehabilitation

Today, expectations concerning the outcome

in PKP. Various kinds of nut-and-bolt configu-

after penetrating keratoplasty are not only re-

rations to fit in the donor including “orientation

stricted towards achieving a clear graft. The

teeth” of the graft in the recipient bed are feasi-

only criterion that counts for the patient is good

ble using a femtosecond laser. We have intro-

vision preferably without the need for contact

duced an inverse mushroom shaped trephina-

lenses but with an easily tolerable need for cor-

tion with the larger diameter of the graft at the

rection using spectacles. Therefore, transplant

level of Descemet’s membrane (Fig. 10.19). Vari-

microsurgeons should not only consider all the

ation of the diameter of the “stipe” and the “cap”

means available to prevent high or irregular

may help to produce the best individual com-

post-PKP astigmatism. Due to the lack of pre-

promise between the amount of transplanted

dictability of the refractive result in an individ-

endothelium and distance to limbal vessels and

ual patient after PKP, they should also familiar-

resistance to intraocular pressure [67].

ize themselves with the surgical techniques for

In addition, posterior lamellar keratoplasty

correcting refractive errors after PKP in order

(PLKP) can be performed more easily with a

to achieve the individually best outcome for a

femtosecond laser [63].

given patient.

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Ophthalmol 110:2162-2167

Infective Complications Following LASIK

Adam Watson, Sheraz Daya

although not common, occurs at least partly due

Core Messages

to the common practice of performing bilateral

∑ Infective complications following LASIK

simultaneous LASIK procedures. Clusters of in-

are a rare, potentially sight-devastating

fection have also been reported [3, 7, 11]. Finally

complication but often have good

it should be noted that it is a vision-threatening

outcomes

complication occurring in people with general-

∑ Early diagnosis helps prevent rapid steroid-

ly high visual expectations, adding to its gravi-

related progression of infection

tas.

∑ Atypical organisms are common, especially

non-tuberculous mycobacteria

∑ Early presenting cases (7-10 days) and late

presenting cases (>10 days) have a different

11.2

microbiological profile

Frequency and Presentation

∑ Intact epithelium inhibits antibiotic pene-

tration. Flap lift, antibiotic soak and epithe-

The reported frequency of infection following

lial defect creation are useful strategies

LASIK ranges from 0 % to 1.5 %, with the fre-

∑ Reculture, biopsy and flap amputation

quency in most large case series being less than

may be necessary for worsening keratitis

0.2 % [4]. Gram-positive and non-tuberculous

despite treatment

mycobacterial infections are commonest, with

∑ Informed consent and attention to risk

these organisms accounting for 26 % and 47 %

factors are crucial

of culture-positive infections respectively in a

review of published cases [4]. Mycobacterial in-

fections are probably overrepresented due to re-

porting bias but do represent a strikingly high

proportion of cases of post-LASIK infection.

11.1

Gram-negative organisms, by contrast, account

Introduction

for very few cases. Fungal and Acanthamoeba

infections have also been described (Table 11.1).

Since its development in 1989 by Pallikaris fol-

There are almost certainly predisposing fac-

lowed later by FDA approval in the United States

tors for post-LASIK infection. Uncontrolled

1999, LASIK

(laser-assisted in situ ker-

meibomian gland dysfunction and blepharitis

atomileusis) has become an extremely com-

probably contribute to staphylococcal infection

monly performed surgical procedure. Infective

[12]. Performing LASIK on eyes that have previ-

complications are rare [4] but present special

ously undergone photorefractive keratectomy

challenges. Infective keratitis following LASIK

(PRK) seems to be a risk factor [4]. Post-LASIK

often involves organisms unusual in other

trauma is undoubtedly associated with infec-

forms of infective keratitis. It usually occurs in

tion. However, the commonest association with

the flap interface and may be relatively inacces-

reported infections is a breakdown in sterility

sible to topical antibiotics. Bilateral infection,

during the procedure, with systematic contami-

154

Chapter 11

Infective Complications Following LASIK

Table 11.1. Organisms reported to have caused post-

ly to be atypical infections, especially non-tu-

LASIK keratitis

berculous mycobacteria but also including fun-

gal infection.

Bacteria

Flap interface infiltrate is the commonest

Staphylococcus aureus

sign evident on examination although infiltrate

Streptococcus pneumoniae

may be confined to the lamellar flap or the un-

Streptococcus viridans

derlying corneal stroma [4]. Other features of

Coagulase-negative staphylococci

Pseudomonas aeruginosa

infection that may be present are those found in

Nocardia asteroides

other forms of infective keratitis, including an-

Mycobacteria

terior chamber reaction, keratic precipitates,

Mycobacterium chelonae

corneal abscess and epithelial defects. Epithelial

Mycobacterium mucogenicum

defects are found far less frequently in post-

Mycobacterium abscessus

LASIK keratitis and tend to be associated with

Mycobacterium szulgai

Gram-positive infection. The lack of an epithe-

Fungi

lial defect has important implications for treat-

Candida albicans

ment, as topical antimicrobial penetration is

Curvularia lunata

poorer in the absence of a defect. An intact ep-

Scedosporium apiospermum

ithelium presents a relatively impermeable bar-

Fusarium solan

rier to topical antibiotic penetration.

Fusarium oxysporum

Colleotrichum (Fusarium-like)

Crystalline keratopathy has also been report-

Other

ed in several cases associated with Mycobacteri-

Acanthamoeba

um chelonae infection [2, 22]. This appearance is

highly suggestive of M. chelonae infection.

nation of the surgical field probably being

11.4

responsible for three reported clusters of myco-

Differential Diagnosis

bacterial infection [3, 7, 11].

11.4.1

Diffuse Lamellar Keratitis

11.3

(DLK,“Sands of the Sahara”)

Characteristics

DLK, a non-infectious inflammation occurring

Patients with post-LASIK infective keratitis

after LASIK in approximately 2-4 % of cases

tend to present with varying combinations of

[13], may present with mild pain, redness and

pain, photophobia, discomfort, redness and dis-

photophobia in the 1st week after surgery. In the

charge. Deterioration in postoperative visual

milder stage 1 and stage 2 forms of DLK the in-

acuity is commonly noted and may be the sole

filtrates are light and diffuse and unlikely to be

presenting symptom. Patients may also be

confused with infection. More severe stages of

asymptomatic with the infection identified at a

DLK involve clumping of cellular infiltrates and,

routine postoperative examination.

in stage 4 cases, stromal melting. The possibili-

The timing of the onset of symptoms varies -

ty of infection should always be considered in

between zero days and several months [4, 12].

these cases and, since treatment of more severe

Post-LASIK infections may usefully be divided

DLK involves flap lift and irrigation, it is pru-

into early and late groups depending on the

dent to take a scrape sample for microbiology

length of time from surgery to the onset of

when lifting the flap [16]. Use of topical steroids

symptoms. Those presenting early occur in the

for presumed DLK may lead to initial apparent

first 7-10 days and are more likely to be caused

improvement in infective keratitis with subse-

by “typical” Gram-positive bacteria. Late infec-

quent rapid progression of infection and de-

tions, presenting beyond 10 days, are more like-

structive stromal necrosis.

11.5

Management

155

11.4.2

11.5.1

Steroid-Induced Intraocular Pressure

Flap Lift

Elevation with Flap Oedema (Pseudo-DLK)

This should be carried out in most circum-

This uncommon phenomenon generally pres-

stances. An exception is if the focus of infection

ents with decreased visual acuity, flap oedema

is very peripheral and associated with overlying

and variable inflammation and may be mistak-

flap necrosis allowing an adequate microbiolog-

en for DLK. Increased frequency of steroid use

ical sample and debridement of infectious ma-

then leads to worsening of the condition. The

terial (Fig. 11.2).

centrally measured intraocular pressure (IOP)

The flap may be lifted completely or partially,

is often normal and careful examination may

depending on the extent and location of infil-

reveal a fluid cleft in the flap interface. Peripher-

trate. Flap lift should be carried out beneath an

al IOP measured with a Tono-Pen (Medtronic-

operating microscope under sterile conditions

Solan) reveals an elevated IOP and the condi-

with or without patient sedation. Some prefer to

tion will resolve with control of IOP, usually

initiate the flap lift at the slit lamp where the flap

with topical agents, and tapering or cessation of

border may be more easily identified. Initiation

steroids [10, 15].

of flap lift is generally with a blunt spatula or

Sinskey hook to break the epithelium and open

the interface for one or two clock hours, then

11.5

completed with non-toothed LASIK flap forceps.

Management

The principles of management are similar to

11.5.2

those in regular infective keratitis, namely:

Specimen Taking

∑ Suspect infection

∑ Obtain a microbiological sample prior to

Gentle scraping of material for microbiological

starting treatment

examination and culture and to debride infec-

∑ Give broad spectrum empirical therapy ini-

tive debris follows this. A hypodermic needle,

tially

number 15 Bard-Parker blade or Kimura spatula

∑ Tailor therapy depending on clinical re-

may be used. The authors prefer to plate the

sponse and microbiological results (Gram

specimens themselves on culture media imme-

and other stains, culture, sensitivities)

diately. We suggest as a minimum, if the amount

∑ If there is a worsening clinical situation and

of material allows, an air-dried slide for im-

no microbiological information to guide,

mediate Gram stain, blood, chocolate and

consider temporary withdrawal of treatment

Sabouraud’s agar plates and brain-heart infu-

for rescrape or corneal biopsy

sion broth. If Mycobacterium is suspected, then

culture on Lowenstein-Jensen medium should

Post-LASIK infective keratitis differs from regu-

be considered. Useful additional stains for late-

lar infective keratitis in that:

presenting cases include auramine-rhodamine

∑ Atypical infections

(non-tuberculous my-

for acid-fast bacilli

[22] and periodic acid-

cobacteria) are relatively common

Schiff (PAS) for fungi [21].

∑ Antibiotic penetration may be poor due to an

Summary for the Clinician

intact epithelium

∑ Flap complications such as striae, epithelial

∑ A microbiological specimen prior to

ingrowth, flap melt and dehiscence may be

treatment is essential

problematic, related to infection or flap lift

∑ Flap lift is usually necessary

We propose a management algorithm that takes

some of these factors into account (Fig. 11.1).

11.5

Management

157

The use of preservative free lubricants to pre-

11.5.3

serve epithelial health should be considered. A

Treatment

cycloplegic (preservative free cyclopentolate or

homatropine) should be added if there is signif-

A moistened lint-free sponge may be used to

icant anterior chamber inflammation.

remove residual debris, followed by “soaking” of

Summary for the Clinician

the flap and stromal bed in antibiotic solution.

The choice of antibiotics may depend on

∑ Choose antibiotics to cover atypical

whether the keratitis falls into the early or late

organisms in late-presenting cases

group (Fig. 11.1). Soaking should be for 2 min or

∑ Antibiotic penetration is aided by an

more with each antibiotic solution in turn, fol-

epithelial defect

lowed by careful relaying of the flap. If there is

∑ Avoid steroid use unless there is unequivo-

little or no epithelial defect overlying the sus-

cal improvement suggesting sterilisation

pected infection, an epithelial defect should be

of infection

created to aid antibiotic penetration (Fig. 11.3).

∑ Avoid steroids in fungal infection and

Intensive topical antibiotics should then be

without concomitant antibiotic use

started (hourly alternating around the clock).

The choice of antibiotics will be partly deter-

mined by the resistance characteristics of bacte-

11.5.4

ria in the local region. Specialist microbiologist

No Improvement

advice should be sought if there is doubt.

Suggestions for treatment choice are given in

Failure of the infection to show signs of im-

Fig. 11.1.

provement after several days of treatment

Topical steroids should be avoided in the

should prompt a re-evaluation. An attempt at

early stages of treatment and only instituted,

reculturing the infective agent is mandatory, by

if at all, when there is clear clinical evidence of

further corneal scrape or corneal biopsy. If the

improvement (e.g. less pain, diminishing and

infection is severe and judged to be threatening

coalescing infiltrate, fewer keratic precipitates,

the eye, flap amputation may be necessary, with

healing epithelial defect), suggesting sterilisa-

half the flap being sent for histological exami-

tion of the offending organism. Introduction of

nation and staining for organisms, the other

any steroid should generally be in low dose (e.g.

half being sent for microbial culture.A high sus-

twice daily prednisolone sodium phosphate

picion for atypical infection exists at this point

0.5 %) and the response closely monitored for

and mycobacteria, fungi and Acanthamoeba

signs of worsening infection, e.g. satellite infil-

should be specifically looked for.

trates. Steroid use without concomitant antibi-

Failure to control the infection despite treat-

otic has been implicated in the recrudescence

ment, as with regular infective keratitis, may re-

of infection after apparent sterilisation of

quire further surgical intervention including

Pseudomonas keratitis [8]. Steroid use should be

therapeutic penetrating keratoplasty, and in-

avoided in cases of fungal keratitis.

traocular instillation of antimicrobial drugs in

Topical antibiotic choice may be altered

the case of perforation with suspected endo-

when microbial sensitivities are available. If the

phthalmitis, with or without lensectomy and

infection is clinically improving, there may be

vitrectomy depending on the involvement of

no need to change the antibiotics other than ta-

intraocular structures.

pering the frequency of use after 2-3 days. If the

infection is improving and sensitivity data are

available, it may be reasonable to discontinue

one of the antibiotics (e.g. gentamicin in a van-

comycin/gentamicin combination when treat-

ing a staphylococcal infection) to minimise ep-

ithelial toxicity and promote healing.

158

Chapter 11

Infective Complications Following LASIK

Fig. 11.5. Subsequent right central flap melt in the

case of M. chelonae keratitis shown in Fig. 11.4

6 months or more with a gradual taper, moni-

toring closely for signs of recurrence. Viable

mycobacteria have been cultured from an am-

putated LASIK flap despite 9 weeks of appropri-

ate treatment for M. chelonae keratitis [19].

Fig. 11.4 A, B. Bilateral central Mycobacterium che-

11.6.2

lonae post-LASIK keratitis (right eye A, left eye B).

Fungal Keratitis

Note central interface infiltrates

Fungal infections comprise about 14 % of re-

ported cases of post-LASIK keratitis [4]. Identi-

11.6

fication of hyphae, pseudohyphae or yeasts may

Special Considerations

be possible from direct microscopic examina-

tion of appropriately stained slide preparations

11.6.1

of a scrape; or culture may yield fungal growth.

Mycobacteria

An additional approach, maybe more applicable

in the future, is PCR testing of specimens for

Topical clarithromycin and amikacin have gen-

fungal DNA, providing a quicker result than

erally been the agents of choice for treatment of

fungal culture. This method, while sensitive,

M. chelonae keratitis. Tobramycin and the fluo-

does suffer from poor specificity [21].

roquinolones are also often effective. There has

Treatment of fungal infections should be de-

been recent interest in the fourth generation flu-

termined in collaboration with a microbiologist

oroquinolones, including moxifloxacin [1] and

and based on the organism’s sensitivities

gatifloxacin, as having greater activity against

when available. Common topical agents are

non-tuberculous mycobacteria. The authors

natamycin 5 % and amphotericin B 0.15 %, both

have experience of treating a case

(unpub-

polyenes with a broad spectrum of activity

lished) of bilateral moxifloxacin-resistant M.

against filamentous fungi and yeasts although

chelonae post-LASIK keratitis (Figs. 11.4, 11.5).

natamycin may be slightly more effective and

This highlights the benefit of using multiple

the preferred choice where available [21]. Topi-

agents to treat infection empirically until the or-

cal econazole 1 % is also being used where ap-

ganism’s sensitivities are known, with contin-

propriate. Topical treatment should generally be

ued use of multiple antibiotics to which the or-

combined with a systemic agent, e.g. one of the

ganism is sensitive to prevent recrudescence.

azoles such as ketoconazole or itraconazole.

Treatment may need to be continued for

Voriconazole, a relatively new triazole agent, has

11.8

Management of Sequelae

159

been reported to have superior activity against

Scedosporium infections [18].

The use of topical steroids may cause fungal

keratitis to progress rapidly to widespread

corneal involvement and perforation. Steroids

should be avoided when treating fungal infec-

tions, at least until effective antifungal treat-

ment has been continued for several weeks. An-

tifungal therapy needs to be prolonged for at

least 6 weeks - agents are generally fungistatic

rather than fungicidal at the concentration

achieved in the corneal stroma, and elimination

Fig. 11.6. Eye 9 months following treatment for M.

of fungus depends ultimately on the host im-

chelonae post-LASIK keratitis. Arrows point to stro-

mune response.

mal scarring (white) and stable interface epithelial

inclusions (yellow). The uncorrected visual acuity is

6/7.5

11.6.3

Viral Keratitis

Case reports of apparent reactivation of Herpes

simplex keratitis following LASIK have been

published [5, 17]. It is not clear whether the

LASIK procedure and/or the postoperative use

of topical steroids were causative. However, ul-

traviolet radiation exposure has been associat-

ed with reactivation of latent Herpes simplex

[20, 6]. In addition to a short-term topical an-

tiviral, consideration should be given to longer-

term systemic antiviral prophylaxis (e.g. oral

acyclovir 400 mg twice daily).

Fig. 11.7. Interface epithelial ingrowth arising from

a flap defect in a case of M. chelonae post-LASIK

keratitis. Tongues of epithelium are progressing pe-

11.7

ripherally

Visual Outcome

The visual outcome following post-LASIK ker-

atitis is highly variable. Approximately 50 % of

11.8

reported cases have no clinically significant

Management of Sequelae

worsening of best-corrected Snellen visual acu-

ity. Twenty-five per cent suffer a severe reduc-

Common sequelae of post-LASIK infection in-

tion [4]. Gram-positive infections are associat-

clude scarring (Fig. 11.6), irregular astigmatism

ed with better visual outcomes while fungal

and varying degrees of epithelial ingrowth aris-

infections

(excluding Candida albicans) are

ing from flap lift or flap melt (Fig. 11.7).

more likely to be associated with severe visual

Once the infection has settled, the goal of

reduction. Reported cases of C. albicans, on the

treatment is to optimise visual acuity in the

other hand, had a good visual outcome - with a

affected eye. How this is achieved will vary

best corrected visual acuity average of 20/25

markedly from case to case. Correction of re-

[16]. Reported mycobacterial cases tend to be

fractive error should initially be explored using

intermediate between Gram-positive and fun-

glasses, soft contact lens and rigid gas perme-

gal infection in terms of visual outcome.

able lenses. Significant epithelial ingrowth in-

160

Chapter 11

Infective Complications Following LASIK

ducing astigmatism needs to be cleared from

Alvarenga L, Freitas D, Hofling-Lima AL et al.

the flap interface prior to any further attempts

(2002) Infectious post-LASIK crystalline ker-

atopathy caused by nontuberculous mycobacte-

at surgical correction. Irregular astigmatism

ria. Cornea 21:426-429

resulting from scarring may be amenable to

Chandra NS, Torres MF, Winthrop KL et al. (2001)

contact lens correction.

Cluster of Mycobacterium chelonae keratitis cas-

Consideration of further excimer laser re-

es following laser in-situ keratomileusis. Am J

fractive surgery should be approached with

Ophthalmol 132:819-830

caution. In addition to likely patient concern

Chang MA, Jain S, Azar DT (2004) Infections fol-

about a repeat procedure, further LASIK will re-

lowing laser in situ keratomileusis: an integration

of the published literature. Surv Ophthalmol

quire recutting of a deeper flap to avoid the

49:269-280

scarred and irregular interface inevitably pres-

Davidorf JM (1998) Herpes simplex keratitis after

ent, and PRK or laser epithelial keratomileusis

LASIK. J Refract Surg 14:667

(LASEK) is associated with a high risk of devel-

Dhaliwal DK, Romanowski EG, Yates KA et al.

opment of haze in an environment with activat-

(2001) Experimental laser-assisted in situ ker-

ed keratocytes.

atomileusis induces the reactivation of latent

Significant opacity affecting the visual axis,

herpes simplex virus. Am J Ophthalmol 131:506-

507

on the other hand, may need to be cleared.

Freitas D, Alvarenga L, Sampaio J et al. (2003) An

Options for this include homoplastic automated

outbreak of Mycobacterium chelonae infection

lamellar therapeutic keratoplasty

(HALTK, a

after LASIK. Ophthalmology 110:276-285

useful technique for opacities limited to the an-

Gritz DC, Kwitko S, Trousdale MD et al. (1992) Re-

terior one-third of the corneal stroma) [9], deep

currence of microbial keratitis concomitant with

anterior lamellar keratoplasty [14] and pene-

antiinflammatory treatment in an animal model.

trating keratoplasty.

Cornea 11:404-408

Hafezi F, Mrochen M, Fankhauser F 2nd et al.

(2003) Anterior lamellar keratoplasty with a mi-

crokeratome: a method for managing complica-

11.9

tions after refractive surgery. J Refract Surg

Prevention

19:52-57

10.

Hamilton DR, Manche EE, Rich LF et al. (2002)

Rare cases of post-LASIK infective keratitis are

Steroid-induced glaucoma after laser in situ

inevitable. Attention to patient eyelid hygiene

keratomileusis associated with interface fluid.

Ophthalmology 109:659-665

with control of blepharitis, careful patient in-

11.

Holmes GP, Bond GB, Fader RC et al. (2002) A

struction regarding pre- and postoperative care

cluster of cases of Mycobacterium szulgai kerati-

and avoidance of trauma, and meticulous atten-

tis that occurred after laser-assisted in situ ker-

tion to equipment sterility and operating envi-

atomileusis. Clin Infect Dis 34:1039-1046

ronment hygiene are likely to lead to fewer cas-

12.

Karp CL, Tuli SS, Yoo SH et al. (2003) Infectious

es. The authors strongly advise that separate

keratitis after LASIK. Ophthalmology 110:503-510

blades and microkeratome heads be used if

13.

McGhee CNJ, Brahma A (2001) Uncommon com-

plications of LASIK: diffuse lamellar keratitis

carrying out simultaneous bilateral LASIK to

and epithelial ingrowth. CME J Ophthalmol 5:52-

diminish the linked risk of bilateral infection.

Above all, careful informed consent of the

14.

Melles GRJ, Lander F, Rietveld FJR et al. (1999) A

patient prior to surgery is mandatory.

new surgical technique for deep stromal, anterior

lamellar keratoplasty. Br J Ophthalmol 83:327-333

15.

Nordlund ML, Grimm S, Lane S et al. (2004) Pres-

References

sure-induced interface keratitis: a late complica-

tion following LASIK. Cornea 23:225-234

16.

Peng Q, Holzer MP, Kaufer PH et al. (2002) Inter-

Abshire R, Cockrum P, Crider J et al. (2004) Topi-

face fungal infection after laser in situ kerato-

cal antibacterial therapy for mycobacterial ker-

mileusis presenting as diffuse lamellar keratitis.

atitis: potential for surgical prophylaxis and

J Cataract Refract Surg 28:1400-1408

treatment. Clin Ther 26:191-196

Treatment of Adenoviral Keratoconjunctivitis

Jost Hillenkamp, Rainer Sundmacher, Thomas Reinhard

Core Messages

∑ The clinical course of adenoviral kerato-

∑ Topical cidofovir is the first antiviral agent

conjunctivitis (AKC) should be divided into

which has effectively reduced the incidence

an acute phase with conjunctival inflamma-

of corneal opacities, but local toxicity rules

tion of varying intensity with or without

out its clinical application

corneal involvement and a chronic phase

∑ Recently, NMSO3, a sulfated sialyl lipid, has

with corneal opacities

demonstrated a greater antiviral potency

∑ AKC is caused by many different serotypes

against adenovirus in vitro than cidofovir

and is highly contagious during the acute

exhibiting minimal cytotoxicity

phase

∑ Topical cyclosporin A (CsA) appears to be

∑ The economic and social price of AKC as

effective in the treatment of persistent

an community epidemic is high

corneal opacities

∑ Corneal opacities, the hallmark of the

∑ Topical interferon might be effective as a

chronic phase, are usually self-limited

prophylaxis of infection

∑ Topical steroids should be avoided because

∑ Topical interferon is currently not commer-

they prolong viral replication, frequently

cially available due to unsettled patent

lead to long-lasting dry-eye symptoms, and

issues

corneal opacities almost always recur after

∑ Adequate infection control measures

discontinuation of topical steroids

should be followed as prevention and to

∑ There is currently no effective and clinically

reduce epidemic AKC outbreaks

applicable topical antiviral agent for the

treatment of the acute phase of AKC

adenovirus subtypes [5]. Overall, many of the

12.1

more than 40 adenovirus serotypes cause infec-

Introduction

tions of the ocular surface with and without

general symptoms. Pharyngoconjunctival fever

12.1.1

is a conjunctivitis with upper respiratory tract

Etiology and Clinical Course of Ocular

involvement caused by serotypes 1, 3-7, and 14.

Adenoviral Infection

The term “epidemic keratoconjunctivitis” de-

scribes an infection of the ocular surface with-

Adenoviral keratoconjunctivitis (AKC) was first

out general symptoms caused by serotypes 2-4,

described by Fuchs in 1889 [7]. In 1955, Jawetz

7-11, 14, 16, 19, 29, and 37. Unspecific follicular

identified adenovirus as the cause of the disease

conjunctivitis is probably caused by all the

[20]. Other authors isolated adenovirus sero-

above and serotype 34. In principle, neither is

types 8,19 and 37 as the most frequent causative

there a clear-cut distinction in this clinical

164

Chapter 12

Treatment of Adenoviral Keratoconjunctivitis

classification nor can the various serotypes be

clearly associated with a distinct clinical pres-

entation [5, 40].

The course of AKC must be divided into an

acute phase and a chronic phase.

Summary for the Clinician

∑ Many different serotypes of adenovirus

cause AKC

12.1.1.1

Fig. 12.1. Acute phase of AKC 1 week after onset of

symptoms. Symptoms obviously first appeared in the

The Acute Phase

right eye; the left eye became involved 2days later

The acute phase has a wide spectrum of dura-

tion and intensity of local symptoms. It is prin-

cipally self-limited. The intensity of symptoms

varies between a picture of a mild unspecific

conjunctivitis and intense conjunctival injec-

tion with marked chemosis and hemorrhagic

involvement of the conjunctiva and the eyelids

(Figs. 12.1-12.5). After an incubation period of

2-14 days symptoms usually begin in one eye

and the other eye becomes symptomatic after

2-4 more days (Fig. 12.1). The mild forms of ade-

noviral conjunctivitis are clinically difficult to

Fig. 12.2. Typical presentation of the acute phase of

differentiate from any other unspecific conjunc-

AKV with serous conjunctivitis

tivitis. The more pronounced cases can be read-

ily diagnosed by the clinician. They present with

a typical picture of conjunctival hyperemia and

chemosis, swelling of the conjunctival plica, and

intense serous or muco-serous tearing (Fig. 12.2).

Conjunctival pseudomembranes occur in some

cases. Ipsilateral preauricular lymphadeno-

pathy is a fairly typical sign observed in many

patients. The more severe cases of ocular aden-

oviral infections are characterized by a highly

distressing morbidity [5] (Figs. 12.3-12.5). The

cornea is not necessarily involved in the acute

phase. If it is, a superficial punctate keratitis

Fig. 12.3. This presentation with severe chemosis is

with small epithelial punctatae or larger stella-

fairly typical for the acute phase of AKC. An infection

ta-type coarse punctatae and subepithelial infil-

with herpesvirus cannot be clinically excluded

whereas this picture clearly differs from allergic

trates may develop [40]. Rarely, the cornea be-

chemosis

comes involved with large epithelial erosions

during the acute phase of the infection. Also

rarely, the corneal endothelium becomes in-

volved in the form of an endotheliitis with

marked temporary corneal edema which usual-

ly subsides spontaneously [40].

12.1

Introduction

165

Fig. 12.4. This presentation with pronounced hem-

Fig. 12.6. Corneal opacities are the hallmark of

orrhagic chemosis is also fairly typical for the acute

the chronic phase of AKC. Histopathology revealed

phase of AKC

subepithelial infiltrates of lymphocytes, histiocytes

and fibroblasts accompanied by a disruption of the

collagen fibers of Bowman’s layer [6, 7]. The patho-

genesis of the nummular opacities most likely in-

cludes a persisting viral replication in subepithelial

keratocytes triggering an immunological host reac-

tion which causes the visible, steroid-sensitive opaci-

ties

neal subepithelial opacities frequently appear

(Fig. 12.6). These nummular opacities or infil-

trates can impair visual function, and may per-

sist for months to years [40, 28]. Histopatholog-

Fig. 12.5. This is an exceptionally severe hemor-

ical investigation of focal biopsies revealed

rhagic involvement of the eyelids in the acute phase of

a case of proven AKC

subepithelial infiltrates of lymphocytes, histio-

cytes and fibroblasts accompanied by a disrup-

tion of the collagen fibers of Bowman’s layer [15,

25]. The pathogenesis of the nummular opaci-

ties most likely includes a persisting viral repli-

Summary for the Clinician

cation in subepithelial keratocytes triggering an

∑ The acute phase of AKC has a wide spectrum

immunological host reaction [40]. This hypoth-

of intensity and duration of symptoms

esis is supported by the clinical observation that

∑ The acute phase of AKC is self-limited

opacities usually resolve with topical steroid

∑ The cornea may or may not be involved

treatment but recur when steroids are discon-

in the acute phase of AKC

tinued [39].

Summary for the Clinician

12.1.1.2

The Chronic Phase

∑ Corneal opacities are the hallmark of the

chronic phase of AKC

It is the corneal involvement during the chronic

∑ Corneal opacities are probably caused by

phase of the disease that sets AKC apart from

an immunological host reaction against

other forms of virus conjunctivitis. Typically,

persisting virus in keratocytes

during the course of the infection, approxi-

∑ Corneal opacities almost invariably sponta-

mately 10 days after onset of symptoms, cor-

neously resolve mostly within 1 year

166

Chapter 12

Treatment of Adenoviral Keratoconjunctivitis

30, 31, 38, 41, 47], topical non-steroidal anti-in-

12.2

flammatories [13, 37], topical cyclosporin A or

Socioeconomic Aspect

topical trifluridine, have been shown not to

be more effective than topical lubrication [16,

AKC is a highly contagious disease which occurs

17, 19, 46].

worldwide sporadically and epidemically. While

not permanently blinding, adenoviral ocular in-

fections remain the most common external oc-

12.3.1

ular viral infection worldwide. The economic

Treatment of the Acute Phase

and social price of this community epidemic

also remains high [6]. Public institutions such

12.3.1.1

as schools or kindergartens must be closed fol-

Topical Steroids

lowing the outbreak of an epidemic. Many work

hours are lost every year.

Treatment of the acute phase of the infection

with topical steroids has been widely recom-

Summary for the Clinician

mended because of the clinical experience that

∑ The economic and social price of AKC

the distressing local symptoms subside earlier

as a community epidemic is high

with steroids. The effect of topical steroids has

been investigated in a prospective randomized

clinical study [42]. The results of this study con-

12.3

firm the clinical impression that the improve-

Treatment

ment of the local symptoms is accelerated with

steroids. Furthermore, the number of corneal

The clinical investigation of candidate treat-

opacities per affected cornea in the chronic

ments of AKC in patient studies has been ham-

phase was reduced as compared to controls

pered in the past by the very variable intensity

[42]. However, the number of patients affected

and duration of the clinical symptoms and the

by corneal opacities was not reduced and

self-limited nature of the acute phase of AKC.

corneal opacities appeared later in the course of

Furthermore, most studies have failed to apply

the disease. This finding suggests that steroids

laboratory tests for adenovirus as the cause of

may prolong the persistence of adenovirus in

the disease and therefore the etiology of the

the cornea. This undesired effect was confirmed

treated diseases remains questionable. A possi-

by Romanowski et al., who found an increased

ble treatment must therefore be evaluated by

viral replication under topical steroids

[32].

adequately designed large prospective con-

Also, a significantly greater number of patients

trolled clinical trials with reliable tests of aden-

treated with topical steroids experienced long-

ovirus as the underlying cause of the treated

lasting, distressing dry eye symptoms as com-

disease. Also, treatment of the acute phase of

pared to controls [42]. These findings lead to the

AKC must be distinguished from treatment and

conclusion that the negative effects of topical

prophylaxis of corneal opacities, the hallmark

steroids outweigh the positive effect of an earli-

of the chronic phase. Currently, no clinically ap-

er relief of the distressing local symptom [32, 40,

plicable specific antiviral therapy is available to

42]. Therefore, topical steroids should be avoid-

shorten the course of the infection, to improve

ed in the treatment of the acute phase of AKC.

the distressful clinical symptoms, to stop viral

Summary for the Clinician

replication, and to prevent the development of

corneal opacities. The effect of several topical

∑ Local symptoms subside with topical

agents has been investigated. Only steroids [42]

steroids but viral replication is probably

and cidofovir [16, 17] have demonstrated a cer-

prolonged

tain therapeutic effect and will therefore be

∑ Topical steroids frequently lead to

further discussed. Other measures, such as

long-lasting dry-eye symptoms

topical povidone-iodine, topical interferon [26,

∑ Topical steroids should be avoided

12.3

Treatment

167

12.3.1.2

Serotype Dependency. Adenovirus demon-

Topical Cidofovir

strated serotype-dependent differences in in-

vitro infectivity titers and clinical course. AD8

Principally, an effective antiviral agent to short-

was the most frequent serotype in a total num-

en the course of the infection, to improve the

ber of 106 positive adenoviral cultures, causing

distressful local symptoms of the acute phase,

significantly more often a severe clinical course

and to prevent the development of corneal

with marked eyelid edema than other sero-

opacities of the chronic phase would represent

types.AD3 and AD4 were associated with high-

the ideal treatment of AKC.

er infectivity titers than other serotypes. Infec-

Topical ganciclovir was only mildly effective

tivity and the clinical course of AKC are

in vitro and in the cotton rat model and was

serotype dependent [33].

thus not further investigated as a potential

Cidofovir proved to be effective against ade-

treatment [43, 44].

novirus types 1, 5 and 6 in the rabbit model [11,

Cidofovir or HPMPC, a broad-spectrum an-

29], but a relative resistance of serotype 19 to

tiviral agent, demonstrated a significant in-

cidofovir was reported [8]. Variants of aden-

hibitory effect on adenovirus types 1, 5, 8 and 19

ovirus serotype 5 with different sensitivity to

isolated from patients with AKC in vitro [8]. The

topical treatment with cidofovir 0.5 % in the

efficacy of cidofovir was also documented in

rabbit ocular model have been described by

vivo. Topical cidofovir demonstrated significant

Araullo-Cruz et al. [1]. Consequently, the effica-

antiviral activity in the AD 5 McEwen/NZ rabbit

cy of cidofovir in patients may also be serotype

ocular model with 0.2% as the lowest effective

dependent. The patients enrolled in clinical

concentration [11, 10]. Gordon et al. first report-

studies were probably infected with various

ed clinical efficacy and safety of topical cido-

adenovirus types.

fovir 0.2 % in the treatment of a single patient

with proven AKC [12].

Pharmacokinetics. Eyedrops may be washed

These results encouraged us to investigate

out by intense tearing; additionally regular

the effect of topical cidofovir in a 0.2 % concen-

conjunctival absorption of eyedrops may be im-

tration in the same preparation as described by

paired in patients with severe conjunctival

Gordon et al. [16] Topical cidofovir 0.2 % proved

chemosis and swelling of the conjunctival plica.

to be a well tolerated drug which did not cause

any discomfort but did not have a statistically

Viral Replication and Onset of Treatment. The

significant effect on the course of the acute or

New Zealand rabbit ocular model of adenovirus

the chronic phase of adenoviral keratoconjunc-

type 5 infection showed a duration of viral repli-

tivitis. In particular, the frequency of corneal in-

cation of 9 days with a peak on day 3. The symp-

filtrates at the end of the 21-day treatment peri-

toms of AKC after the phase of viral replication

od was not altered by cidofovir 0.2 % [16].

are thought to be caused by the host’s immune

There are several possible explanations for

response [9]. Treatment with cidofovir in the

the failure of cidofovir 0.2 % to show the clinical

rabbit model began 24 h after inoculation [29].

efficacy that may have been expected from the

The treatment of study patients began 1-7 days

antiviral activity demonstrated in vitro [8] and

(mean: 3.5 days) after onset of symptoms when

in the rabbit ocular model [11, 10].

they first presented to the clinic [16]. Cidofovir

may have been effective in the rabbit ocular

Concentration of Cidofovir. Cidofovir

0.2 %

model because treatment began early in the

was administered 4 times daily. Gordon and

course of the infection while treatment of our

Romanowski et al. showed that cidofovir 0.2 %

patients may have failed because treatment did

administered 4-5 times daily limited adenoviral

not start until the phase of viral replication had

replication in the adenovirus type 5/New Zea-

almost been completed.

land rabbit ocular model. Cidofovir 0.5 % and

The fivefold concentration of cidofovir test-

1 % administered only twice daily was not supe-

ed in our second pilot study did not alter the

rior, but equally effective [11].

course of the acute phase but appeared to be

168

Chapter 12

Treatment of Adenoviral Keratoconjunctivitis

Summary for the Clinician

∑ Topical cidofovir is the first antiviral agent

which effectively reduces the incidence

of corneal opacities

∑ Local toxicity rules out the clinical applica-

tion of topical cidofovir in an antivirally

effective concentration

Fig. 12.7. Local side effects of topical cidofovir 1 %:

conjunctivitis and erythematous inflammation of the

12.3.2

eyelids

Treatment of the Chronic Phase

12.3.2.1

Topical Steroids

effective in the prevention of severe corneal

The pathogenesis of the nummular corneal

opacities [17]. So far, only topical immunosup-

opacities of the chronic phase most likely in-

pressants such as steroids [39] or cyclosporin A

cludes a persisting viral replication in sub-

[28] have been shown to be effective in the treat-

epithelial keratocytes triggering an immunolog-

ment of existing corneal opacities. These agents

ical host reaction. Topical steroids suppress the

probably act by suppressing the immunological

host reaction and thus lead to a quick disappear-

response directed against viral antigens which

ance of the opacities. Unfortunately, opacities al-

persist in the cornea. By contrast, as an antiviral

most invariably reappear when topical steroids

agent, cidofovir treats the underlying cause of

are discontinued. The opacities frequently recur

corneal opacities. As opposed to a treatment

probably because steroids effectively suppress

with steroids, or less so with topical cyclosporin

the immunological host reaction but lack a con-

A [28], it could therefore possibly prevent their

comitant antiviral effect [40, 42]. Suppressing

occurrence, thereby avoiding the problem of re-

the immunological host reaction may therefore

currences after discontinuation of treatment

even enhance and prolong viral persistence

[39]. Unfortunately, local toxicity forbids the

within keratocytes. Even prolonged tapering of

clinical application of cidofovir in the 1 % con-

topical steroids failed to prevent recurrences of

centration. We observed an increased preva-

corneal opacities, even years after the initial

lence of conjunctival pseudomembranes as well

acute phase of the disease [39]. For this reason

as conjunctivitis and erythematous inflamma-

and because of unwanted side effects of pro-

tion of the skin of the eyelids. These changes

longed use of topical steroids such as cataract

subsided completely after

7-28 days

(mean

formation and secondary glaucoma, steroid

13.7 days) with topical lubrication and dexpan-

treatment for the chronic phase of AKC cannot

thenol ointment applied to the affected skin

be recommended. There are no controlled clini-

areas [17] (Fig. 12.7). Others have additionally

cal studies documenting the natural course of

described lacrimal blockade following the ap-

corneal opacities in AKC, but in the pre-steroid

plication of topical cidofovir [34]. These results

era Thygeson reported that corneal opacities in

are disappointing, however, the study demon-

AKC invariably spontaneously disappear within

strated for the first time the principal efficacy of

1 year. This is in accordance with our own clini-

a topical antiviral agent as a treatment of AKC.

cal experience. Persistence of opacities without

This positive aspect of the study encourages

treatment for more than 1 year is a rarity. Also

further research for an effective yet tolerable

rarely, persistent, scarred corneal opacities cause

antiviral agent.

irregular astigmatism which can be effectively

corrected with hard contact lenses. Penetrating

keratoplasty is usually not required [40].

12.3

Treatment

169

Summary for the Clinician

12.3.3

∑ Corneal opacities almost always recur after

Prophylaxis

discontinuation of topical steroids

∑ Even careful and prolonged tapering

12.3.3.1

of topical steroids failed to prevent the

Topical Interferon

recurrence of corneal opacities

∑ Corneal opacities mostly resolve sponta-

Interferons have multiple immunomodulative

neously within 1 year

effects and therefore their effect on the course of

viral infections is difficult to predict [40]. Treat-

12.3.2.2

ment of the acute phase of AKC with topical in-

Topical Cyclosporin A

terferon has been shown not to be effective [26,

30, 31, 38, 41, 47]. However, the consequent appli-

Cyclosporin A (CsA) is a well-established im-

cation of one drop per day of topical interferon

munosuppressant which has been used in the

(Berofor®) to all unaffected hospital staff and

prevention of transplant rejection for 25 years

inpatients in a nosocomial viral epidemic kera-

[3]. Topical CsA was used effectively in the treat-

toconjunctivitis which was recurrent in spite

ment of Mooren’s ulcer [48], vernal keratocon-

of hygienic prophylactic measures seemed to

junctivitis

[2], ulcerative keratitis associated

effectively prevent further spread of the infec-

with rheumatoid arthritis [24], anterior uveitis

tion [35]. In view of the epidemic character of

[18], and Thygeson’s punctate keratitis [27]. Side

AKC, prophylactic topical interferon seems to

effects of topical CsA have not been described.

be an effective measure to protect yet unaffect-

CsA may also have some antiviral potency as

ed individuals. However, topical interferon is

CsA has been shown to inhibit herpes simplex

currently not commercially available. Berofor®

virus in vitro [45]. In a non-controlled study

has been removed from the market due to

topical CsA

2 % led to a disappearance of

unsettled patent issues.

corneal opacities in two-thirds of 56 treated pa-

Summary for the Clinician

tients with persisting opacity [28]. The opacities

slowly responded to this treatment over several

∑ Interferons have multiple immunomodula-

weeks, a response significantly slower than the

tive properties

rapid response to topical steroids [28]. Topical

∑ Topical interferon might be effective as a

CsA was well tolerated, and only 7 % of all treat-

prophylaxis of infection

ed patients discontinued the medication be-

cause of a local burning sensation. After slow

12.3.3.2

tapering of topical CsA, one-third of the initial

Infection Control and Hygienic Measures

responders suffered a recurrence of the opaci-

ties. In all of these patients the opacities could

It has been shown that infection control pro-

be effectively abolished with another course of

grams including specified methods of patient

topical CsA on a low maintenance level of 1-2

screening and isolation, handwashing, instru-

drops/day [28].

ment disinfection, medication distribution, and

furlough of infected employees are associated

Summary for the Clinician

with decreased rates of nosocomial AKC out-

∑ Topical CsA appears to be effective in the

breaks and outbreak morbidity in a large teach-

treatment of persistent corneal opacities

ing eye institute [14]. A number of precautions

∑ So far,no side effects of topical CsA have

should be observed to ensure safety in any oph-

been described

thalmologist’s office. Paramount among these is

hand washing, both immediately after contact

with patient’s eyes and again between patients

[4]. Highly concentrated alcohol-based hand

rubs and hand gels have been demonstrated to

170

Chapter 12

Treatment of Adenoviral Keratoconjunctivitis

inactivate adenovirus within 2 min [21], povi-

tion of NMSO3 10 % also effectively inhibited

done-iodine, peracetic acid, and formaldehyde

viral replication in the established Ad5/NZW

have also demonstrated antiviral activity as dis-

rabbit ocular model although to a lesser degree

infectant agents against adenovirus although

than cidofovir [34]. Further dosage and toxicity

the resistance of individual serotypes varies and

studies of NMSO3 are required before this agent

the genomes of adenoviruses showed consider-

can be tested in humans.

ably more chemical resistance than the com-

plete viral particle [36].

Careful disinfection of contact tonometry

12.5

devices between patients is important. Further-

Current Clinical Practice

more, contact lens fitting is a typical procedure

and Recommendations

carrying the risk of transmitting adenovirus

[4, 21]. A recent study investigated the effect of

Until treatment with an adequate antiviral agent

chemical, hydrogen peroxide, and heat steriliza-

has been established the following therapeutic

tion systems on contaminated hard and soft

strategies can be recommended:

contact lenses. Only heat sterilization was effec-

∑ The acute phase of AKC may be treated with

tive. As heat sterilization is not readily available,

copious topical lubrication alone to alleviate

it may be prudent for patients with AKC to dis-

distressful local symptoms.

pose of unclean contact lenses [21].

∑ Topical cyclosporin A may be applied in pa-

tients with functionally relevant corneal

opacities that fail to spontaneously disappear

12.4

after months. This treatment must be ta-

Conclusion and Outlook

pered very slowly over several weeks to avoid

recurrences.

Following topical cidofovir’s failure in clinical

∑ Prophylaxis of infection of exposed, yet unaf-

development, the need for an antiviral to treat

fected individuals with topical interferon can

AKC persists.

be recommended.

It must be the aim of future work to investi-

∑ Topical steroids should be avoided in both

gate the therapeutic properties of an effective

the acute and the chronic phase of the dis-

yet non-toxic topical antiviral agent for the

ease.

treatment of all non-herpetic viral infections

∑ Adequate infection control measures should

of the ocular surface. The acute phase of AKC

be followed as prevention and to reduce epi-

calls for an antiviral monotherapy whereas the

demic AKC outbreaks.

chronic phase with corneal opacities may re-

quire supplementation with an immunosup-

pressant such as topical cyclosporin A.

References

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(2000) The efficacy of topical cidofovir treatment

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thalmol Vis Sci 41:ARVO Abstract, S59

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BenEzra D, Pe’er J, Brodsky M, Cohen E (1986)

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Recently, NMSO3, a sulfated sialyl lipid,

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9:S41-43

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Holland EJ, Olsen TW, Ketcham JM et al. (1993)

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In Vivo Micromorphology of the Cornea:

Confocal Microscopy Principles and Clinical Applications

Rudolf F. Guthoff, Joachim Stave

Core Messages

13.1

∑ In vivo confocal microscopy permits cell

Introduction

density quantification for all corneal

subpopulations

With good reason, biomicroscopy in ophthal-

∑ Tear film dynamics can be studied using

mology is dominated by slit-lamp examina-

non-contact confocal biomicroscopy,

tions. The foundations laid by Gullstrand were

and the generation of dry spots can be

outlined in exemplary fashion in Vogt’s Lehr-

evaluated

buch und Atlas der Spaltlampenmikroskopie des

∑ Corneal epithelial thickness can be

lebenden Auges [Textbook and Atlas of Slit-

measured with high precision

Lamp Microscopy of the Living Eye], first pub-

∑ Dendritic cells can be displayed and

lished in 1921 [19]. The second (1930) edition of

quantified

that standard textbook placed particular em-

∑ The influence of contact lenses on epithe-

phasis on information yielded by focal illumi-

lial cell densities can be evaluated

nation of scatters, described as Hornhautkör-

∑ Inflammatory cells associated with bacteri-

perchen (corpusculi corneae) [78].However,the

al and viral infections of the cornea can be

maximum magnification achievable with this

displayed and quantified

technique - approx. ¥40 - limited further subd-

∑ Follow-up after refractive corneal surgery

ifferentiation and did not reveal clinicomor-

permits exact measurement of flap thick-

phologic correlations at the cellular level.

ness and wound healing reactions in the

Specular microscopy was also first described

interface

by Vogt [78], but did not gain popularity until

∑ Functional imaging at the cellular level

photographic and video techniques allowed

using sodium fluorescein may be helpful

documentation and quantification of corneal

for understanding metabolic activities in

endothelial cells [5, 7, 8, 31, 41]. Although the

corneal epithelium

specular microscope is useful for the in vivo

∑ Corneal nerves can be displayed three-

examination of the cornea, its applications are

dimensionally and perhaps quantified

restricted to the endothelial cell layer.

on the basis of nerve fiber density

In 1968, the same year that Maurice de-

scribed the first high-powered specular micro-

scope [41], the first scanning confocal micro-

scope was proposed

[54]. This device was

characterized by high z-axis resolution and pro-

vided high-resolution microscopic images of

cells within living tissues of patients without the

need for fixation or staining.

174

Chapter 13

In Vivo Micromorphology of the Cornea: Confocal Microscopy Principles and Clinical Applications

Alongside this tandem scanning confocal

level of illumination. The precision of laser-

microscope, a slit-scanning confocal design has

scanning ophthalmoscopy in this context is

also been produced [35, 58, 64, 73]. Currently, the

based on the principle of the confocality of the

tandem scanning design is manufactured by the

examined object with the light source and the

Advanced Scanning Corporation (New Orleans,

detector plane. A laser light source is focused

LA) and the most recently developed version of

through a pinhole diaphragm to one point on

the slit-scanning design is produced by Nidek

the object. The reflected laser light is separated

Technologies Srl (Vigonza, Italy). The authors

by a beam splitter from the incident laser beam

have been accumulating experience with the

path and deflected through a second confocal

slit-scanning design since 1994 [58, 64] and have

diaphragm to reach a photosensitive detector.

attempted to modify the system in order to

Because of the confocal design,light originating

achieve reproducible and quantifiable results

from outside of the focal plane is highly sup-

[4].

pressed, and only the object layer located at the

focal plane contributes to the image. In order to

build up a two-dimensional image perpendicu-

13.2

lar to the optical axis of the device, the laser

Principle of In Vivo Confocal Microscopy

beam has to scan the sample point by point. This

Based on the Laser-Scanning Technique

is achieved by introducing two oscillating mir-

rors into the beam path. Figure 13.1 provides

The development of in vivo confocal laser-scan-

a schematic illustration of this principle. By

ning microscopy in the late 1980s permitted

moving the focal plane optically, an image can

precise three-dimensional (3-D) visualization of

be acquired from a deeper layer of the examined

microstructures of the ocular fundus in partic-

object, thus enabling a data cube to be built up

ular, with its optic nerve head and the peripap-

in a successive series.

illary retina. Modern digital image processing

By contrast, in slit-lamp biomicroscopy

technology enables quantitative data to be col-

examination of the cornea, an optical section

lected non-invasively, rapidly and with a low

that is essentially perpendicular to the corneal

Fig. 13.1. Principle of confocal laser-scanning microscopy

13.2

Principle of In Vivo Confocal Microscopy Based on the Laser-Scanning Technique

175

surface is seen in up to ¥50 magnification or,

with an additional lens for endothelial viewing

(specular microscopy), in up to ¥200 magnifi-

cation. Nowadays, documentation is generally

performed using digital photography. All other

cellular structures, e.g., the epithelium, cannot

be imaged with this technique because of the

high proportion of scattered light.

Optical tomography perpendicular to the in-

cident light path has only become possible with

the adaptation of confocal microscopy, as de-

scribed above, for the examination of the living

eye [6, 9, 10, 28, 43, 73, 74]. This yields images of

the endothelium, for example, that are com-

parable to those obtained with specular mi-

croscopy. In this case the most pronounced

source of scattered light is the cytosol of the

endothelial cells, with the result that the cell

borders appear dark. Only light reflected from

the focal plane contributes to the image. In this

way, cell structures in the stroma, nerves and

corneal epithelium [81] can also be imaged in

fine optical sections.

Fig. 13.2.

Confocal slit-scanning microscope

13.2.1

(ConfoScan 3/NIDEK)

Slit-Scanning Techniques

Techniques based on the principles of the rotat-

cal axis (z-scan) is accompanied by a loss of res-

ing NIPKOW disk or tandem slit-scanning were

olution. Loose optical coupling to the cornea us-

used initially for confocal microscopy of the

ing a gel (Fig. 13.4) limits the precision of depth

anterior segment of the eye. Figure 13.2 shows a

information relating to the optical section in the

slit-scanning microscope of this type incorpo-

cornea, and hence the 3-D reconstruction of cell

rating the use of a halogen lamp. For the pur-

structures [44, 53, 64]. Systematic errors such as

poses of corneal assessment these microscopes

inhomogeneous image illumination and image

can be used to image confocal sections with an

distortion also arise as a result of the electro-

optical layer thickness of approx.

5-10 mm.

mechanical slit-scanning technique used. Lin-

Synchronization of slit-scanning

[35,

73,

80]

ear movement along the z-axis during individ-

with the video rate of a residual light camera

ual image acquisition also produces distortion

yields sharp and motion-independent image

along the z-axis. These fundamental sources of

sequences at 25 frames/s [34, 36] (Fig. 13.3).

error can only be prevented by a rapid laser

Three-dimensional image distortion due to

scanning system unhampered by mass inertia

eye movements with this non-contact micro-

and incorporating a serial dot raster technique

scopy technique can only be minimized by

with stepwise advance of the confocal plane

faster image acquisition. However, more rapid

during the z-scan.

movement of the confocal plane along the opti-

176

Chapter 13

In Vivo Micromorphology of the Cornea: Confocal Microscopy Principles and Clinical Applications

Fig. 13.3 A-D. Slit-scanning microscopy: A superficial and B basal cells; C nerve plexus with keratocytes and

D endothelium

13.2.2

Laser-Scanning Microscopy

and Pachymetry

As an alternative to confocal slit-scanning mi-

croscopes, a confocal laser-scanning micro-

scope for the anterior segment of the eye was

developed at the Rostock Eye Clinic on the basis

of an already commercially available laser-scan-

ning system. Not least because of its compact

construction, the Heidelberg Retina Tomograph

(HRT II, Heidelberg Engineering GmbH,

Fig. 13.4. Gel coupling between lens and eye

Germany) was selected as the basic device for a

(ConfoScan/NIDEK)

digital confocal corneal laser-scanning micro-

scope.

In laser-scanning ophthalmoscopy of the

posterior segment, the optically refractive me-

dia of the eye forms part of the optical imaging

system. For anterior segment applications, a

high-quality microscope lens is positioned be-

tween the eye and the device, providing a laser

13.2

Principle of In Vivo Confocal Microscopy Based on the Laser-Scanning Technique

177

Fig. 13.5. Rostock Cornea Module (RCM)

Fig. 13.6. TOMOCAP contact cap

(confocal laser-scanning microscope)

(Rostock Cornea Module)

focus less than 1 mm in diameter. The result is a

tient and image data administration, also deliv-

high-resolution, high-speed, digital confocal

ering rapid access to, and hence comparison

laser-scanning microscope permitting in vivo

with, data from previous examinations.

investigation of the cornea (Fig. 13.5). Move-

The Heidelberg Retina Tomograph HRT II

ment of the confocal image plane inside the

has been modified with a lens system attach-

cornea can be achieved manually at the micro-

ment known as the Rostock Cornea Module

scope lens or by using the automatic internal

(RCM; J. Stave, utility model no. 296 19 361.5,

z-scan function of the HRT II. Laser-scanning

licensed to Heidelberg Engineering GmbH).

tomography is consequently possible in the

The module is combined with a manual z-axis

anterior segment of the eye.

drive to move the focal plane inside the cornea.

This technique permits rapid and reliable vi-

This enables a cell layer at any depth to be im-

sualization and evaluation of all the microstruc-

aged and, for example, selected as the starting

tures in the cornea, including the epithelium,

plane for the automated internal z-scan. During

nerves, and keratocytes, as well as the endothe-

the examination, pressure-free and centered

lium and bulbar conjunctiva. For the first time,

contact with the cornea can be monitored visu-

the dendritic (or Langerhans’) cells can now

ally using a color camera.

also be visualized in vivo with an image quality

The distance from the cornea to the micro-

that permits quantification [70, 83]. In principle,

scope is kept stable using a single-use contact

any body surface that can be reached by the lens

element in sterile packaging (TomoCap). Opti-

system is a suitable candidate for examination,

cal coupling is achieved via the tear film or by

with the result that potential applications also

applying protective gel to the eye (Fig. 13.6). The

exist outside ophthalmology

(e.g., the skin,

TomoCap is a thin cap with a planar contact sur-

tongue surface and oral mucosae).

face made from PMMA and is coupled optically

The original functions of the basic HRT II

to the lens with the aid of a gel.

device for evaluating the optic nerve head in

In the 3-D imaging mode, the distance be-

glaucoma are fully retained when the system is

tween two subsequent image planes is approxi-

modified into the confocal laser microscope.

mately 2 mm in the cornea. A 3-D image consists

Software adapted to the special requirements of

of 40 image planes, thus covering a depth range

scanning microscopy of the cornea has also

of 80 mm. The acquisition time for a volume im-

been developed. This permits the acquisition of

age is 6 s, and each individual section image is

individual section images, image sequences of

recorded in 0.024 s. In the image sequence ac-

section images, and volume images with inter-

quisition mode up to 100 images can be stored

nal z-scan over a distance of approx. 80 mm. The

with variable frame rates (1-30 frames/s). It thus

digital properties of the device offer good pa-

becomes possible to document dynamic pro-

178

Chapter 13

In Vivo Micromorphology of the Cornea: Confocal Microscopy Principles and Clinical Applications

Fig. 13.7. Rostock Cornea Module (RCM)

cesses in the tissue (e.g., blood flow in the scle-

flection coating appropriate for the laser wave-

ra).When the Rostock Cornea Module is used to

length.

set a plane manually at a desired depth, e.g., at

With the aid of an additional lens positioned

the LASIK interface after laser surgery to cor-

between the HRT II and the microscope lens, the

rect refraction, image series from this depth can

field of view of the scanning system (fixed at 15°

be acquired with almost 100 % image yield and

with the HRT II) is reduced to approx. 7.5° to

precise depth allocation [32, 45, 46, 77].

allow for the necessary magnification (Fig. 13.7).

With the HRT II, z-axis movement between

Depending on the microscope lens and addi-

images in the internal z-scan is performed - for

tional lens used, the size of the field of view in

the first time - in a stepwise manner, i.e., during

the contact technique can be 250 mm ¥ 250 mm,

acquisition of one section image the z-setting

400 mm ¥ 400 mm, or 500 mm ¥ 500 mm. Dry mi-

remains constant. This is a major advance and

croscope lenses can be used in non-contact, for

a prerequisite for generating distortion-free

example to image the tear film [66].

images of structure from one plane during

In particular, the compact construction of

the z-scan. A crucial prerequisite for undistort-

the HRT II simplifies its use as a confocal in vivo

3-D reconstructions has therefore been

microscope because the view is virtually unob-

achieved [26].

scured when monitoring the patient and bring-

A short focal length water immersion micro-

ing the microscope up to the cornea.

scope lens with a high numerical aperture was

The precise perpendicular positioning of the

used to achieve high magnification (Achroplan

cornea in front of the microscope within the mi-

63¥ W/NA 0.95/AA 2.00 mm, 670 nm, Carl Zeiss;

crometer range is facilitated by color camera

alternatively, LUMPLFL

60¥ W/NA 0.90/AA

control. By observing the laser reflex on the

2.00 mm, Olympus).

cornea, even when bringing the microscope to

To optimize image quality, the Zeiss micro-

the eye, it is possible to make a lateral or vertical

scope lens was customized with special anti-re-

correction so that contact with the cornea is

13.2

Principle of In Vivo Confocal Microscopy Based on the Laser-Scanning Technique

179

microscope and cornea. The precise movement

of the focal plane through the cornea with si-

multaneous digital recording of depth position

relative to the superficial cells of the epithelium

(at the corneal surface) thus makes exact

pachymetry possible.

13.2.3

Fundamentals of Image Formation

in In Vivo Confocal Microscopy

The laser light emitted constitutes an electro-

magnetic wave with a defined wavelength lamb-

da (l). This light wave is modified on passage

through the cornea. Part of it migrates un-

changed through all layers (transmission). At

interfaces with changing refraction indices

(n_D), the wave alters direction due to scatter and

refraction. Light scattering is the basis of image

formation in confocal microscopy where only

backscattered light is used for the image. The

amount of backscattered light is very small be-

cause scatter occurs predominantly in a forward

direction

[14]. Light-scattering interfaces are

found in the cornea, for example, at the junction

between cytoplasm or extracellular fluid with

n_D=1.35-1.38 and lipid-rich membranes with

n_D=1.47 in the form of cell borders, cell nucleus

membranes and mitochondrial membranes

[63] (Fig. 13.9). The amount of backscattered

light depends on the structure of the interface

surfaces. Rough surfaces scatter light in a broad-

ly diffuse pattern, whereas directed waves with

narrow scatter cones are formed on smooth

structures. In addition, the confocal image is in-

fluenced both by the number as well as the size

and orientation of scattering organelles or par-

ticles. Objects whose diameters are of the same

order of magnitude as the wavelength of the

Fig. 13.8 A-C. Camera control: laser reflexes on the

laser light display Mie scatter, and very much

cornea - producing the immersion gel bridge be-

smaller molecules display Rayleigh scatter,

tween the objective lens/cap and the cornea

which is directed backwards to a greater extent

than Mie scatter. Different orientations of elon-

gated cell organelles can place different particle

exactly in the optical axis (Fig. 13.8). As a result,

cross-sections in the path of the incident light

images are captured only of cell structures that

beam, the result being that the light is backscat-

are in a plane parallel to the surface, i.e., trans-

tered to varying degrees. A high proportion of

verse sectional images are acquired. The contact

cell organelles also increases the amount of

technique guarantees a fixed distance between

backscattered light [14].

180

Chapter 13

In Vivo Micromorphology of the Cornea: Confocal Microscopy Principles and Clinical Applications

Fig. 13.9. The confocal image of one section (x, y) is

produced by the sum of the backscattered light inten-

sities (I_R) from the focal depth range (z) (I_V forward

scatter, I_T transmission)

13.3

General Anatomical Considerations

Fig. 13.10. Schematic illustration of the corneal epi-

thelium and upper corneal stroma

The corneal epithelium consists of five to six lay-

ers of nucleated cells that can be subdivided

functionally and morphologically into three

amorphous on light microscopy. Its location on

zones:

in vivo confocal microscopy is well defined by

∑ Superficial cells: approx. 50 mm frontal diam-

the subepithelial plexus (SEP).

eter and approx. 5 mm thick. About 1/7 of

The stroma accounts for some 90 % of total

these cells is lost by desquamation within

corneal volume. Ninety-five percent of the stro-

24h. Before detachment the cytoplasm and

ma consists of amorphous ground substance

nucleus undergo a change in their optical

(glycoproteins, glycosaminoglycans: keratan

characteristics.

sulfate and chondroitin sulfate) and collagen

∑ Intermediate cells:

50 mm diameter and

fibers. The remaining 5 % of stromal volume is

10 mm thick. These cells form a contiguous

accounted for by cellular structures known as

polygonal, wing-shaped pattern (wing cells).

keratocytes, which are specialized fibroblasts.

∑ Columnar basal cells have a flat basal surface,

Besides the nerves, their irregularly shaped

adjacent to Bowman’s membrane, a frontal

nuclei are the only well-defined sources of scat-

height of approx. 20 mm and a frontal diame-

tered light in corneal stroma detected on confo-

ter of 8-10 mm. Like endothelial cells, they

cal microscopy. Their widely branching cyto-

can be quantified accurately because of their

plasmic extensions are not visible in the healthy

defined location in relation to the basement

cornea (Fig. 13.11).

membrane (Fig. 13.10).

The cornea is the most densely innervated

tissue in the human body. It is supplied by the

Bowman’s membrane - which is clearly distinct

terminal branches of the ophthalmic nerve in

histologically from the epithelial basement

the form of 30-60 non-myelinated ciliary nerves

membrane - is 10-16 mm thick and remains

(Fig. 13.12).

13.4

In Vivo Confocal Laser-Scanning Microscopy

181

In the limbus region these are seen as

whitish, filigree-like structures; their complex

stromal and epithelial branchings are not visi-

ble by slit-lamp microscopy, but are relatively

clear on confocal microscopy.

Like Bowman’s membrane, Descemet’s mem-

brane, which should be regarded as the base-

ment membrane of the endothelium, remains

amorphous on light microscopy. It is 6-10 mm

thick. On confocal microscopy it is defined opti-

cally by the easily identifiable endothelial cells.

The endothelium consists of about 500,000

hexagonal cells approx. 20 mm in diameter, 5 mm

thick and with large, flattened, central nuclei.

The high concentration of cell organelles is in-

dicative of very intensive metabolic activity.

13.4

In Vivo Confocal Laser-Scanning

Microscopy

Fig. 13.11. Schematic illustration of the layered

The layered structure of the epithelium of the

structure of the human cornea. The differently

eye can be visualized with high contrast using

shaped keratocyte nuclei can be distinguished on in

the Rostock Cornea Module (RCM) attachment

vivo confocal microscopy (adapted from Krstiè)

with the HRT II and, because of the good quali-

ty of depth resolution, can be imaged in optical

sections a few micrometers thick. The same is

true for the subepithelial plexus,the entire stro-

ma including the keratocytes, and the endothe-

lial fine structure (Fig. 13.13).

Fig. 13.12. Schematic illustra-

tion of the subepithelial plexus

(SEP) and its branches in the

corneal epithelial layers (BEP

basal epithelial plexus)

182

Chapter 13

In Vivo Micromorphology of the Cornea: Confocal Microscopy Principles and Clinical Applications

Fig. 13.13. A Superficial cells; B wing cells; C basal cells;

D nerve plexus; E keratocytes/anterior stroma;

F keratocytes/posterior stroma; G endothelium

The water content of the cornea is regulated by

13.4.1

evaporation and the resultant osmotic gradient.

Confocal Laser-Scanning Imaging

The oxygen in the air is dissolved in the tear flu-

of Normal Structures

id and thus supports the aerobic metabolism of

the epithelium.

13.4.1.1

The tear film is 7-10 mm thick and is charac-

Tear Film

terized by a three-layered structure. The ex-

ternal lipid layer, which is produced chiefly by

The pre-ocular tear film with its complex fluid

the meibomian glands close to the margin

structure bathes the cornea and conjunctiva.

of the eyelids, prevents rapid evaporation of

Tear film structure and function are maintained

the aqueous layer and renders the surface

by a highly differentiated system of secretory,

hydrophobic. The inner mucin layer consists of

distributive and excretory interactions [49, 60].

glycoproteins. Its task is to make the epithelial

In particular, these function to smooth the

surface hydrophobic and thus to guarantee

corneal surface and maintain its optical clarity.

wettability.

13.4

In Vivo Confocal Laser-Scanning Microscopy

183

Replacing the contact system in the confocal

13.4.1.2

laser-scanning microscope with a dry objective

Epithelial Layer

lens (Fig. 13.14) enables the fine structure of the

tear film to be imaged (Fig. 13.15). The rapid

13.4.1.2.1

imaging sequence in the device also permits

Superficial Cells

dynamic processes to be recorded [39, 65, 75].

(up to approx. 50 µm in Diameter)

In the case of the most superficial epithelial

cells, bright cell borders and a dark cell nucleus

and cytoplasm are readily visualized on con-

focal laser-scanning microscopy. The cells

characteristically display a polygonal - often

hexagonal - shape. Cells undergoing desquama-

tion are characterized by a highly reflective

cytoplasm, in the center of which the brightly

appearing (pyknic) cell nucleus with its dark

perinuclear space is clearly visible (Fig. 13.16).

The average density of superficial cells in the

central and peripheral cornea is approx. 850

cells/mm².

Fig. 13.14. Non-contact microscopy: laser reflex on

the cornea

Fig. 13.16. Superficial cells: the cytoplasm and cell

nuclei are visualized; cells in the process of desqua-

mation possess a highly reflective cytoplasm, in the

center of which the bright (pyknic) cell nucleus with

Fig. 13.15. Normal tear film

its dark perinuclear space is clearly visible (z = 50 mm)

184

Chapter 13

In Vivo Micromorphology of the Cornea: Confocal Microscopy Principles and Clinical Applications

Fig. 13.17. Intermediate cells: the cells of the interme-

Fig. 13.18. Basal cells: these are regularly arranged

diate layers are characterized by bright cell borders

cells with bright borders, but the cell nucleus is not

and a dark cytoplasm. The cell nucleus can be identi-

visualized. Intercellular comparison reveals inhomo-

fied only with difficulty. The wing cells display only

geneous cytoplasmic reflectivity

minimal variation in terms of size and appearance

13.4.1.2.2

ratio between superficial cells, intermediate

Intermediate Cells/Wing Cells

cells and basal cells is 1:5:10.

(up to Approx. 20 µm in Diameter)

13.4.1.3

The cells of the intermediate layers are charac-

Langerhans’ Cells

terized by bright cell borders and a dark cyto-

plasm. The cell nucleus can be distinguished

Confocal microscopy permits in vivo evaluation

only with difficulty. In terms of size and appear-

of Langerhans’ cells (LCs) within the human

ance, wing cells in healthy subjects exhibit only

cornea, with a particular emphasis on cell mor-

minimal variation (Fig. 13.17). The average cell

phology and cell distribution.

density is approx. 5,000 cells/mm² in the central

LCs present as bright corpuscular particles

cornea and approx. 5,500 cells/mm² in the pe-

with dendritic cell

(DC) morphology and a

riphery.

diameter of up to 15 mm. LC distribution follows

a gradient from low numbers in the center to

13.4.1.2.3

higher cell densities in the periphery of the

Basal Cells

cornea. Moreover, in vivo confocal microscopy

(up to Approx. 10 µm in Diameter)

permits differentiation of LC bodies lacking

dendrites, LCs with small dendritic processes

The basal cells are located immediately above

forming a local network, and LCs forming a wire

Bowman’s membrane. They present as brightly

net via long interdigitating dendrites (Fig. 13.19).

bordered cells in which the cell nucleus is not

While almost all the cells located in the periph-

visible. Between-cell comparison reveals inho-

ery of the cornea demonstrate long processes in-

mogeneous reflectivity of the cytoplasm. Like

terdigitating with the corneal epithelium, those

the wing cells above them, the basal cells display

in the center of the cornea often lack dendrites,

only minimal variation in shape and size

most probably underlining their immature phe-

(Fig. 13.18). The average cell density is approx.

notype [21]. Immature LCs are equipped to cap-

9,000 cells/mm² in the center of the cornea and

ture antigens, while mature forms are able to

10,000 cells/mm² in the periphery. In normal

sensitize naive T-cells through MHC molecules

subjects, therefore, in terms of cell densities, the

and secretion of interleukin-12 as well as costim-

13.4

In Vivo Confocal Laser-Scanning Microscopy

185

Fig. 13.19. In vivo confocal microscopic images, representing different forms of Langerhans’ cells: A individ-

ual cell bodies without processes; B cells bearing dendrites; C cells arranged in a network via long interdigitat-

ing dendrites

ulatory molecules, and thus represent an integral

In vivo visualization of these nerve struc-

part of the immune system [3].

tures is possible by confocal corneal micro-

The average density of LCs in normal sub-

scopy.

jects is 34±3 cells/mm² (range: 0-64 cells/mm²)

The cornea is innervated primarily by senso-

in the central cornea and

98±8 cells/mm²

ry fibers arising from the ophthalmic nerve, a

(range: 0-208 cells/mm²) in the periphery [83].

side branch of the trigeminal nerve. Human

In contact lens wearers, LC density varies from

corneal nerves are non-myelinated and vary in

60±16 cells/mm² (range: 0-600 cells/mm²) in

thickness between 0.2 and 10 mm.

the central cornea to 159±18 cells/mm² (range:

The nerve fiber bundles, which enter the an-

0-700 cells/mm²) in the periphery. LC densities

terior and central stroma in the corneal periph-

differ significantly between healthy volunteers

ery, run parallel to the corneal surface in a radi-

and contact lens wearers both in the central

al pattern before making an abrupt 90° turn in

(p=0.03) and in the peripheral cornea

the direction of Bowman’s membrane [47]. On

(p=0.001), while the gradient of LC density from

confocal corneal microscopy these nerve fibers

periphery to center was almost identical in both

mostly present as thick, almost always stretched,

groups (unpublished data).

highly reflective structures

(Fig. 13.20). Fre-

It has been suggested that LCs participate in

quently, the stromal nerves are found in close

immune and inflammatory responses, thereby

proximity to keratocytes. The deep stroma is

determining cell-mediated immunity. In light of

devoid of nerves that can be visualized on con-

this theory, the present data on LCs in the hu-

focal microscopy.

man cornea provide a helpful basis for further

In the anterior stroma, immediately before

investigations in ocular pathology.

Bowman’s membrane, the nerve fiber bundles

display three different patterns. Some of the

13.4.1.4

nerve fibers ramify before reaching Bowman’s

Corneal Nerves

membrane without penetrating it and form the

subepithelial plexus [51] (see schematic illustra-

The cornea is one of the most sensitive struc-

tion in Fig. 13.12). Other nerves penetrate Bow-

tures in the human body, and even the most

man’s membrane either directly following a

minimal contact provokes the lid reflex to pro-

perpendicular or slightly oblique course, or just

tect the eye. This sensitivity is attributable to the

before penetration they ramify into several fine

large numbers of nerve fibers that pass through

branchlets. After they have penetrated Bow-

the cornea. Furthermore, the corneal nerves

man’s membrane, they again make a 90° direc-

exert an influence on the regulation of epithelial

tional change and pass between the basal cell

integrity and on wound healing.

layer of the epithelium and Bowman’s mem-

186

Chapter 13

In Vivo Micromorphology of the Cornea: Confocal Microscopy Principles and Clinical Applications

brane toward the corneal center and form the

basal epithelial plexus (see schematic illustra-

tion in Fig. 13.12). In so doing, they give off many

small side branchlets directed both toward the

corneal surface, where they end freely, and to-

ward the center [47, 48]. The nerve fibers of the

basal epithelial plexus mostly run parallel to

each other and often form Y- or T-shaped

branches. Their predominantly granular,“string

of pearl” structure is characteristic; more rarely

they display a smooth surface. Unlike the stro-

mal nerves, they are characterized by lesser re-

flectivity and frequently follow a meandering

path (Fig. 13.21). Occasionally, a thicker nerve

fiber bundle will divide into two finer nerve

fibers, before these then reunite after a short

distance into a single nerve fiber with the same

Fig. 13.20. Nerve fibers (arrowed) in the anterior

thickness as before

(Fig. 13.21 A). The finer

corneal stroma. The stretched pattern of the stromal

branchlets also form connections between larg-

nerves is characteristic. The keratocyte nuclei are

er nerve fibers (Fig. 13.21 A, B).

identifiable as hyperreflective oval structures, some

of which are in close proximity to the nerve (star)

Fig. 13.21 A, B. Highly reflective nerves from the

granular “string of pearl” appearance. In most cases

basal epithelial plexus located between Bowman’s

they run parallel to each other and show T (circle)-

membrane and the basal cell layer of the corneal ep-

and Y (star)-shaped nerve branchings that may pro-

ithelium. The nerve fibers have their characteristic

duce connections between larger nerve fibers

13.4

In Vivo Confocal Laser-Scanning Microscopy

187

Fig. 13.22. Subepithelial nerve

Fig. 13.23. Anterior stroma: in corneal stroma only

the keratocyte nuclei are visualized; the density of the

cell nuclei is highest of all in the anterior stroma (see

Fig. 13.24); the size of the cell nuclei shown is approx.

15 mm

13.4.1.5

Bowman’s Membrane

The anterior limiting membrane has an amor-

phous appearance. Its location can be estab-

lished from the nerves of the basal epithelial

plexus, which ramify there (Fig. 13.22).

13.4.1.6

Stroma

Apart from neural structures, only the highly

reflective, sharply demarcated cell nuclei of the

keratocytes are visualized on examination of

the stroma. The cytoplasm of this fibroblast

subpopulation and the collagen fibers produced

by them are not visible. Keratocyte nucleus den-

sity is higher in the anterior stroma close to

Bowman’s membrane than in the central and

deep stroma

(Figs. 13.23,

13.24). Keratocyte

density is highest in the anterior stroma, clearly

declines toward the central stroma, and increas-

Fig. 13.24. Central stroma: clearly demarcated,

es again slightly in the region immediately

highly reflective, oval-shaped nuclei of keratocytes in

the central stroma; here cell nucleus density is the

before Descemet’s membrane.

lowest in corneal stroma

188

Chapter 13

In Vivo Micromorphology of the Cornea: Confocal Microscopy Principles and Clinical Applications

13.4.1.9

Limbal Region

Because it forms a junctional zone with the con-

junctiva, the limbal region is especially impor-

tant. Inflammatory cells migrate across it into

the cornea in immunological disease, it is the

source of new inbudding corneal vessels and,

not least, it also plays an important role in

corneal regeneration as the site of origin of

corneal stem cells.

The limbal region is where the corneal

epithelium forms a junction with the conjuncti-

val epithelium which comprises approx. 10-12

cell layers. This region also contains a radial

arrangement of trabecular conjunctival pro-

cesses (the limbal palisades of Vogt) that are

Fig. 13.25. Endothelium: a monolayer of regularly

considered to be the site of origin of corneal

arranged hexagonal cells completely covering the

stem cells [12, 61]. Overall, the organization of

posterior surface of the cornea. Unlike the basal cells

the conjunctival epithelium is less uniform be-

(see Fig. 13.18), these cells have a brightly reflecting

cytoplasm and dark cell borders. The cell nucleus is

cause different epithelial cell types (e.g., goblet

not visible

cells) occur here and the arrangement of the in-

dividual cell layers is also not so strictly parallel

with the surface [13].

On confocal microscopy, the epithelial cells

of the conjunctiva, unlike those of the cornea,

are more reflective, smaller and less well demar-

cated. Their nucleus is relatively large and

13.4.1.7

bright. The junctional zone is characterized by

Descemet’s Membrane

inhomogeneous reflectivity and marked varia-

tion in cell shape and size (Fig. 13.26). The lim-

Like Bowman’s membrane, Descemet’s mem-

bal palisades of Vogt can often be visualized as

brane has an amorphous appearance and is

parallel trabecular extensions of the conjunc-

therefore not visualized in healthy subjects.

tival epithelium (Fig. 13.27). In the immediate

junctional zone the conjunctival epithelium

13.4.1.8

also commonly exhibits tongue-like extensions

Endothelial Cells

which are mostly well demarcated, especially in

the deeper layers, and at the end of which are

The endothelium consists of a regular pattern of

located isolated cells or cell groups with very

hexagonal reflective cells. The cell nuclei cannot

bright cell borders and a bright cytoplasm

usually be visualized. The cell borders reflect

(Fig. 13.28). These may be secretory cells. Sub-

less light than the cytoplasm, with the result that

epithelially, in the region of the conjunctiva

a network of dark cell borders appears between

close to the limbus, are the blood vessels of the

areas of bright cytoplasm. Endothelial cell den-

limbal vascular plexus, in the lumen of which

sity can be determined by counting (Fig. 13.25).

flowing blood cells can be seen (Fig. 13.29).

190

Chapter 13

In Vivo Micromorphology of the Cornea: Confocal Microscopy Principles and Clinical Applications

The Heidelberg Retina Angiograph (HRA) in

combination with the Rostock Cornea Module

can be used for confocal laser-scanning fluores-

cence microscopy of the microstructure of the

corneal epithelium and tear film using contact

and non-contact techniques (Fig. 13.31) with a

lateral resolution of 1 mm and up to ¥1,000 mag-

nification. The red-free reflection and fluores-

cence images display the intercellular mi-

crostructure with stained cell nuclei and altered

cell surfaces and borders. The same area exam-

ined on the cornea can be visualized simultane-

ously in reflection and fluorescence mode. The

penetration profile of NaF can be measured

with precise depth resolution over a prolonged

time using the contact technique. Autofluores-

cence measurements are also possible. Fig-

ure 13.32 shows the tear film (a) and epithelium

Fig. 13.30. Tear film/dry spots

(b) in fluorescence mode.

13.5.2

13.5

Meesmann’s Dystrophy

Clinical Findings

Meesmann’s dystrophy is a rare, bilaterally sym-

13.5.1

metrical epithelial condition inherited as an

Dry Eye

autosomal dominant trait and attributable to

mutations in the keratin 3 (K3) gene [11] or

Disturbances of tear film secretion or tear film

keratin 12 (K12) gene [82] on chromosome 12

structure give rise to a condition known as dry

[27] or chromosome 17 [71]. Due to the rupture

eye. On microscopy such disturbances are evi-

of mainly interpalpebral epithelial cysts, the

dent as altered reflection or dry spots on the

condition causes episodic pain, photophobia,

epithelium (Fig. 13.30).

epiphora, blepharospasm and fluctuating visual

As the most important component of the

acuity or a moderate decline in visual acuity,

corneal diffusion barrier, the corneal epitheli-

even in young children. No causal therapy

um displays differing permeability for aqueous

exists.

ionic substances such as sodium fluorescein

(NaF). Patients with diabetes, for example,

13.5.2.1

have significantly increased permeability for

Summary Evaluation

NaF. NaF also penetrates areas of micro-ero-

sions and pathologically altered cells. The liter-

Clinically, Meesmann’s dystrophy is character-

ature reveals discrepant views concerning the

ized bilaterally by small cystic changes in

nature of the penetration process. Most authors

the corneal epithelium, particularly in the

subscribe to the view that the fluorescein fills

interpalpebral zone, and individual superficial

the “footprint” spaces vacated by cells that have

punctate opacities. Histological assessment and

been lost. However, others assume that fluores-

electron microscopy reveal exclusively intra-

cein fills the intercellular space. At present only

epithelial cysts with cell debris

(clumped

confocal slit-scanning microscopes and fluo-

keratin) which migrate to the corneal surface

rophotometers are used to analyze this phe-

during normal epithelial regeneration and

nomenon.

rupture there (Fig. 13.33). In addition, there are

192

Chapter 13

In Vivo Micromorphology of the Cornea: Confocal Microscopy Principles and Clinical Applications

Fig. 13.33. A B.N., 12 years old: microcystic epithelial changes in the interpalpebral zone, isolated superficial

punctate opacities, otherwise normal corneal structures. B Retroillumination (cf. A)

Fig. 13.34. A B.N., 12 years old: confocal microscopy

sections (see Fig. 13.35). B B.N., 12 years old: confocal

of the epithelium in vicinity of the superficial cells

microscopy of the epithelium at a depth of 30 mm with

(depth: 5 mm) showing cystic structures with spheri-

increased visualization of spherical highly reflective

cal, highly reflective contents similar to the histologic and cystic structures

13.5.3

Epithelium in Contact Lens Wearers

Distinct changes in corneal morphology,

pachymetry and structure in contact lens wear-

ers can be demonstrated by in vivo confocal

laser scanning microscopy. These findings are

best interpreted as resulting from mechanical

or metabolic disturbances of the cornea.

All cell layers (superficial, intermediate and

Fig. 13.35. Histologic findings in Meesmann’s dys-

basal cells) are present and characterized by

trophy (after Naumann: Pathologie des Auges [49])

bright cell borders and uniformly dark cyto-

with intraepithelial cysts containing cellular debris

13.5

Clinical Findings

193

Fig. 13.36. Epithelium in contact lens wearers: A oblique corneal section: superficial, intermediate and basal

cells, Bowman’s membrane and anterior stroma; B superficial cells; C intermediate cells; D basal cells

plasm. The cell count increases with layer depth

Our data (unpublished results) show a sig-

due to a decrease in cell diameter. Bowman’s

nificant increase in superficial cell density

membrane and the subepithelial plexus display

(p<0.05) both centrally and peripherally.

border structures between epithelium and stro-

The intermediate cells do not show any

ma (Fig. 13.36 A).

morphological changes by comparison with

Superficial cells are characterized by a dark

findings in normal subjects: pale cell borders,

nucleus, and the cytoplasm is generally darker

invisible nucleus and dark cytoplasm were

than in the normal cornea. The polygonal struc-

detected in both the lower and upper wing

ture is retained, but cell bodies are generally

cells (Fig. 13.36 C). A significant reduction in the

smaller (30 mm in contact lens wearers and up to

cell count was noted only in the periphery

50 mm in the normal cornea) (Fig. 13.36 B).

(p<0.05).

194

Chapter 13

In Vivo Micromorphology of the Cornea: Confocal Microscopy Principles and Clinical Applications

Fig. 13.37. Micromorphologic changes in the

cornea of contact lens wearers: A corneal micro-

deposits; B signs of polymegathism, pleomorphism

and endothelium precipitates; C signs of poly-

megathism and pleomorphism

Basal cell structure is characterized by an in-

est impact. Corneal microdeposits in stroma

homogeneous cytoplasm and invisible nucleus;

(Fig. 13.37 A) and signs of polymegathism, pleo-

cell diameters are approx. 8-10 mm (Fig. 13.36 D).

morphism

(Fig. 13.37 B, C) and endothelium

A significant reduction in the cell count was also

precipitates (Fig. 13.37 B) are the most common

detected in the peripheral cornea (p<0.05).

findings.

Analysis of the pachymetry data revealed re-

As demonstrated in Fig. 13.38, alterations in

duced corneal thickness in the periphery com-

Langerhans’ cells also occur due to contact lens

pared to that in normal volunteers, especially in

wearing.

patients who had worn contact lenses for longer

In light of this, in investigations of the cornea

than 10 years. There were no age-related changes

in contact lens wearers, attention must focus on

in cell count or epithelial thickness, but stromal

the cell density of each layer and on the thick-

thickness was reduced.

ness of the corneal epithelium, and results must

The type of contact lens (hard vs. soft) has no

always be compared between the center and

influence on corneal morphology; duration of

periphery.

contact lens wear was the factor with the great-

13.5

Clinical Findings

195

Fig. 13.38. Langerhans’ cells and reflective keratocytes after contact lens wearing (3 years)

form the slit-lamp microscopic substrate of the

13.5.4

nummular lesions [57] (Fig. 13.39 A, B).

Epidemic Keratoconjunctivitis

Hyperreflective punctate structures can be

visualized in the intermediate layer of the epi-

Epidemic keratoconjunctivitis (EKC) is a highly

thelium on confocal Rostock laser-scanning mi-

contagious infection caused by type 8, 19, 37

croscopy (Fig. 13.40 A). These may be lympho-

adenoviruses; one of its chief complications is

cytes,histiocytes and/or fibroblasts [33].

the development of nummular areas of sub-

By contrast with physiologic findings, the

epithelial corneal opacity which, in exceptional

basal cell layer is hardly distinguishable as such.

cases, may lead to years of reduced visual acuity

In addition to a network of hyperreflective

and to increased glare sensitivity

[30,

69].

dendritic structures

(Fig. 13.40 B), which be-

Histopathologic examination reveals that the

comes clearly less dense with increasing depth

nummular lesions consist of an accumulation of

(Fig. 13.40 C, D), corpuscular changes with den-

cells from the monocyte-macrophage system,

dritic extensions are visualized (Fig. 13.40 C, D),

such as lymphocytes, histiocytes and fibro-

some of which appear to be spread out between

blasts [18].

the nerve fibers (Fig. 13.40 E). Considering their

Viral persistence in the keratocytes is sus-

location, size and shape, these are most proba-

pected as the cause for the continuing presence

bly the antigen-presenting Langerhans’ cells

of the nummular lesions. The immune response

[59], which are responsible for the induction of

induces focal infiltration of immune cells

cell-mediated delayed-type immune responses.

around the infected keratocytes. The complexes

They assume an important role in triggering

196

Chapter 13

In Vivo Micromorphology of the Cornea: Confocal Microscopy Principles and Clinical Applications

Fig. 13.39 A, B. Slit-lamp

microscopy photograph: right

eye of a 28-year-old female

patient on day 14 after the onset

of symptoms of epidemic kera-

toconjunctivitis, showing the

subepithelial nummular lesions

as fleecy-fused areas of opacity

with unclear margins: A slit-

lamp microscopy and B with

the “Pentacam” Scheimpflug

camera (Oculus)

Fig. 13.40 a-e. Confocal image of the central cornea

located between the cells; b basal cell layer with hy-

in epidemic keratoconjunctivitis; edge length of the

perreflective dendritic network; c, d transition from

image in vivo, 250 mm; focal planes moved axially from

basal cell layer to nerve plexus layer with dendritic

epithelium to endothelium. a Intermediate epithelial

cell structures, some of which appear to be spread out

layer with isolated hyperreflective round structures between the nerve fibers; e Bowman’s membrane

contact allergies, rejection reactions and viral

defense, and in the healthy cornea they are lo-

13.5.5

cated in the epithelial layers of the conjunctiva,

Acanthamoeba Keratitis

the limbus and peripheral cornea, but not in the

central cornea. Migration of the Langerhans’

Numerous free-living phagotrophic amoebae

cells into the central cornea may occur in re-

cause opportunistic infection in humans. Acan-

sponse to traumatic, chemical or inflammatory

thamoeba keratitis has been recognized as a

stimuli [1, 18].

potentially blinding disease, which is often only

The changes visible beneath the nerve fiber

diagnosed at a late stage. The condition is some-

layer are possibly scatter artifacts in the vicinity

times confused with other types of infectious

of the ruptured Bowman’s membrane [33].

keratitis, particularly those of fungal and her-

The superficial epithelial layer, stroma and

petic origin.

endothelium do not display any abnormalities.

13.5

Clinical Findings

197

Fig. 13.41. Slit-lamp photograph from a 42-year-old female patient with a unilateral red, painful eye:

A with epithelial defects, stromal ring infiltrate; B fluorescein staining positive; sensibility decreased. PCR

(herpes zoster) and corneal scrapings (pathological agents including Acanthamoeba) negative

Fig. 13.42 A, B. Corneal microcysts (cystic stage of life cycle, round in shape, up to 10 mm, double wall)

are visible at the level of the deep intermediate and basal cells (z = 32 mm) (A) and in the anterior stroma

(z = 93 mm) (B)

Although not widely available, the confocal

In vivo confocal microscopy permits identi-

microscope can be helpful in establishing the

fication of Acanthamoeba cysts in the cornea

diagnosis of Acanthamoeba keratitis, based on

[40, 2]. The identity of findings with those from

the visualization of pear-shaped cysts approx.

conventional ex vivo microscopy and PCR pro-

10 mm in length and irregular trophozoites [38,

vides a basis for simple and reliable in vivo

55] (Figs. 13.41-13.43).

diagnosis (Fig. 13.44).

13.5

Clinical Findings

199

superficial stromal region. Figure 13.45 is a slit-

13.5.6

lamp photograph and Fig. 13.46 shows confocal

Corneal Ulcer

microscopy. In vivo confocal microscopy of

corneal ulcers provides additional information

Little experience has been gained with confocal

about corneal healing processes, and permits

microscopy in unspecific corneal ulcers. Leuko-

evaluation of epithelialization and reinnerva-

cyte infiltration may be demonstrated in the

tion at the cellular level.

ulcer margins in both the epithelial and the

Fig. 13.46. A Oblique section of central cornea near

cytes; C distortion of basal cell layer and other struc-

the ulcer: regular epithelial structure, absence of

tures of the subepithelial plexus; D anterior stroma in

subepithelial plexus, and distortion of anterior stro-

ulcer area: keratocytes or cell nuclei are not visible,

ma; B at the level of deeper intermediate and basal

severe destruction of stromal structure

cells: bright cellular structures, most probably leuko-

200

Chapter 13

In Vivo Micromorphology of the Cornea: Confocal Microscopy Principles and Clinical Applications

13.5.7

Refractive Corneal Surgery

The different methods of refractive corneal sur-

gery are designed to reduce ametropia, where

present. Depending on the technique used, re-

fractive corneal surgery may result in morpho-

logic changes and sometimes also in irritation

and complications in the vicinity of the corneal

epithelium or stroma that may lead to sub-

jective disorders

[56,

50]. The morphology

and mechanism of wound healing processes

following refractive corneal surgery are there-

Fig. 13.47. Slit-lamp photograph from a 25-year-old

fore of particular interest in this context [25, 15]

male patient 1 year after laser in-situ keratomileusis

(Figs. 13.47-13.49).

(LASIK): uncorrected visual acuity 20/20, normal

In vivo confocal microscopy of the cornea af-

corneal morphology apart from a small circular stro-

ter refractive surgery yields information about

mal scar, representing the border of the former flap

zone (arrow)

the functional status of the keratocytes and the

reinnervation of stroma and epithelium [37]. It

is possible to define the precise depth location

for corneal opacities and to measure changes in

corneal thickness

[45]. Even years later, the

depth of the interface zone following laser in-

situ keratomileusis (LASIK) can be identified

on the basis of the morphologic changes visible

there.

Fig. 13.48 A, B. Confocal images from the same patient: A level of the interface zone with diffuse hyperreflec-

tion and “microdot” structures; B region of the circular stromal scar with evidence of reinnervation of the flap

(arrow) (z = 170 mm (A); z = 85 mm (B))

13.6

Future Developments

201

Fig. 13.49. Epithelium and keratocytes after LASIK

the detailed, 3-D investigation of the human

13.6

cornea. The further development of the confo-

Future Developments

cal microscope [79] took the form of a modified

confocal laser-scanning ophthalmoscope [66]

13.6.1

based on a commercially available instrument

Three-Dimensional Confocal

(Heidelberg Retina Tomograph II, Heidelberg

Laser-Scanning Microscopy

Engineering GmbH, Germany) [62]. A water-

immersion microscope lens (Achroplan 63¥/

Many researchers have investigated the cornea

0.95W/AA 2.00 mm, Carl Zeiss, Germany) with

with in vivo confocal microscopy [4, 22, 23, 24,

a long working distance and high numerical

37, 72]. This sophisticated tool has been useful in

aperture was used and coupled to the cornea via

augmenting our understanding of anatomy in

a PMMA cap by interposing a transparent gel

the healthy and diseased human cornea. The

(Vidisic, Mann Pharma, Germany) for in vivo

limitations in magnification due to slight,

imaging [66]. For 3-D imaging, an internal scan-

unavoidable eye movements are obvious and

ning device moves the focal plane perpendi-

therefore 3-D reconstruction is restricted on

cularly to the x-y plane, in the same way as

practical grounds. The step size is too coarse

in optic disk tomography performed with the

and magnification is too small. However, 3-D vi-

original HRT II configuration. During image

sualization and modeling would improve our

capture the z-movement is stopped and the

understanding of the morphology of corneal

image plane is exactly perpendicularly to the

architecture, e.g., of epithelial nerve structure.

z-axis. For the investigations presented, an

This was our motivation for developing a

acquisition time of 1 s with a scanning depth

fast, non-invasive, high-resolution method for of

30 mm was used for all subjects; this is

202

Chapter 13

In Vivo Micromorphology of the Cornea: Confocal Microscopy Principles and Clinical Applications

Fig. 13.50 A-D. Schematic illustration (A) and 3-D

virtual removal of the epithelium. Thin nerves run-

reconstruction (B-D) of the corneal epithelium with

ning parallel to Bowman’s membrane in the basal

anterior stroma and nerves (healthy human subject):

epithelial plexus. Thicker fibers originating from the

B anterior view, C posterior view, D anterior view with subepithelial plexus

currently thought to be the maximum when pa-

trum. Shadows and illumination were manipu-

tient and examiner movements are taken into

lated after assigning density values to gray

account.

values to more clearly visualize the spatial

Images are presented in the form of a series

arrangement without loss of information.

of 2-D grayscale images (384¥384 pixels, 8 bit)

As a first in vivo application of the new de-

representing optical sections through the

vice in combination with 3-D reconstruction

cornea. The original raw image stacks were con-

techniques, nerve fiber distribution was charac-

verted using ImageJ (NIH, USA) for 3-D recon-

terized in healthy human corneal epithelium.

struction using Amira 3.1 (TGS Inc., USA). The

The spatial arrangement of epithelium, nerves

voxel size is around 0.8¥0.8

¥0.9 mm using the

and keratocytes was visualized by in vivo 3-D

above-mentioned acquisition parameters. The

confocal laser-scanning microscopy

(CLSM)

Amira volume-rendering software package pro-

(Fig. 13.50). The

3-D reconstruction of the

vides an interactive environment allowing fea-

cornea in healthy volunteers yielded a picture of

tures such as volume orientation for viewing

the nerves in the central part of the human

planes and 3-D perspectives, segmentation and

cornea. Thick fibers arise from the subepithelial

determination of distances and surfaces. The

plexus, and the nerves further subdivide di-

image stacks were carefully aligned and modi-

and trichotomously, resulting in five to six

fied to eliminate unspecific information by

thinner fibers arranged parallel to Bowman’s

adapting the gray values in the depicted spec-

membrane and with partial interconnections

13.6

Future Developments

203

Fig. 13.51. Nerve fibers of the basal epithelial plexus,

in strict alignment parallel to Bowman’s membrane

(Fig. 13.51). Branches penetrating the anterior

epithelial cell layer cannot be visualized.

In conclusion, 3-D CLSM is the first tech-

nique to permit visualization and analysis of the

spatial arrangement of the epithelium, nerves

and keratocytes in the living human cornea. The

method developed provides a basis for further

device refinements and for studies of changes in

cellular arrangement and epithelial innervation

in corneal disease. For example, CLSM may help

to clarify gross variations of nerve fiber pat-

terns under various clinical and experimental

conditions.

13.6.2

Functional Imaging

In conventional microscopy the possibility of

using dyes to visualize specific anatomic struc-

tures yields major information gains. This is es-

pecially true when techniques of fluorescence

Fig. 13.52 A, B. Fluorescence micrographs of the su-

microscopy or immunohistochemistry are used

perficial corneal epithelial cells: A intact corneal epi-

in combination with confocal techniques [67].

thelium without appreciable fluorescence; B stippled

Because these methods are well suited for inves-

epithelium after contact glass examination, blue

tigating the functional status of tissues,they are

marked area with fluorescein-stained cells, orange

also interesting for in vivo microscopy in hu-

marked area with unstained intact cells

mans, for example, for studies of wound healing

or inflammation processes. However, problems

arise due to the necessity for “real time” investi-

these may be regarded as a further enhance-

gation because of involuntary movements on

ment of corneal assessments by slit-lamp mi-

the part of the subjects and due to the selection

croscopy following fluorescein or rose bengal

of suitable non-toxic vital stains. Nevertheless,

staining [68, 17, 16, 42, 52].

successful initial steps have already been taken

To achieve this, a Heidelberg Retina Angio-

toward confocal in vivo fluorescence micro-

graph (HRA/C, Heidelberg Engineering GmbH,

scopy of the anterior eye segments [20, 29], and

Germany) has been modified with a lens attach-

204

Chapter 13

In Vivo Micromorphology of the Cornea: Confocal Microscopy Principles and Clinical Applications

Fig. 13.53 A-D. Patient with intact corneal epithelium: A intact corneal epithelium, slit-lamp microscopy

photograph; B reflection mode, tear film still intact 20 s after eyelid opening; C fluorescence mode, superficial

corneal epithelium, only minimal fluorescence of individual cells; D fluorescence mode, higher magnification

ment (Rostock Cornea Module) so that the laser

The result is a technique that enables fur-

focus is shifted to the anterior eye segments.

ther-reaching investigations of damaged corneal

This enables fluorescence microscopy images to

epithelium and of the associated wound healing

be obtained after staining with the non-specific

processes. In future, confocal fluorescence mi-

stain sodium fluorescein and excitation with

croscopes specially designed for in vivo investi-

blue argon laser light (wavelength 488 nm) and

gations in humans will perhaps permit high-

addition of a barrier filter (500 nm). Using a

quality functional imaging that is even more

green argon laser (514 nm) in reflection mode

comprehensive and specific.

with the same device, it is also possible to visu-

alize break-up phenomena of the tear film [29,

75, 76] (Figs. 13.52-13.54).

13.6

Future Developments

205

Fig. 13.54 A-D. Same patient as in Fig. 13.52 after ap-

defect (dry spot) just 3 s after eyelid opening; C fluo-

plication of a local anesthetic and following applana-

rescence mode, same area as in B, superficial corneal

tion tonometry: A slit-lamp microscopy photograph

epithelium, area with marked fluorescence; D fluores-

with corneal stippling; B reflection mode, tear film cence mode, higher magnification

∑ This will generate enhanced quality in clini-

Summary for the Clinician

cal evaluation

∑ Confocal high-resolution biomicroscopy

∑ The use of vital staining substances, e.g.,

will be used for the in vivo description of

sodium fluorescein or etidium homodimer

corneal pathology at the cellular level

or calcein, may give insights into the meta-

∑ It will enable degeneration and repair

bolic activities of a variety of cells under

mechanisms under various conditions to be

different wound healing or degenerative

examined so that the findings can be corre-

conditions

lated with those from conventional slit-

lamp biomicroscopy

206

Chapter 13

In Vivo Micromorphology of the Cornea: Confocal Microscopy Principles and Clinical Applications

Acknowledgements. The authors are grateful

13.

Duke-Elder S, Wybar KC (eds) (1958) System of

for the cooperation and detailed contributions

ophthalmology, vol II. Henry Kimpton, London,

pp 113-127; 543-557

of Alexander Eckard, Steffi Knappe, Robert

14.

Dunn A, Smithpeter C, Welch AJ, Richards-Kor-

Kraak, Petra Schröder, Oliver Stachs, Hans-Peter

tum R (1997) Finite-difference time-domain sim-

Vick, and Andrej Zhivov.

ulation of light scattering from single cells. J Bio-

med Opt 2:262-266

15.

Fagerholm P (2000) Wound healing after pho-

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Ophthalmol (in press)

Allergic Eye Disease:

Pathophysiology, Clinical Manifestations and Treatment

Bita Manzouri, Thomas Flynn, Santa Jeremy Ono

inflammation and is a problem that is wide-

Core Messages

spread among individuals who suffer with aller-

∑ Allergic eye disease affects a reported

gies. Although the incidence of allergic eye

20 % of the population worldwide and

disease varies by geographical location, its

may be increasing in line with other atopic

prevalence is difficult to gauge as allergies tend

diseases, such as asthma, as a result of

to be underreported. A recent survey conducted

environmental factors

by the American College of Allergy, Asthma and

∑ Other pathological mechanisms,in addi-

Immunology found that 35 % of families inter-

tion to the standard type I hypersensitivity

viewed in the United States experienced aller-

reaction, have been recently implicated in

gies,

50 % of whom reported associated eye

the pathogenesis of allergic eye disease

symptoms [48]. However, this prevalence is set

∑ Established treatments have targeted mast

to increase probably as a result of environmen-

cells, but as a result of our greater under-

tal factors. For example, the morbidity and mor-

standing of the mechanisms involved in

tality of asthma have increased with this, coin-

eye allergy, researchers are now concentrat-

ciding with the increase in house dust mite

ing on other cell types, such as eosinophils

levels, and are greatest in communities exposed

and dendritic cells, as potential targets for

to high allergen levels [32].

immunomodulation

Geographical variations, the lack of any

∑ Other areas of investigation to elucidate

clear-cut objective diagnostic criteria and the

novel treatment strategies include the

difficulty over the diagnosis - especially when it

study of the genetics of ocular allergy,

is the sole manifestation of atopy - have made it

the role of environmental factors in the

difficult to report the incidence rates for differ-

pathogenesis of ocular allergy, and the

ent forms of allergic eye disease. In the past,

use of immunostimulatory DNA sequences

clinical features were used to classify allergic

that can inhibit the allergic response

eye disease, but recent work that has defined the

underlying pathogenic mechanisms has provid-

ed an understanding of the cellular and media-

tor mechanisms involved, thereby enabling a

14.1

better understanding of the disease process and

Introduction

the development of more effective treatments.

Allergic conjunctivitis is typically divided

Owing to the fact that the eye is one of the first

into five types: seasonal allergic conjunctivitis

organs to encounter environmental allergens,

(SAC), perennial allergic conjunctivitis (PAC),

allergic eye disease has become a common ocu-

vernal keratoconjunctivitis (VKC), atopic kera-

lar problem, estimated to affect about 20 % of

toconjunctivitis (AKC) and giant papillary con-

the population worldwide [51]. Allergic eye dis-

junctivitis (GPC). The latter is an iatrogenic dis-

ease is one of a spectrum of diseases that share

ease associated with foreign bodies on the eye,

a common initiating mechanism and pattern of

such as contact lenses and ocular prostheses.

210

Chapter 14

Allergic Eye Disease: Pathophysiology, Clinical Manifestations and Treatment

Although not always included in this grouping,

such as monocyte chemoattractant protein-1

it is thought to have a possible allergic mecha-

(MCP-1), eotaxin-1, or the protein regulated on

nism because of the predominance of mast

activation normal T-cell expressed and secreted

cells. GPC invariably resolves when the cause is

(RANTES), resulting in the migration of inflam-

removed and keratopathy is rare.

matory cells into the site of allergen exposure

The aim of this review will be to focus on the

[5].

underlying mechanisms of allergic eye disease

This sensitized mast cell mediated response

and the current classification of the various dis-

is responsible for many of the symptoms seen in

ease manifestations. Treatment modalities, both

SAC and PAC - such as itching, redness and eye-

well established and new innovations, will also

lid swelling - with most of these patients having

be discussed.

a positive family history of atopy and raised lev-

els of allergen specific IgE in the serum and

tears [32]. Immunohistochemical studies have

14.2

shown that in SAC there is a significant increase

Pathophysiology

in the numbers of conjunctival mast cells, which

correlates with the patient’s severity of symp-

Ocular allergic disease is typically associated

toms [32]. A number of proinflammatory cy-

with immunoglobulin E mediated mast cell

tokines are released by mast cells and these in-

activation (type I immediate hypersensitivity

clude histamine, leukotriene C₄, prostaglandin

reaction) in the conjunctival tissue. However,

D₂, platelet-activating factor

(PAF), tryptase,

recent data from several groups indicate that

chymase, cathepsin G and other eosinophil and

other additional mechanisms can also be in-

neutrophil chemoattractants in what is termed

volved in causing a red, allergic eye.

the early phase response [32]. This response

lasts for a maximum of 20 min after allergen

activation and includes enhanced tear levels of

14.2.1

histamine, protease tryptase, and leukotrienes,

Type I Hypersensitivity

and an increase in the number of eosinophils

[46]. At about 6 h a late phase response occurs

The allergic response begins when allergen is

which includes a second peak of tear histamine

encountered by an antigen presenting cell (APC),

(without an increase in tryptase) and an in-

either directly or as part of an immune complex

crease in tissue adhesion molecules E-selectin

with immunoglobulin. The APCs then process

and interstitial cell adhesion molecule 1 (ICAM-

and present the allergen to CD4+ T cells as a

1), which is followed by an influx of inflammato-

peptide fragment in association with the major

ry cells such as neutrophils, T cells, basophils

histocompatibility

(MHC) class II molecule.

and eosinophils [46]. The presence of tear hista-

These T cells are then polarized into T helper

mine and the absence of tear tryptase in the late

type 1 (Th1) cells and T helper type 2 (Th2) cells.

phase response may indicate that basophils, as

The Th2 cells produce a variety of interleukins,

opposed to mast cells, are involved.

two of which - IL-4 and IL-13 - stimulate im-

Mast cells are also known to synthesize, store

munoglobulin class switching of B cells from

and release a number of cytokines such as IL-4,

producing IgM to producing IgE. This im-

IL-5, IL-8, IL-13 and TNFa [46]. Cytokine in-

munoglobulin binds to high affinity receptors

volvement, particularly the Th2 cytokines, has

(FceRI) on the surface of mast cells and ba-

been the focus of many studies recently looking

sophils. Subsequent encounter with this aller-

into the mechanisms of ocular allergy. It is

gen results in the cross linkage of IgE bound to

known, for example, that IL-4 plays a key role in

FceRI on the surface of mast cells and a cascade

allergic inflammation by promoting T-cell

of signal transduction with a resultant release of

growth, by inducing the production of IgE from

preformed and newly synthesized mediators.

B cells, by upregulating the adhesion molecule

Tissue fibroblasts and epithelial cells are also

vascular cell adhesion molecule 1 (VCAM 1),

triggered by Th2 cells to produce chemokines

and by regulating the differentiation of the Th2

14.2

Pathophysiology

211

subset, which is essential for the allergic reac-

more persistent and progressed to a late-phase

tion [19, 31].

reaction. Typically, high doses of allergen in-

Physiologically, mast cells represent a hetero-

duced a continuous reaction manifested by

geneous population. They are subdivided on the

burning, redness, itching, tearing and a foreign

basis of their ultrastructural characteristics,

body sensation that began 4-8h after challenge

protease content, and T-lymphocyte dependen-

and persisted for up to 24 h. This clinical reac-

cy [49]. In humans, mast cells that contain

tion was accompanied by a significant recruit-

tryptases, chymases, carboxypeptidase A, and

ment of inflammatory cells in tears. Neutrophils

cathepsin G are designated MC_TC and those that

first appeared about

20 min after challenge,

contain tryptase only are designated MC_T. Al-

with eosinophils and lymphocytes increasing in

though both subtypes develop from the same

prominence 6-24 h after challenge.

CD34+ mononuclear precursor, the MC_T sub-

The eosinophil predominates in the late

type is dependent on the presence of T lympho-

phase reaction. It is a powerful effector cell,

cytes, present at mucosal surfaces, and increas-

releasing arginine rich toxic proteins capable of

es in number in aeroallergen driven allergic

causing corneal epithelial damage [32]. Normal-

disease, whilst the MC_TC subtype appears to be

ly, eosinophils are not found in the conjunctival

independent of T cells but its development

epithelium of non-atopic subjects but the num-

requires fibroblastic derived growth factors,

bers are increased in the conjunctival epitheli-

which are predominant in connective and

um, subepithelium and tears of patients with

perivascular tissues, and is characteristic of

AKC and, to a greater extent, VKC patients.

fibrotic processes [32]. Normally, approximately

Furthermore, this increase in eosinophils and

80 % of conjunctival mast cells are of the MC_TC

eosinophil products

[e.g. eosinophil peroxi-

phenotype and are mainly subepithelial in dis-

dase, eosinophil cationic protein (ECP)] is also

tribution, with the rest being MC_T, but during

present in both skin test positive and skin test

allergic inflammation such as that seen in SAC,

negative VKC and is not confined to ocular tis-

VKC or AKC, the numbers of the latter type in-

sues. This suggests that, in at least some forms of

crease in the epithelial and subepithelial layers

allergic conjunctivitis such as VKC, eosinophilic

[37]. In the chronic and fibrosing condition

infiltration - and not IgE sensitization - is the

AKC, however, the MC_TC subtype predominates,

more relevant feature of the disease and is asso-

perhaps indicating an important transition

ciated with signs of systemic activation of

from a simple mediator driven disorder to that

eosinophils [10].

of chronic inflammation leading to conjunctival

fibrosis [37].

14.2.3

Non-specific Conjunctival Hyperreactivity

14.2.2

Ocular Inflammatory Reaction:

Non-specific stimuli can also cause target organ

Late Phase

hyperreactivity and this is thought to play a role

in allergic diseases of the eye. It is postulated

A late phase reaction sustained by a complex

that “non-specific conjunctival hyperreactivity”

network of inflammatory cells and mediators

may represent a distinct pathophysiological ab-

can also occur in the eye. This has been demon-

normality in allergic eye disease [10]. The vari-

strated in humans using allergen for conjuncti-

ability of symptoms experienced in allergic

val provocation of allergic subjects [10]. Aller-

conjunctivitis which do not correlate with envi-

gen challenge caused the typical early-phase

ronmental changes such as the levels of sensitiz-

reaction within 20 min, with the initial reaction

ing allergens, as well as the ocular reaction in-

being dose dependent. With smaller doses of

duced by non-sensitizing stimuli, may well be

allergen the reaction was not so pronounced

explained by this non-specific hyperreactivity.

and spontaneous recovery occurred within a

Natural non-specific stimulation with agents

brief period. With larger doses, the reaction was

such as wind, dust, and sunlight may act only as

212

Chapter 14

Allergic Eye Disease: Pathophysiology, Clinical Manifestations and Treatment

triggers of an abnormal non-specific reactivity

CD4+ T cells can be further subdivided into

of the conjunctiva in allergic patients [10].

two distinct subsets based on their pattern of

Furthermore, multiple physical, chemical, in-

cytokine production. The first subset, Th1 cells,

fectious, or antigenic factors may stimulate the

produce IL-2, IL-3, TNFb and interferon g

biological responses of mast cells, leading to the

(IFNg) and are more associated with classic de-

release of several mediators. Rubbing of the

layed type hypersensitivity. The second subset,

eyes, exposure to UV light, and increase of ocu-

Th2 cells, produce a range of cytokines encoded

lar surface temperature may lead to acute de-

on chromosome 5, such as IL-4 and IL-5, which

granulation of the mast cells and release of their

promote immediate hypersensitivity responses

mediators. The local generation of stimuli that

through their ability to stimulate proliferation,

induce different patterns of mast cell cytokine

B cell IgE production and eosinophil produc-

release may represent another method of bio-

tion, activation and survival [32]. It has been

logical, non-specific activation of mast cells

shown that in AKC there is increased numbers

[42]. It has been observed that whenever a pa-

of both Th1 and Th2 lymphocytes as opposed to

tient with VKC is exposed to the sun, signs and

in VKC where lymphocytes secreting cytokines

symptoms recur. Furthermore, the symptoms of

typical of the Th2 subset are found. This ob-

allergy become most severe in children with

servation suggests that VKC results from a mat-

VKC who develop bacterial conjunctivitis. Cer-

uration shift of CD4+ T cells towards a pattern

tain types of lipopolysaccharides of bacteria

of secretion of cytokines which drives a mast

may cause degranulation of mast cells, leading

cell and eosinophil mediated inflammatory

to the release of their mediators that cause exac-

response [34].

erbation of the allergic process.

14.3

14.2.4

Clinical Syndromes of Allergic Eye Disease

T-Cell-Mediated Hypersensitivity

in Allergic Eye Disease

Allergic diseases of the eye comprise a number

of different inflammatory conditions that share

Both CD4+ and CD8+ T cells populate the

common features such as seasonal variation,

subepithelial tissue of the normal human con-

association with atopic disease and presumed

junctiva. In the active forms of SAC and PAC, the

involvement, to a greater or lesser extent, of the

T cell profile remains virtually unchanged com-

type I hypersensitivity mechanism in their

pared to the normal milieu, but in chronic aller-

pathophysiology. They are traditionally classi-

gic disorders such as VKC, AKC and GPC, CD4+

fied, as outlined above, into five distinct entities:

T cells but not CD8+ T cell numbers are in-

SAC, PAC, VKC, AKC, and GPC (Fig. 14.1).

creased, with a mixed cellular infiltrate contain-

As previously mentioned, recent evidence

ing many mast cells, eosinophils, neutrophils,

suggests that the traditional type I hypersensi-

and macrophages [32]. In chronic allergic dis-

tivity reaction may be less important in some of

eases there is no clear-cut difference between

these diseases than others. However, these dis-

the allergen specific IgE responses and the

eases share many symptoms in common and it

nature and severity of the allergic responses;

is therefore reasonable to group them in the

hence it is likely that non-IgE mechanisms are

same broad category of “allergic eye disease”.

contributory, with the involvement of cell medi-

The cardinal feature of all allergic eye disease is

ated responses [32].

itching - in the absence of this symptom one

Most of the T cells in normal conjunctiva are

should be wary of making this diagnosis. Other

naïve, but in chronic allergic conditions 90 % of

symptoms such as tearing, burning and foreign

the T cells are memory T cells [35]. Correspond-

body sensation may be present in variable de-

ing with this rise in activated T cells, there is

grees in all of these conditions. Despite similar-

also upregulation of markers present on antigen

ities in the symptoms, it is important to distin-

presenting cells.

guish, where possible, between the different

14.3

Clinical Syndromes of Allergic Eye Disease

213

Fig. 14.1. A Normal bulbar conjunctiva; B giant papillae in GPC; C typical appearance of superior tarsal con-

junctiva in a severe case of SAC; D corneal ulcer in VKC; E early stages of corneal pannus in AKC; F Horner-

Trantas dots seen in AKC. (Pictures courtesy of Dr. Mohammed Siddique, Institute of Ophthalmology, London)

types of allergic eye disease as each of them has

to lead to visual impairment, with AKC being

a different visual prognosis. Accurate diagnosis

the most destructive disease and having the

will allow appropriate counselling of patients.

worst visual prognosis. The emergence of new-

The most common type of allergic eye dis-

er treatments based on an increasing under-

ease, seasonal allergic conjunctivitis (hay fever

standing of the individual pathogenic mecha-

conjunctivitis), is also the least serious in terms

nisms of each disease also underlines the

of visual outcome. SAC and PAC together ac-

importance of accurate diagnosis. The different

count for 98 % of allergic eye disease [41]. VKC

types of allergic eye disease can usually be dis-

and AKC, although much rarer, are more likely

tinguished by history and examination alone.

214

Chapter 14

Allergic Eye Disease: Pathophysiology, Clinical Manifestations and Treatment

symptomatic.“Household” allergens such as the

14.3.1

dust mite or pet dander are the usual offenders

Seasonal Allergic Conjunctivitis

in PAC. These patients may also be sensitive to

seasonal allergens and so there may be a super-

Of the allergic eye diseases, SAC represents the

imposed seasonal element to their symptoms.

most “pure” form of type I hypersensitivity. As

the name suggests, the symptoms and signs are

intermittent and occur rapidly following expo-

14.3.3

sure to a specific allergen, with patients often

Vernal Keratoconjunctivitis

having a personal or family history of atopy. In

the absence of prolonged exposure to allergen,

A disease of childhood, VKC accounts for 0.5 %

attacks are short lived. The commonest season-

of allergic eye disease [32]. Like AKC it has a

al allergen is pollen, with tree pollen predomi-

male preponderance but onset is much earlier,

nating in spring, grass pollen in summer and

typically late in the first decade. It is seen most

ragweed pollen in autumn. Symptoms are typi-

commonly in temperate climates such as those

cally absent during winter. The severity of signs

of the Mediterranean, South Africa and North

and symptoms varies from patient to patient

America. However, genetic as well as environ-

depending on the specific allergen and the ex-

mental factors are important. Even in cooler

posure.

northern climates the disease is more common-

ly seen in people of African or Asian descent

14.3.1.1

[39]. There is frequently a personal or family

Symptoms

history of atopy but this association is not as

strong as in other types of allergic eye disease,

Patients usually complain of intense itching of

with a large proportion of VKC patients having

the eyes associated with a watery discharge.

no such history.

In the majority of cases the disease shows

14.3.1.2

seasonal variation with symptoms typically ap-

Signs

pearing in spring and lasting about 6 months.

Additional recurrences in winter are common.

There may be eyelid oedema. Conjunctival ves-

In some cases the disease evolves over time into

sels may be injected and conjunctival chemosis

a more chronic, perennial form of inflammation

may give the conjunctiva a “milky” appearance.

with up to one-quarter of VKC patients having a

Symptoms and signs are usually bilateral al-

perennial form of the disease from the outset

though they may be asymmetrical. Young chil-

[11]. Although serious visual complications may

dren can present with dramatic unilateral lid

occur, VKC is a less destructive disease than

oedema and chemosis.

AKC and usually burns itself out by the early

twenties [30].

14.3.2

14.3.3.1

Perennial Allergic Conjunctivitis

Symptoms

PAC is less common than SAC. Although the

Symptoms are usually bilateral but may be

symptoms and signs of these diseases are the

asymmetrical and, like all allergic eye diseases,

same, the distinction between them lies in the

itching is a cardinal feature. Photophobia is also

timing of the symptoms. Whereas SAC sufferers

prominent and patients may complain of tear-

have symptoms for a defined period of time,

ing and a mucoid discharge. Depending on the

PAC sufferers are sensitive to allergens that are

severity of corneal involvement, they may also

present year-round and so are perennially

complain of a foreign body sensation or pain.

14.3

Clinical Syndromes of Allergic Eye Disease

215

14.3.3.2

Cornea. Sight-threatening complications occur

Signs

less frequently in the cornea than in AKC. How-

ever, both non-specific and pathognomonic

In contrast to AKC, the periorbital skin is usual-

corneal signs are seen. In a follow-up series of

ly unaffected. The disease is further classified

195 patients with VKC, 9.7 % developed corneal

into tarsal, limbal or mixed VKC depending on

ulcers and 6 % developed a permanent decrease

the location of the conjunctival inflammatory

in visual acuity [11]. Abnormalities of the cen-

signs.

tral and superior cornea are most commonly

seen in tarsal disease. In its earliest form there

Tarsal. The inflammation is predominantly in

may be only punctuate epithelial erosions.

the superior tarsal conjunctiva although the

These may, with time, coalesce to form larger

bulbar conjunctiva may show non-specific signs

erosions that may in turn evolve into the charac-

such as injection or chemosis. The superior

teristic “shield” ulcer of VKC. Shield ulcers are

tarsal conjunctiva develops a papillary reaction.

non-infectious and occur in the central/superi-

Papillae are typically large (>1 mm) and diffuse,

or cornea. At first they are shallow with a trans-

giving a “cobblestone” appearance. These tarsal

parent base. Over time the ulcer becomes filled

papillae tend to persist even when the disease is

with inflammatory debris and the base opaci-

quiescent but become hyperaemic and oedema-

fies. Further accumulation of inflammatory de-

tous during periods of disease activity. The

bris leads to plaque formation. The pathogene-

presence of a thick, mucoid, white secretion as-

sis of these ulcers is incompletely understood.

sociated with these papillae is another indicator

Mechanical abrasion of the epithelium by large

of disease activity. Papillae may enlarge to sev-

papillae on the superior tarsal conjunctiva is

eral millimetres in diameter and may give rise

thought to play a role, as is epithelial corrosion

to ptosis. In severe forms of the disease, linear

by toxic granule proteins released from eosino-

subepithelial scars

(Arlt’s lines) may appear

phils in the tarsal conjunctiva and tear film. In

parallel to the lid margin.

persistent or recurrent limbal disease, peripher-

al corneal signs such as pannus or opacification

Limbal. Limbal VKC is characterized by single

(pseudogerontoxon) may develop. Limbal le-

or multiple gelatinous, pale infiltrates in the

sions may also cause significant astigmatism.

limbal conjunctiva. The extent of limbal in-

volvement is variable. Infrequently, there may

be 360° limbal inflammation. There is usually

14.3.4

injection of the surrounding bulbar conjuncti-

Atopic Keratoconjunctivitis

val vessels. Aggregates of degenerating eosino-

phils at the apex of the infiltrates are seen as

First described in 1952 [22], AKC constitutes a

small white spots (Horner-Trantas dots) - both

more relentless form of conjunctival inflamma-

the limbal infiltrate and the Horner-Trantas

tion than either SAC or VKC. Atopic dermatitis

dots are transient.

(eczema), a pruritic skin condition that affects

In mixed VKC both limbal and tarsal signs

3 % of the population, is present in 95 % of pa-

may be observed. Although limbal and tarsal

tients with AKC [7]. Conversely, 25-40 % of

VKC are believed to be variants of the same dis-

atopic dermatitis patients have AKC [18]. Typi-

ease, certain differences have been observed in

cally patients have had atopic dermatitis since

their demographics and natural history. Limbal

childhood with ocular symptoms developing at

VKC is particularly common in people of

a later stage. Symptoms may begin in the late

African or Asian descent. There is mixed evi-

teens or early twenties but the peak incidence is

dence as to which, if either, of the variants is

between the ages of 30 and 50. Males are more

more responsive to treatment [11, 52]. Patients

commonly affected than females and there is of-

with tarsal disease are certainly more likely to

ten a personal or family history of other atopic

develop sight-threatening corneal ulceration

diseases. Unlike SAC, and most cases of VKC, the

[52].

symptoms are perennial. It differs from PAC in

216

Chapter 14

Allergic Eye Disease: Pathophysiology, Clinical Manifestations and Treatment

that the symptoms are less intermittent. Al-

hyperaemia or chemosis. Rarely, papillary

though there may be periods of relative quies-

hyperplasia of the limbal conjunctiva occurs,

cence, signs of disease activity are usually pres-

resulting in a gelatinous limbal nodule similar

ent to some degree.

to those seen in limbal VKC. Associated Horner-

Trantas dots have been seen. Prolonged or

14.3.4.1

severe inflammation may result in conjunctival

Symptoms

cicatrization. This is most commonly seen in the

lower fornix and may result in shallowing of the

Bilateral itching of the eyelids and periorbital

fornix and symblepharon. Activation of fibro-

skin is the most frequent symptom. Patients

blasts by mast cells has been proposed as a

also complain of tearing, photophobia, burning

mechanism for conjunctival scarring in allergic

and blurred vision. Increased mucus and in-

disease [47]. Several cases of squamous cell car-

flammatory debris may thicken the tear film

cinoma/CIN have been reported in patients

and contribute to a stringy discharge. Depend-

with atopic dermatitis or AKC [20, 24] although

ing on the severity of corneal involvement, pa-

the mechanism of tumourigenesis remains

tients may complain of a foreign body sensation

unclear.

and pain.

Cornea. Visual deterioration in AKC is most

14.3.4.2

commonly caused by corneal complications.

Signs

Corneal scarring in AKC may result from vascu-

larization, infection or keratoconus. A broad

Invariably there are signs of disease on the eye-

spectrum of corneal disease may be seen de-

lids and periorbital skin. Ocular surface inflam-

pending on the severity and chronicity of

mation in AKC may, as the name suggests, affect

inflammation. Punctate epithelial erosions are

the conjunctiva and cornea. In many cases the

seen early in the course of the disease. The

disease is mild and corneal signs may actually

severity of the corneal erosions correlates with

be absent or minimal. Such cases have been

the number of inflammatory cells (especially

termed atopic blepharoconjunctivitis (ABC) [53].

eosinophils) in brush cytology samples from

the superior tarsal conjunctiva [50]. Peripheral

Eyelids. The periorbital skin typically has the

corneal vascularization, which may be associat-

dry, indurated and scaly appearance of eczema.

ed with opacification, is common. These

Eyelid swelling may contribute to the general-

changes may occur as a result of limbal stem cell

ized wrinkling of the skin and the development

deficiency. Rarely, corneal vascularization may

of a fold in the lower lid skin (Dennie-Morgan

encroach on the visual axis and cause visual im-

fold). In severe cases there may be fissures at the

pairment. Epithelial erosion may coalesce to

lateral canthus and/or absence of the lateral

form non-infectious corneal ulcers. Toxic gran-

part of the eyebrow (Herthoge’s sign). The latter

ule proteins derived from conjunctival eosino-

signs may be induced or aggravated by vigorous

phils have been implicated in the pathogenesis

eyelid rubbing. Lid margins may be thickened

of these ulcers [33]. Staphylococcal colonization

(tylosis) and may develop meibomian gland

of the lid margins coupled with a decrease in

dysfunction. Colonization of the lid margin

barrier function [56] also puts AKC patients at

with staphylococcus with resultant staphylo-

increased risk of developing bacterial infectious

coccal blepharitis is common [54].

corneal ulcers. They are particularly vulnerable

to herpes simplex keratitis [16]. Chronic eye

Conjunctiva. There is typically a papillary re-

rubbing may be an important factor in the asso-

action on the tarsal conjunctiva, which, in con-

ciation between AKC and keratoconus [6].

trast to VKC, is usually more prominent on the

inferior, rather than the superior, tarsal con-

Other Causes of Visual Deterioration in AKC.

junctiva. The bulbar conjunctiva may show

AKC is associated with the development of pre-

non-specific signs of inflammation such as

mature bilateral cataracts. Typically the lens

14.4

Treatment of Allergic Eye Disease

217

opacity develops in the anterior subcapsular

14.3.5.2

region and has well defined margins. It is often

Signs

referred to as a “shield” cataract. A rarer cause

of visual impairment in AKC is that of retinal

Giant papillary conjunctivitis is characterized,

detachment [57]. The reasons for this associa-

in the late stages, by the presence of abnormally

tion are not well understood. Finally, chronic

large (>0.3 mm) papillae on the superior tarsal

use of topical steroids in the treatment of AKC

conjunctiva. In the earliest stage, however, when

may result in posterior subcapsular cataracts

the patient first becomes symptomatic, the con-

and glaucoma (see below).

junctiva may appear normal. As the disease pro-

gresses the superior tarsal conjunctiva becomes

thickened and hyperaemic. Small papillae de-

14.3.5

velop first which increase in size and number

Giant Papillary Conjunctivitis

over time. The distribution of giant papillae

varies according to the type of lens worn. In

The term giant papillary conjunctivitis de-

wearers of soft lenses papillae emerge first at

scribes the advanced stages of the conjunctival

the superior edge of the tarsal plate. Wearers of

response to the prolonged presence of a foreign

hard lenses, which are smaller, develop papillae

body on the ocular surface.It was first observed

closer to the superior lid margin [23]. The bul-

and characterized in contact lens wearers [3]

bar conjunctiva and inferior fornix are usually

and was later reported in patients with ocular

normal.

prostheses and exposed suture ends. Nowadays,

The symptoms and signs of this disease may

it is seen commonly in contact lens wearers, and

resemble those of VKC. Important factors in the

most of the knowledge of this condition arises

history, which could help to distinguish these

from experience with these patients. Wearers of

conditions, include contact lens history and

soft contact lenses are most likely to develop

patient age since VKC is seldom seen after the

giant papillary conjunctivitis, but it has been

early twenties.

estimated that 1-5 % of rigid gas-permeable lens

wearers may also be affected [26, 27]. The condi-

tion shows no age or gender preference and

14.4

there does not appear to be a strong association

Treatment of Allergic Eye Disease

with allergy [28].

The mainstays of treatment for the majority of

14.3.5.1

allergic eye disease symptoms are topical eye

Symptoms

drops, and for this purpose a wide range of top-

ically administered agents have been developed

Earliest symptoms are of mucus discharge in

to treat the milder disease varieties. These

the morning and itching on removal of the lens-

include antihistamines, mast cell stabilizing

es. As the disease progresses these symptoms

agents and anti-inflammatory agents. Addition-

become more marked and may be associated

ally, topical nasal decongestants are also avail-

with a foreign body sensation. Patients com-

able. Of the topical eye drops, it is antihista-

plain of blurred vision as a result of coating of

mines and mast cell stabilizers that have been

the lens with mucus and increasing lens mobil-

extensively studied to assess their therapeutic

ity and instability. As the disease advances pa-

value in a large number of comparative clinical

tients become increasingly intolerant of their

trials over the years. Furthermore, as the chem-

contact lenses.

ical and cellular infiltrates in both acute and

chronic allergic eye disease become better char-

acterized, there are significant implications for

treatment of these conditions. Efficacy of all of

these agents varies from patient to patient and

the choice of agent used depends on a number

218

Chapter 14

Allergic Eye Disease: Pathophysiology, Clinical Manifestations and Treatment

of variables, such as the underlying state of

gic conjunctivitis, combining an antihistamine

health of the eye being treated, drug costs and

with a vasoconstrictor is more effective than

availability, contact lens wear, and the potential

either agent alone. The vasoconstrictors com-

for compliance [8].

monly used in combination with topical anti-

The preferred treatment modality in mild

histamines are phenylephrine or naphazoline

diseases such as SAC and PAC is topical therapy,

[8].

since neither is sight threatening, and their

pathogenesis involves mast cell degranulation

and the release of histamine. Topical treatment

14.4.2

offers several advantages: the ease of applica-

Mast Cell Stabilizing Agents

tion directly to the site affected by the disease

process, the general lack of systemic side effects,

The most common topical drugs invariably

and the washout effect of the drops themselves

used by ophthalmologists for all forms of aller-

aiding the removal of the inflammatory media-

gic conjunctivitis are the mast cell stabilizing

tors.

agents. These include sodium cromoglygate,

lodoxamide, ketotifen, nedocromil sodium and

the newly introduced olopatadine. Mast cell sta-

14.4.1

bilizers are effective in the milder forms of aller-

Antihistamines

gic eye disease and have very few side effects,

either locally or systemically, but for patients to

The first line of treatment of ocular allergy in-

receive long-term benefit from them such that

cludes the avoidance of allergens, the use of cold

expected exposure to allergen reduces the

compresses for symptom relief (especially itch-

tryptase and inflammatory cells after allergen

ing), and regular lubrication of the eye to wash

challenge, treatment is needed for many years

out tear histamine and other inflammatory me-

[46].

diators, thus diluting their effects and aiding the

Sodium cromoglygate is the prototypic mast

patient’s comfort. Topical therapy may start

cell secretion inhibitor. It is the oldest and most

with the use of antihistamines or mast cell sta-

widely used agent of this family of drugs. How-

bilizers. Considering the former, the stimulation

ever, despite its extensive use, the mechanisms

of H1 receptors in the conjunctiva mediates

of its action are still unclear. The efficacy of the

the symptom of itching whereas H2 receptor

medication appears to be dependent on the con-

activation results in vasodilation. Second gener-

centration of the solution used [9]. Nedocromil

ation H1 receptor antagonists are used for the

sodium has been shown to be able to inhibit

topical treatment of the benign forms of allergic

chloride ion flux in mast cells, epithelial cells

conjunctivitis, and these include levocabastine,

and neurons. This feature may explain how it

azelastine and emedastine. They all bind selec-

can prevent responses such as mast cell degran-

tively to H1 receptors in the conjunctiva and

ulation. Others have suggested the inhibition

have little or no effect on dopaminergic, adren-

of IgE production by B cells as an alternative

ergic or sertotoninergic receptors [46]. Of this

mechanism [46]. Newer agents such as lodox-

new generation H1 receptor antagonists, topical

amide have become available, which are faster

azelastine has been shown to be a powerful

acting and approximately 2,500 times more

topical antihistamine, decreasing eosinophil

potent than sodium cromoglycate in the pre-

and T lymphocyte activation, having an in-

vention of histamine release, that also act to re-

hibitory effect on a broad array of other media-

duce tear tryptase and inflammatory cells after

tors, and being a potent suppressor of itching

allergen challenge [8]. In a comparative trial

and conjunctival hyperaemia after conjunctival

with sodium cromoglygate and lodoxamide in

provocation with an allergen, with an onset

subjects with the more severe forms of allergic

of action seen within 3 min and a duration of

eye disease (VKC, AKC and GPC), lodaxamide

effect of at least 8-10 h [32, 46]. Although topical

was found to be superior for symptom relief. It

antihistamines can be used alone to treat aller-

was also found to be effective in the long-term

14.4

Treatment of Allergic Eye Disease

219

treatment of VKC especially in cases with an

indomethacin. These agents, unlike corticos-

epitheliopathy [17, 43].

teroids, do not mask ocular infections, affect

wound healing, increase intraocular pressure,

or contribute to cataract formation [8]. How-

14.4.3

ever, of these agents, only ketorolac trometh-

Dual-Acting Agents

amine (Acular) has been approved by the Food

and Drug Administration for the management

Dual-acting agents are named for their antihis-

of acute SAC [15]. It acts to significantly reduce

tamine effects and their inhibition of mediator

tear tryptase levels and the number of eosino-

release. They are the newest generation of an-

phils and lymphocytes in tear specimens after

tiallergic agents. The advantages of these drugs

conjunctival provocation [29].

lie in the rapidity of symptomatic relief given by

Ocular NSAIDs have been associated with a

immediate histamine receptor antagonism cou-

low-to-moderate incidence of burning and

pled with the long-term disease modifying ben-

stinging [9]. The concern of NSAID-induced

efit of mast cell stabilization. Not all of these

asthma does not appear to be a problem except

agents are equivalent and in selecting a dual-ac-

in patients who have the triad of asthma, nasal

tion agent, one should look for a potent and

polyposis and aspirin sensitivity [45].

long-lasting agent that relieves the signs and

symptoms of allergy, including itching, redness,

lid swelling and chemosis [41].

14.4.5

Clinical studies have demonstrated the effi-

Topical Corticosteroids

cacy and tolerance of olopatadine for the man-

agement of allergic conjunctivitis or in a con-

Topical steroid preparations are the most effec-

junctival allergen model [1, 4, 13]. This agent

tive therapy for moderate to severe forms of

both acts as a mast cell stabilizer and has anti-

VKC, but their use should be strictly limited for

histamine activity. This dual mode of action has

severe cases and carefully monitored since their

been shown to be advantageous for the manage-

long-term use is associated with an increased

ment of allergic conjunctivitis, and as a topical

risk for the development of cataracts and glau-

preparation has been subjectively preferred by

coma and can potentiate ocular herpetic infec-

patients

[4,

44]. Furthermore, a direct anti-

tions. In fact, topical steroids are responsible for

inflammatory property for this drug has been

the 2 % incidence of glaucoma in VKC patients

suggested by a study which showed that

[12]. In T cell dependent AKC and VKC, sodium

olopatadine inhibited the anti-IgE antibody-

cromoglycate has been used either prophylacti-

mediated release of TNFa from human con-

cally or as maintenance therapy to control mild

junctival mast cells [14].

symptoms only, but is ineffective in acute exac-

erbations. In acute exacerbations, even the new-

er class of mast cell stabilizers may not be

14.4.4

enough, and under these circumstances steroids

Non-steroidal Anti-inflammatory Drugs

(fluoromethalone or dexamethasone) tend to be

(NSAIDs)

used in doses of up to one drop hourly to

reverse corneal epitheliopathy caused by the

Prostaglandins, especially PGE₂ and PGI2, lower

release of epithelial toxic mediators from

the threshold of the human skin and conjuncti-

eosinophils and neutrophils [32]. Once control

va to histamine-induced itching. NSAIDs, by in-

of the acute phase of the disease has been

hibiting the production of prostaglandins, help

achieved, steroids should be discontinued and

to alleviate this itching but also reduce pain and

alternative topical treatment, as outlined previ-

inflammation of the eye associated with allergic

ously, should be started [12].

reactions [9, 46]. NSAIDs used in the topical

Two modified corticosteroids have recently

treatment of allergic ocular conditions in-

been investigated for their efficacy in allergic

clude ketorolac, diclofenac, fluribrofen and

conjunctivitis: rimexoline (a derivative of pred-

220

Chapter 14

Allergic Eye Disease: Pathophysiology, Clinical Manifestations and Treatment

nisolone) that is quickly inactivated in the ante-

tients with severe, steroid resistant AKC [2].

rior chamber of the eye, thus improving efficacy

Patients were randomly assigned to treat-

and decreasing the safety concerns, e.g. raised

ment with topical CsA 0.05 % or placebo for

intraocular pressure; and both low-dose and

a period of 28 days with the symptoms and

high-dose loteprednol etabonate are highly ef-

signs of AKC recorded on the day of enroll-

fective as prophylaxis against, and in the acute

ment and at the end of the treatment period.

phase of, allergic conjunctivitis [8].

The results, recorded by a composite score

computed by summing the severity grade of

all five symptoms and six signs of AKC,

14.4.6

showed a greater improvement in the CsA

Calcineurin Inhibitors

group relative to the placebo group at the end

of the treatment period. It was hence con-

Two calcineurin inhibitors are currently in clin-

cluded that topical CsA 0.05 % is safe, and

ical use:

may actually have some effect in alleviating

Cyclosporin A (CsA) is a fungal antimetabo-

the signs and symptoms, in severe AKC that

lite and anti-CD4+ agent that decreases the

is resistant to topical steroid treatment.

clinical signs and symptoms of the chronic

Tacrolimus (FK-506) is a macrolide antibiot-

forms of VKC and AKC. It acts to control

ic with potent immunomodulatory proper-

ocular inflammation by blocking Th2 lym-

ties which has already been used to treat the

phocyte proliferation and IL-2 production,

immune mediated problems encountered

by inhibiting histamine release from mast

with corneal graft rejection, ocular pem-

cells and basophils, and by reducing the pro-

phigoid and uveitis. It acts on T lymphocytes

duction of IL-5, thereby reducing the recruit-

to block the production of lymphokines,

ment and effects of eosinophils on the con-

such as IL-2, IL-2, IL-5, TNFa and interferon-

junctiva

[12]. Although systemic CsA has

g a. It also blocks the degranulation of mast

been used for the treatment of severe AKC

cells and several mast cell cytokines, such as

and keratoconjunctivitis sicca, topical cyclo-

IL-3 and IL-5 [8].

sporin causes ocular irritation with burning,

tearing, erythema and itching. This is due to

the fact that since the drug is lipophilic, it has

14.4.7

to be dissolved in an alcohol base which

Future Drug Developments

causes the ocular irritation [8]. However, the

topical form of this drug is not yet generally

The aims of future drug development will focus

available.

on steroid-sparing agents that control the

CsA has been evaluated in patients with

immune response. These may be administered

steroid dependent AKC. In one study, 12 pa-

alone, or in combination with newer drugs that

tients were randomized to treatment with

have already demonstrated their efficacy in the

CsA and 9 patients to a vehicle treatment

management of these conditions, such as anti-

group. The results showed that in the CsA

histamines and mast cell stabilizers.

group, 9 out of 12 patients were able to cease

Our understanding of the pathophysiology

steroid therapy as compared to 1 out of 9 in

of allergic conjunctivitis has increased greatly

the vehicle group [21]. Furthermore, the final

over the last 3 years. New areas of investigation

steroid use was significantly lower in the CsA

to elucidate novel treatment strategies include

group versus the vehicle group. This study

the study of the genetics of ocular allergy, since

concluded that CsA is an effective and safe

it has been known for some time that different

steroid sparing agent in AKC and is also ca-

mouse strains are more or less responsive to

pable of improving the symptoms and signs

specific allergen challenge in the eye, and link-

of AKC. In another randomized trial the

age analysis of these mice is being pursued to

short-term efficacy and safety of topical CsA

define disease susceptibility genes for ocular

0.05 % was evaluated in the treatment of pa-

allergy [41]. A few studies have addressed the

14.5

Conclusion

221

role of environmental factors in the pathogene-

[46]. Miyazaki et al. evaluated the therapeutic

sis of ocular allergy. For example, it has been

potential of immunostimulatory sequence

shown that there is a positive association be-

oligodeoxynucleotide

(ISS-ODN) administra-

tween the dietary intake of n-6 polyunsaturated

tion in ocular allergy using a mouse model of

fatty acids and seasonal allergic rhinoconjunc-

ragweed-specific conjunctivitis [36]. They con-

tivitis [55]. Other studies have focused on the

cluded that ISS-ODN was an effective treatment

genetics of allergic conjunctivitis. One of the

for ocular allergy when administered systemi-

earliest published studied approximately

117

cally or conjunctivally. Systemic treatment

families with probands with allergic conjunc-

markedly inhibited clinical parameters of SAC

tivitis [40]. Evidence was found, by analysis of

and blocked conjunctival eosinophilia in the

the genomic DNA, for genetic linkage of allergic

late phase reaction. Additionally, it also effec-

conjunctivitis for chromosomes 5, 16 and 17.

tively blocked neutrophilia, which is a hallmark

This genetic linkage for allergic conjunctivitis

of the late phase reaction.

was shown to differ from that reported for

Other areas of potential therapeutic value

atopic asthma, and hence it was concluded that

which require further research include the use

there were likely to be organ specific disease

of antagonists of the action of macrophage in-

susceptibility genes, which, together with gener-

flammatory protein-1a (MIP-1a) and the use of

al atopy genes, target the allergic response to

IL-1 receptor antagonists. Data have shown that

specific mucosal tissues.

MIP-1a constitutes an important second signal

Resident dendritic cells in the conjunctiva

for mast cell degranulation in the conjunctiva in

have also been the focus of recent research since

vivo and consequently for acute phase disease

it has been shown that dendritic cell activation

[38]. Therefore, antagonizing the interaction of

by an allergen is a very early step in disease

MIP-1a with its receptor (CCR1) or signal trans-

pathogenesis, with dermal allergy being used as

duction from this receptor may hold promise

the prototype [41]. Other areas of interest lie in

for future treatment of both acute and late

the activation and mediator release from hu-

phase reactions. Similarly, in a mouse model of

man conjunctival mast cells on FceRI cross-

allergic eye disease, IL-1 inhibition using an

linking. A recombinant humanized monoclonal

IL-1 receptor antagonist was found to downreg-

anti-IgE antibody, omalizumab, was recently

ulate the recruitment of eosinophils and inflam-

developed which binds specifically to the IgE

matory cells by decreasing the concentration of

binding site on human FceRI and thereby

attractant chemokines [25]. This research also

blocks the binding of IgE to mast cells and baso-

offers a potential novel treatment for the pre-

phils [5]. Studies have shown that this agent

vention and treatment of allergic eye disease.

benefits patients with moderate to severe aller-

gic asthma who remain symptomatic despite

treatment with systemic or inhaled corticos-

14.5

teroids [5]. Additionally, omalizumab has been

Conclusion

shown to be safe and well tolerated.

One of the most innovative treatment ad-

Allergic eye disease represents a heterogeneous

vances has been in the use of immunostimula-

group of diseases that share a common sympto-

tory DNA sequences that can inhibit the allergic

mology but different pathogenesis. They are

response. Both bacterial DNA and synthetic

further distinguished by their long-term visual

oligodeoxynucleotides containing specific mo-

prognosis, with diseases such as SAC and PAC

tifs centered on a CpG dinucleotide have been

having no long-term effects on sight whereas

shown to be potent immunostimulatory agents

VKC and AKC, through corneal involvement

[46]. It is likely that these sequences represent a

and subsequent scarring reactions, can adverse-

signal to the immune system, resulting in a

ly affect visual prognosis. Future work needs to

powerful Th1 response and this can be used to

increase our understanding of the genetics and

switch an allergic response from a Th2 domi-

mechanisms of mast cell cytokine expression

nated immune profile towards a Th1 profile

and mediator release, the regulation of the

222

Chapter 14

Allergic Eye Disease: Pathophysiology, Clinical Manifestations and Treatment

cellular inflammatory response and the B cell

regulation of IgE secretion. Armed with this

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Subject Index

Autologous serum

ABCG2

- eyedrops

4, 52

ACAID (Anterior chamber associated immune

Autologous transplantation

deviation)

110

Avastin

Acanthamoeba keratitis

153, 196

Azathioprine

112

Adenoviral keratoconjunctivitis

see keratoconjunctivitis

Air drying

Bandage contact lens

Airdissection

Basal cells

184

Albumin

Basement membrane

24, 58

Allergen

214

Basiliximab

112, 117

Allergic eye disease

209

Bioengineering

Allograft rejection

Biomicroscopy, slit-lamp

174

Allorecognition

110

Blood group antigen

106

– direct pathway

76, 110

Blood product

– indirect pathway

110

Bowman’s membrane

187

Amnion

Broad antigen

102

– composition

Buccal mucosa graft

– structure

Bullous keratopathy

Amniotic epithelium

Amniotic membrane

21, 49, 51, 52, 57, 58

Amphotericin B 0.15%

158

Calcineurin inhibitor

220

Anecortave acetate

Candida albicans

159

Angiogenesis

Caustication

Angiogenic growth factor see growth factor

CD4+ T cells

110, 212

Aniridia

CD8+ T cells

110, 212

Anterior chamber associated immune deviation

Centration

123, 134

see ACAID

110

Certican

116

Antiangiogenic therapy

Chemical burns

Anticoagulant

Chronic limbitis

Antigen presenting cells (APCs)

76, 105

Cidofovir

163, 168

Antihistamine

217, 218

– topical

167

Antilymphangiogenic therapy

Collagen

22, 52

Antithymocyte globulins

117

Conjunctiva

177

APCs (antigen presenting cells)

105

Conjunctival hyperreactivity

Arlt’s lines

215

- non-specific

211

Artificial anterior chamber

129

Conjunctivalisation

Asthma

209

Conjunctivitis

Astigmatism

123, 136

- giant papillary

209, 217

– irregular

123, 124

– perennial allergic

209, 214

Atopic keratoconjunctivitis see kerato-

– seasonal allergic

209, 213, 214

conjunctivitis

Connexin 43

Auto-limbal transplantation

48, 50

Contact lens

83, 90, 173, 192

226

Subject Index

Contamination

Epitheliotrophic factor

1, 2, 3, 5

Corneal angiogenic privilege

Epithelium

42, 57, 181

Corneal basement membrane dystrophy

– conjunctival

Corneal dissection

– corneal see corneal epithelium

- laser

Erbium:YAG Laser

147

– manual

Everolimus

109, 116

Corneal epithelial thickness

173

Excimer Laser 193-nm

144

Corneal epithelium

58, 180

Extracapsular cataract extraction

138

– basal cells

180

Extracellular matrix

– Bowman’s membrane

180

– intermediate cells

180

– stroma

180

Fibrin

– superficial cells

180

Fibroblast feeder layer

Corneal lymphatic vessel

Fibronectin

2, 22

Corneal nerve

173, 185

FK506

112, 114, 220

Corneal oedema

Flap lift

153, 155

Corneal opacity

Flieringa ring

133

- nummular

165

Fluoroquinolone

158

– persistence

168

FTY 720

116

– subepithelial

165

Fuchs’s endothelial dystrophy

111, 129, 145

Corneal perforation

Functional imaging

173, 203

Corneal surgery, refractive

173, 200

Fungal infection

153

Corneal transplantation

Corneal ulcer

144,199

Corneoscleral tissue

Gas-permeable lens

217

Corticosteroid

52, 114

Gatifloxacin

158

– topical

219

Giant papillary conjunctivitis

Cortisone

112

see conjunctivitis

Cryopreservation

Glutaraldehyde

Cut angle

129

Goblet cell

Cyclophosphamide

112

Graft failure

144

Cyclosporin A (CsA)

52, 109, 112, 114, 118, 163, 220

Graft rejection

105

– topical

169

Graft size

123, 129, 133

– eyedrops

Graft survival

102

Cystic epithelial change

191

Gram-negative organism

153

Cytokeratin profile

Growth factor

5, 23, 24

Cytotoxic T lymphocyte

– angiogenic

- TGF-b1

Guided trephine system see trephine

Daclizumab

112, 117

Delayed type hypersensitivity

Dendritic cells

173, 209

HA-3

105

Dennie-Morgan fold

216

Hand-held trephine see trephine

Descemet’s membrane

188

Hanna trephine see trephine

Digital photography

175

Hay fever (see also conjunctivits,

Donor

123

seasonal allergic)

213

Dry eye

5, 8, 10, 11, 190

Herpes simplex keratitis see keratitis

Dystrophy

144

Hertoge’s sign

216

– stromal

144

HLA

102

- class I molecules

102

– class II molecules

102

Embryonic stem cells

– matching

52, 61, 78, 102

Endothelial cells

188

– typing

52, 102

– density

HLAMatchmaker

102

Epidemic keratoconjunctivitis

– algorithm

104, 107

see keratoconjunctivitis

Horizontal torsion

127

Subject Index

227

Horner-Trantas dot

215

– penetrating

65, 69

H-Y

105

- posterior lamellar

129, 148

Hypersensitivity type I

210

Keratoprothesis

Keratotomy

– radial

138

IL (Interleukin)-1

114

– inhibition

221

IL-2

Laminin

- antagonist

112

Langerhans cells

52, 105, 177, 184

– inhibitor

109

Laser

123

IL-6

114

– femtosecond

123, 148

Immune privilege

LASIK

123, 153

Immune rejection

Leflunomide

112

Immunoglobulin E

210

Limbal stem cells

Immunomodulatory cytokine

– culture

Immunosuppressive molecule

– deficiency

35, 111

Immunosuppressive strategy

109

– causes

Impression cytology

39, 60

– ex vivo expansion

52, 57

Infiltrates

165

Limbal epithelial crypt

Inflammation

Limbal palisades of Vogt

38, 44

Inflammatory cells

173

Limbal region

188

Instrastromal ring segment

Limbal transplantation

Interface haze

65, 68

Lymphangiogenesis

Interferon

163, 169

Lymphocytes

Interleukin (see also IL)

210

- CD4+ T cells

110, 212

Intermediate cells

184

– CD8+ T cells

110, 212

Intraoperative adjustment

123

– Cytotoxic T

Irradiation

Lyophilisation

Keratectomy, phototherapeutic (PTK)

67, 129

Macrolide antibiotic

220

Keratinisation

Macrophage inflammatory protein-1a

Keratitis

83, 158, 159

(MIP-1a)

221

– acanthamoeba

196

Mapping

125

– diffuse lamellar (sands of the Sahara)

154

Mast cell 210

– fungal

158

- degranulation

212

– herpes simplex

69, 91, 118, 159, 216

– stabilizer

217

– infective

153

– agents

218

Keratoconjunctivis

Mechnical barrier

- adenoviral

163

Meesmann’s dystrophy

190

– atopic

209, 215

Metalloprotease

– epidemic

195

– inhibitors

– limbal

215

Methotrexate

112

– superior limbic

MHC (major histocompatibility

– vernal

209, 214

complex)

102, 110

Keratoconus

67, 69, 111, 123, 136, 144, 216

– class II molecule

76, 210

Keratocytes

Microbiological examination

155

- viral persistence in

195

– scraping of material

155

Keratometric refractive power

124

Microenvironment

Keratometry

125

Microscopy

173

Keratopathy

129

– confocal

173, 175

– aphakic/pseudophakic bullous

129, 144

– laser-scanning

176

Keratoplasty

64, 129

– specular

173

– deep anterior lamellar

65, 69

Minor H mismatch

105

– high-risk

90, 109

Minor matching

102

– indications

144

MIP-1a (Macrophage inflammatory protein-1a)

221

– low-risk

94, 104

Mismatches, class I and II

228

Subject Index

MMF see mycophenolate mofetil

Rubbing

212, 216

Molecular typing

107

- degranulation of the mast cells

212

Moxifloxacin

158

Muromonab-CD3

117

Mycobacteria

158

Sands of the Sahara

154

– non-tuberculous

153

Scarring

Mycophenolate mofetil

52, 109, 112, 114

Seasonal allergic conjunctivitis see conjunctivitis

Sequential sector conjunctival epitheliectomy

Severe atopic dermatitis

111

Natamycin 5%

158

Shield cataract

217

Neovascularisation

29, 88

Shield ulcer

215

Neurotrophic keratitis

8, 9, 41

Sirolimus

112, 115, 116

– diabetic

Sjögren’s syndrome

– postherpetic

Slit-scanning technique

175

NMSO3 (sulphated sialyl lipid)

163

– confocal microscopy

175

Non-steroidal anti-inflammatory drug (NSAID)

219

Stem cells, embryonic

NSAID (Non-steroidal anti-inflammatory drug)

219

Stem cell niche

37, 40

Nummular subepithelial infiltrates

Sodium cromoglygate

218

Split antigen

102

Steroid

109

Ocular surface burn

Steroid, topical

111, 166, 167

Ocular surface reconstruction

Stevens-Johnson syndrome

Superficial cells

183

Suture removal

125

p63 (transcriptional factor)

Symblepharon

Pachymetry

194

Systemic immunosuppression

PCIOL (Posterior chamber intraocular lens)

138

– toric

138

Pemphigoid

Tacrolimus

52, 109, 112, 114, 118, 220

Perennial allergic conjunctivitis

T-cell activation

114

see conjunctivitis

Tear film

42, 182

Peritomy

– dynamics

173

Plasma

3, 17

Tear substitute

Posterior chamber intraocular lens (PCIOL)

138

Topical steroid

Postoperative treatment

Topography

125

Progenitor cell

– regularity of

123

Prograf see tacrolismus

TOR inhibitor

109

Proinflammatory cytokine

Toxicity, local

168

Prostaglandin

25, 219

Trachoma

Proteoglycan

Transcriptional factor p63

Pseudogerontoxon

215

Transplant

Pterygium surgery

– allo-limbal

Punctual occlusion

– auto-limbal

Trephination

129

– nonmechanical

129

RAD (Everolimus, Certican)

116

– techniques

130

Rapamune

115

– elliptical

137

Rapamycin

52, 109, 115, 118

– nonmechanical laser

143

RCM (Rostock cornea module)

178

– host

123

Recurrent erosion syndrome

Trephine

138

Regularity

123

– guided system

138, 142

Rejection

– Hanna

143

– acute

110

– hand-held

141

– chronic

110

– motor

141

– endothelial

73, 75

– suction

142

– epithelial

Triple procedure

138

– stromal

Triplet

104

Rheumatoid arthritis

Tylosis

216