AGE-RELATED

MACULAR
DEGENERATION
 SECOND EDITION

AGE-RELATED MACULAR
DEGENERATION
EDITORS

D. VIRGIL ALFARO III, MD

Attending Surgeon
Retina Consultants of Charleston
Faculty Advisor
The Citadel
Charleston, South Carolina

ERIC P. JABLON, MD
Partner
Retina Consultants of Charleston
Charleston, South Carolina

JOHN BARNWELL KERRISON, MD

Partner
Retina Consultants of Charleston
Assistant Clinical Professor
Department of Ophthalmology
Medical University of South Carolina
Charleston, South Carolina

KENNETH A. SHARPE, MD
Partner
Retina Consultants of Charleston
Charleston, South Carolina

MONICA RODRIGUEZ FONTAL, MD

Clinical Trial Director
Retina Consultants of Charleston
Charleston, South Carolina
Acquisitions Editor: Ryan Shaw
Product Development Editor: Kate Marshall
Production Project Manager: Bridgett Dougherty
Senior Manufacturing Coordinator: Beth Welsh
Marketing Manager: Stephanie Manzo
Design Coordinator: Holly McLaughlin
Production Service: SPi Global

Second Edition

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First Edition © 2006, Lippincott Williams & Wilkins

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Library of Congress Cataloging-in-Publication Data

Age-related macular degeneration (Alfaro)
Age-related macular degeneration / editors, D. Virgil Alfaro, III, Eric P. Jablon, John B. Kerrison, Kenneth
A. Sharpe, Monica Rodriguez-Fontal. — Second edition.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-1-4511-5169-5
I. Alfaro, D. Virgil, III, editor of compilation.II. Jablon, Eric P., editor of compilation. III. Kerrison, John
B., editor of compilation.IV. Sharpe, Kenneth A., editor of
compilation.V. Rodriguez-Fontal, Monica, editor of compilation.VI. Title.
[DNLM:1. Macular Degeneration.2. Choroidal Neovascularization.WW 270]
RE661.D3
618.97'7735—dc23
2014000607

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10 9 8 7 6 5 4 3 2 1
 DEDICATION

This textbook is dedicated to Stephen J. Ryan, MD. Steve succumbed to cancer

this year, reminding us all of life's fragility and fleeting nature.
Steve's introduction to ophthalmology and retina began at the Wilmer Eye
Institute at Johns Hopkins University in the early 1970s. Dr. Edward Maumenee
influenced many young ophthalmologists during his tenure as professor and
chairman. Dr. Morton F. Goldberg, who was Steve's chief resident and became
chairman of the Wilmer Eye Institute, recalls that Steve was a glutton for work
and highly motivated to do research. Steve had a severe allergy to cats, rabbits,
and other animals; however, this did not impair his research or home life where
his landlady had pets. In fact, he wore a surgeon's mask at work in the lab and at
home. This illustrates how energetic he was, allowing nothing to get in his way.
Steve served as chief resident at Wilmer and went on to become chairman of
ophthalmology at the Los Angeles County Hospital and the University of
Southern California School of Medicine.
For nearly 40 years, Steve dedicated his professional life to the University of
Southern California and the Doheny Eye Institute. Under his leadership and
guidance, the reputation of the institute soared, and it has been listed as one of
the top 10 eye centers in the country for two decades.
Steve had many passions in the academic world of ophthalmology, but his
name will always be remembered as one considers management of the severely
injured eye. Using animal models, he defined the importance of the vitreous as it
relates to proliferative vitreoretinopathy after penetrating eye injury. He and his
international fellow from Ireland, Paul Cleary, published numerous papers that
became widely known as the Ryan-Cleary model of ocular trauma.
As an editor of this textbook, it would be an oversight not to mention the
great personal impact that Steve had on my professional life. His compelling
personality and ability to affect those whom he trained has had a life-long
influence. I have no doubt that there are many, many ophthalmologists around
the world who have also been blessed by his mentorship.
D. Virgil Alfaro III, MD
CONTRIBUTORS

D. Virgil Alfaro III, MD

Attending Surgeon
Retina Consultants of Charleston
Faculty Advisor
The Citadel
Charleston, South Carolina

Madhu Amara, MD
University Retina
Oak Forest, Illinois

Gisela Barcat Angelelli, MD

Ophthalmologist
Department of Retina
Santa Lucia Ophthalmologic Hospital
Buenos Aires, Argentina

Richard J. Antcliff, MD, FRCOphth

Consultant Ophthalmic Surgeon
Department of Ophthalmology
Royal United Hospital
Bath, Somerset, England

J. Fernando Arévalo, MD, FACS

Professor
Wilmer Eye Institute
Johns Hopkins University
Baltimore, Maryland
Chief
Vitreoretinal Division
The King Khaled Eye Specialist Hospital
Riyadh, Kingdom of Saudi Arabia
Irene A. Barbazetto, MD
Clinical Assistant Professor
Department of Ophthalmology
School of Medicine
New York University
Attending Physician
Department of Ophthalmology
Manhattan Eye and Ear Hospital, NSLIJ
New York, New York

Harit K. Bhatt, MD
Clinical Assistant Professor
Illinois Eye and Ear Infirmary
University of Illinois at Chicago
Chicago, Illinois
Attending Physician/Partner
University Retina and Macula Associates
Bedford Park, Illinois

Antonio López Bolaños, MD

Associate Professor
Department of Retina
Instituto de Oftalmologia
Hospital Conde de Valenciana
Ciudad de México, Mexico

Jamel L. F. Brown, BS, MA

Retina Consultants of Charleston
Ladson, South Carolina

Wissam Charafeddin, MD
Retina Fellow
Retina Department
Centro de Oftalmologia Barraquer
Barcelona, Spain

Yan Chen, PhD

Postdoctoral Fellow
The Vanderbilt Eye Institute
Vanderbilt University
Nashville, Tennessee

Lucienne C. Collet, MD
Admiravisión
Department of Ophthalmology
Barcelona, Spain

Borja F. Corcóstegui, MD
Professor
Department of Ophthalmology
Universitat Autonomous Barcelona
Barcelona, Spain

Michelle L. Crouse, MA
Clinical Trial Coordinator
Retina/Vitreous
Retina Consultants of Charleston
Charleston, South Carolina

Matías E. Czerwonko, MD
Advisor
Department of Quality and Patient Safety
Hospital Universitario Austral
Buenos Aires, Spain

Mary Alexander Deas-Hamrick

Clinical Trial Coordinator
Retina Consultants of Charleston
Charleston, South Carolina

Francis Char DeCroos, MD

Retina Fellow
Department of Ophthalmology
Wills Eye Institute/Mid Atlantic Retina
Philadelphia, Pennsylvania

Diana V. Do, MD
Associate Professor of Ophthalmology
Wilmer Eye Institute
School of Medicine
Johns Hopkins University
Baltimore, Maryland

Jay S. Duker, MD
Professor
Department of Ophthalmology
School of Medicine
Tufts University
Chairman
Department of Ophthalmology
Tufts Medical Center
Boston, Massachusetts

Carlos F. Fernández, MD
Chief
Department of Ophthalmology
Clinica Oftalmolaser
Lima, Peru

Monica Rodriguez Fontal, MD

Clinical Trial Director
Retina Consultants of Charleston
Charleston, South Carolina

Reinaldo A. Garcia, MD
Associate Professor
Retina and Vitreous Disease
Clinica Oftalmologica El Vinedo
Valencia, Edo. Carabobo, Venezuela

Gerardo Garcia-Aguirre, MD
Attending Physician
Retina Department
Asociacion para Evitar la Ceguera en Mexico
Clinical Professor of Ophthalmology
Escuela de Medicina
Instituto Tecnológico y de Estudios Superiores de Monterrey
Mexico City, Mexico
Jose M. Garcia-Gonzalez, MD
Retina Fellow
Section of Ophthalmology
University of Chicago
Chicago, Illinois

Peter L. Gehlbach, MD, PhD

Associate Professor
Department of Ophthalmology
School of Medicine
Johns Hopkins University
Associate Professor
Biomedical Engineering
School of Medicine
Johns Hopkins University
Baltimore, Maryland

Isobel V.L. Goldsmith

Clinical Trial Coordinator
Retina Consultants of Charleston
Charleston, South Carolina

JoAnna L. Goulah, PhD

Clinical Trial Research Coordinator
Retina Department
Charleston Neuroscience Institute
Charleston, South Carolina

Sundeep Grandhe, MD
University Retina
Oak Forest, Illinois

Gabriela E. Granella, MD
Director and Retina Specialist
Oftalmológico Santa Lucía
Buenos Aires, Argentina

Craig M. Greven, MD
Professor and Chairman
Department of Ophthalmology
School of Medicine
Wake Forest University
Winston-Salem, North Carolina

Mariana Ingolotti, MD
Assistant Professor
Department of Pathophysiology
Austral University
Buenos Aires, Argentina

Eric P. Jablon, MD
Partner
Retina Consultants of Charleston
Charleston, South Carolina

Timothy L. Jackson, PhD, FRCOphth

HEFCE Senior Clinical Lecturer
School of Medicine
King's College London
Consultant Ophthalmic Surgeon
Department of Ophthalmology
King's College Hospital
London, United Kingdom

Rama D. Jager, MD
Attending Physician/Partner
University Retina and Macula Associates
Bedford Park, Illinois

John Barnwell Kerrison, MD

Partner
Retina Consultants of Charleston
Assistant Clinical Professor
Department of Ophthalmology
Medical University of South Carolina
Charleston, South Carolina

Claudia M. Krispel, MD, PhD

Vitreoretinal Fellow
Department of Ophthalmology
Wilmer Eye Institute
Johns Hopkins Hospital
Baltimore, Maryland

Nancy Kunjukunju, MD
Assistant Professor
School of Medicine
University of Missouri–Kansas City
Assistant Program Director
Department of Ophthalmology
University of Missouri–Kansas City
Kansas City, Missouri

Henry Alexander Leder, MD

Assistant of Ophthalmology
Department of Ophthalmology
School of Medicine
Johns Hopkins University
Baltimore, Maryland

Maria Lozano-Vazquez, MD
Ophthalmology Attending
Department of Surgery
University of Santiago de Compostela
Santiago de Compostela, Spain
Ophthalmology Attending
Department of Surgery
Hospital Naval
Ferral, Spain

Ana Machín Mahave, MD

Attending Physician
Department of Ophthalmology
Sierrallana Hospital
Torrelavega, Cantabria, Spain

Mariana Mata-Plathy, MD
Glaucoma Specialist
Glaucoma Service
Clinica Oftalmologica El Viñedo
Valencia, Carabobo State, Venezuela

Pablo Carnota Méndez, MD

Attending Physician
Department of Retina and Vitreous Surgery
Centro de Ojos de La Coruña
La Coruña, Spain

Keisuke Mori, MD, PhD

Professor
Department of Ophthalmology
Saitama Medical University
Iruma, Saitama, Japan

Amelia Nelson
Student
Retina Consultants of Charleston
Charleston, South Carolina

Quan Dong Nguyen, MD, MSc

Associate Professor of Ophthalmology
Diseases of the Retina and Vitreous, and Uveitis
Wilmer Eye Institute
School of Medicine
Johns Hopkins University
Baltimore, Maryland

Maximiliano Olivera, MD
Assistant Lecturer
Molecular and Cell Medicine
Facultad de Ciencias Biomédicas
Universidad Austral
Pilar, Buenos Aires, Argentina

Veronica Oria, MD
Retina and Vitreous Service
Clínica Oftalmológica El Vinedo
Valencia, Carabobo State, Venezuela

Carlos E. Ortiz, BS
Retina Consultants of Charleston
Charleston, South Carolina

Gabriela Papa-Oliva, MD
Associate Professor
Department of Ophthalmology
Hospital Miguel Pérez Carreño. IVSS
Caracas, Venezuela

Samir Patel, MD
Co-founder and President
Ophthotech Corporation
New York, New York

Robert Petrarca, MBBS, BSc (Hons), MCOptom

Ophthalmologist
Department of Ophthalmology
King's College Hospital NHS Foundation Trust
Honorary Research Fellow
King's College London
London, United Kingdom

Hugo Quiroz-Mercado, MD
Director of Ophthalmology
Vitreo-Retina Specialist
Denver Health Medical Center
Professor of Ophthalmology
School of Medicine
University of Colorado
Denver, Colorado

Ernesto Romera Redondo, MD

Ophthalmologist
Department of Ophthalmology
Hospital Sierrallana
Torrelavega, Cantabria, Spain

Caio V. Regatieri, MD, PhD

Assistant Professor
Department of Ophthalmology
Tufts University
Boston, Massachusetts
Adjunct Professor
Department of Ophthalmology
Federal University of São Paulo
São Paulo, Brazil

Carl D. Regillo, MD, FACS

Director of Retina Research
Wills Eye Institute
Professor of Ophthalmology
Thomas Jefferson University
Philadelphia, Pennsylvania

Richard B. Rosen, MD, FACS, FASRS

Professor and Vice Chair
Director of Research
Surgeon Director and Chief
Retinal Services
Department of Ophthalmology
New York Eye and Ear Infirmary
New York, New York

Scott D. Schoenberger, MD
Instructor
Department of Ophthalmology
Vanderbilt University Medical Center
Nashville, Tennessee

Kenneth A. Sharpe, MD
Partner
Retina Consultants of Charleston
Charleston, South Carolina

Veeral Sheth, MD
Clinical Assistant Professor
Department of Ophthalmology
University of Illinois at Chicago
Chicago, Illinois
Director
Scientific Affairs
University Retina and Macula Associates
Lemont, Illinois

Jason S. Slakter, MD
Clinical Professor
Department of Ophthalmology
School of Medicine
New York University
Partner
Vitreous–Retina–Macula Consultants of New York
New York, New York

Paul Sternberg Jr., MD

G.W. Hale Professor and Chairman
Department of Ophthalmology
Vanderbilt University Medical Center
Nashville, Tennessee

Luis M. Suárez Tata, MD

Chief
Retina-Vitreo
Clínica oftalmológica El Viñedo
Valencia, Carabobo State, Venezuela

Carlos Méndez Vázquez, MD

Director
Department of Retina and Vitreous Surgery
Centro de Ojos de La Coruña
La Coruña, Spain

Robin A. Vora, MD
Fellow, Medical Retina
Department of Ophthalmology
New England Eye Center
Boston, Massachusetts
Attending Physician
Department of Ophthalmology
Kaiser Permanente
Oakland, California
Andre J. Witkin, MD
Retina Fellow
Retina Department
Wills Eye Institute
Philadelphia, Pennsylvania

Lawrence Yannuzzi, MD
Professor of Clinical Ophthalmology
Columbia University Medical School
Founder and Chairman
Vitreous–Retina–Macula Consultants of New York
New York, New York
FOREWORD

It is an honor for me to write the foreword to the second edition of Age-Related

Macular Degeneration, by Dr. Virgil Alfaro and his colleagues at Retina
Consultants of Charleston.
Our colleagues at Retina Consultants of Charleston represent the ideal model
to emulate, as they are academic physicians in private practice. Collectively, they
have published eight textbooks. Two of these books, The Essentials of
Neuroophthalmology and Vitreoretinal Surgery of the Injured Eye, are regarded
as true classics in their field. On Index Medicus, I find many peer-reviewed
articles that they have authored in the most selective ophthalmology journals.
The publication of this textbook comes at a poignant time, as we see historic
and significant advances in the diagnosis and treatment of age-related macular
degeneration (AMD). The book has excellent chapters written on intravenous
fluorescein angiography, indocyanine green angiography, and optical coherence
tomography. These narratives provide classic teaching and instruction for
residents and fellows to obtain a solid foundation in their use to diagnose and
treat AMD.
Certainly, one of the most significant changes that we have noted in our field
is the classification system of choroidal neovascular membranes in AMD. Age-
Related Macular Degeneration, second edition, provides a concise and
understandable discussion of this classification system.
The advent of anti-VEGF therapy has revolutionized the treatment of wet
AMD. Newer treatments and newer treatment regimens have contributed to
improvement of vision in these patients and in prevention of visual loss.
Macugen, Avastin, and Lucentis remain potent and reliable sources of anti-
VEGF therapy, and newer drugs such as Eylea appear to provide patients with
more sustained efficacy necessitating fewer injections.
Lastly, their “Frontiers of Therapy” section provides up-to-date information
for treating AMD secondary to geographic atrophy through cell transplantation
and pharmacologic therapies.
Francisco Gomez-Ulla, MD
 Dean of Ophthalmology
Universidad de Santiago de Compostela
Santiago de Compostela, Spain
PREFACE

We should not forget our mentors, those who went before us and influenced us to
our own personal successes. Just as we are busy and our schedules are saturated
with patients, surgeries, meetings, and the like, so, too, our teachers lived and
flourished. Somehow, they managed to find time to guide and teach us. Those
who influenced me were and are personally involved with the current status of
the diagnosis and treatment of age-related macular degeneration.
In 1984, Drs. Stanley Chang, Harvey Lincoff, and D. Jackson Coleman
walked the eighth floor of Cornell University Medical School and the New York
Hospital and took me under their wing. These iconic figures in the world of
vitreoretinal diseases taught me and countless other students, residents, and
fellows the great balance of clinical practice and significant research.
Stephen J. Ryan served as chairman of the Los Angeles County Hospital
department of ophthalmology and president of the Doheny Eye Institute. During
my residency, he had put together a world class group of retina and macula
specialists, including Peter E. Liggett, John Lean, Edgar Thomas, and others.
They all served as investigators in the Macular Photocoagulation Studies that
were to provide important clinical guidelines for years to follow. Coresidents
Baruch Kuppermann, Pravin Dugel, Victor Gonzales, Keith Pince, Vinh Tran,
Colin Ma, David Wagner, James Tsai, and others immersed themselves in this
great clinical pearl, called the L.A. County Hospital.
Marvin Sears and Peter Liggett provided the leadership and mentorship
during my fellowship at the Yale Eye Center. Marvin was proudest of his great
teaching skills and his publication in Science that showed for the first time the
presence of prostaglandins in the eye.
Age-Related Macular Degeneration represents 2 years of hard work by my
fellow editors and authors. Our plan was to author, edit, and publish a body of
work that would be a source of salient information for our students and
colleagues. Each book chapter has been written to stand alone as a significant
and inclusive text that the reader can use to learn and understand the topic at
hand. It is our hope that patients around the world will benefit from our labor, as
their doctors read and study this book.
D. Virgil Alfaro III, MD
Charleston, South Carolina
ACKNOWLEDGMENTS

The editors are indebted to the hard work and dedication of Sarah M. Granlund,
who served as our project manager during all phases of this textbook. Indeed,
her great diplomacy and administrative skills provided the editorial team with
someone to turn to for help to bring this project to completion.
CONTENTS

Dedication
Contributors
Foreword
Preface
Acknowledgments

SECTION 1 ANATOMY, PHYSIOLOGY, AND PATHOLOGY OF

THE MACULA
1 Normal Anatomy of the Macula
J. Fernando Arévalo, Carlos F. Fernández

2 Animal Models for Age-Related Macular Degeneration

Matías E. Czerwonko, Mariana Ingolotti, Amelia Nelson, Jamel L. F. Brown, D. Virgil Alfaro III

3 Pathogenesis and Pathophysiology of Age-Related Macular

Degeneration
Ana Machín Mahave, Ernesto Romera Redondo, John Barnwell Kerrison

4 Etiology of Late Age-Related Macular Disease

Maximiliano Olivera

5 Histopathology of Age-Related Macular Degeneration

Mariana Ingolotti, Eric P. Jablon, John Barnwell Kerrison, Hugo Quiroz-Mercado, D. Virgil Alfaro III

SECTION 2 CLASSIFICATION OF AGE-RELATED MACULAR

DEGENERATION
6 Classification of Exudative Age-Related Macular Degeneration
Michelle L. Crouse, Wissam Charafeddin, Gerardo Garcia-Aguirre, Gabriela E. Granella, D. Virgil
Alfaro III, John Barnwell Kerrison, Eric P. Jablon

7 Polypoidal Choroidal Vasculopathy

Gisela Barcat Angelelli, D. Virgil Alfaro III, Lawrence Yannuzzi, Richard J. Antcliff

8 Retinal Angiomatous Proliferation in Age-Related Macular

Degeneration
Gabriela E. Granella, Eric P. Jablon

9 Differential Diagnosis of Age-Related Macular Degeneration

Gabriela Papa-Oliva, Monica Rodriguez Fontal, D. Virgil Alfaro III
SECTION 3 IMAGING
10 Fluorescein Angiography
Gisela Barcat Angelelli, Eric P. Jablon, Richard B. Rosen, D. Virgil Alfaro III

11 Indocyanine Green Angiography in Age-Related Macular Degeneration

Irene A. Barbazetto, Jason S. Slakter

12 Fundus Autofluorescence Imaging in Age-Related Macular

Degeneration
Monica Rodriguez Fontal, Craig M. Greven, D. Virgil Alfaro III, Eric P. Jablon

13 Optical Coherence Tomography

Caio V. Regatieri, Robin A. Vora, Jay S. Duker

SECTION 4 NONEXUDATIVE AGE-RELATED MACULAR

DEGENERATION
14 Antioxidants and Age-Related Macular Degeneration: Clinical
Associations and Basic Mechanisms
Yan Chen, Paul Sternberg Jr.

SECTION 5 EXUDATIVE AGE-RELATED MACULAR

DEGENERATION
15 Pre–Anti-VEGF Era
Reinaldo A. Garcia, Veronica Oria, Luis M. Suárez Tata, Mariana Mata-Plathy

16 Macugen for Age-Related Macular Degeneration

Pablo Carnota Méndez, Carlos Méndez Vázquez

17 Lucentis (Ranibizumab)
JoAnna L. Goulah, D. Virgil Alfaro III, Carlos E. Ortiz

18 Bevacizumab for the Treatment of Exudative Age-Related Macular

Degeneration
Lucienne C. Collet, Borja F. Corcóstegui

19 VEGF Trap-Eye for Neovascular Age-Related Macular Degeneration

Claudia M. Krispel, Quan Dong Nguyen, Diana V. Do

20 Radiation for the Treatment of Wet Age-Related Macular Degeneration

Robert Petrarca, Timothy L. Jackson

21 Combination Therapy for Neovascular Age-Related Macular

Degeneration
Francis Char DeCroos, Andre J. Witkin, Carl D. Regillo
 22 Fovista, a Pegylated Anti-PDGF Aptamer in Wet AMD
Mary Alexander Deas-Hamrick, Samir Patel, Isobel V.L. Goldsmith, D. Virgil Alfaro III

23 Complications of Intravitreal Injections

Maria Lozano-Vazquez, Jose M. Garcia-Gonzalez, Veeral Sheth, Harit K. Bhatt, Sundeep Grandhe,
Madhu Amara, Rama D. Jager

SECTION 6 FRONTIERS OF THERAPY

24 Introduction to Gene Therapy and Related Techniques for Retinal
Disorders and Age-Related Macular Degeneration
Henry Alexander Leder, Keisuke Mori, Peter L. Gehlbach

25 New Pharmacotherapies for Age-Related Macular Degeneration

Scott D. Schoenberger, Paul Sternberg Jr.

26 Artificial Vision, Retinal Implants, and Implantable Telescope

Nancy Kunjukunju

27 Retinal Transplantation
Antonio López Bolaños
Index
SECTION 1

ANATOMY, PHYSIOLOGY, AND PATHOLOGY

OF THE MACULA
 1
NORMAL ANATOMY OF THE
MACULA
J. FERNANDO ARÉVALO • CARLOS F. FERNÁNDEZ

INTRODUCTION
Francisco Buzzi in Milan, Italy, was the first to anatomically define the macula
at the end of the 18th century (1782–1784). He described it as the yellow portion
of the posterior retina, lateral to the optic nerve, with a depression in its center.
Also in the 18th century (1797), Fragonard, a French ophthalmologist, made a
very detailed description of the foveal zone, but he failed to mention the central
foveola. In addition, Soemmering (1795–1798) described the macula lutea;
however, he thought that the foveola was a hole or foramen (foraminulum
centrale retinae) and correlated it to the blind spot of the visual field. Finally,
Michaelis in 1838 established the role of the macula, and it was confirmed by
Müller in 1856. At the end of the 19th century, Tratuferi in Italy showed the first
schematic drawings with the topographic localization of the retinal layers. The
relationship between the retinal axonal layer, cones, and rods was established by
Ramon y Cajal in 1894 (1).
The macula is recognized as the specialized region of the retina in charge of
high-resolution visual acuity. Anatomically, it can be defined as the central part
of the posterior retina that contains xanthophyll pigment and two or more layers
of ganglion cells. In this chapter, we focus primarily on the aspects of the normal
anatomy of the macula as an introduction to the understanding and management
of age-related macular degeneration.

EMBRYOLOGY
The neural components of the eye are an extension of the forebrain, and thus part
of the central nervous system (Table 1.1).

Table 1.1 DEVELOPMENT OF THE SENSORY RETINAa

aThe marginal layer free from nuclei (not included) appears between the 4th and 5th wk, after which it
disappears. In addition, the transient layer of Chievitz (not included) appears between the 6th wk and 3rd
mo and disappears after the 3rd mo.

The Neuroretina
The embryogenesis of the neuroretina occurs during the first month of life. The
forebrain consists of a single layer of neuroectodermal cells. The optic vesicle
extends laterally from the forebrain and then invaginates to form the optic cup.
There is a double layer of neuroectodermal cells in the optic cup; the apices are
together and basal aspects apart. The macular area appears at the end of the 4th
week.
The inner layer of neuroectoderm becomes the sensory retina posteriorly.
Part of the inner limiting membrane is the basement membrane of the sensory
retina, next to the vitreous. The foveal pit forms late in embryonic life, and
morphologic maturity does not occur until the age when it is possible to obtain
20/20 visual acuity in small children (2). The induction of the foveal pit requires
a normal pigment epithelium (3). The inner layer of neuroectoderm is divided
into the inner neuroblastic layer (Müller, amacrine, and ganglion cells) and the
outer neuroblastic layer (cone and rod, horizontal, and bipolar cells). Between
the inner neuroblastic layer and the outer neuroblastic layer is the transient fiber
layer of Chievitz; this layer will progressively disappear.
The central and peripheral retinae start to differentiate between the first and
third months. The ganglion cell layer becomes thicker, as do the inner plexiform
layer and the amacrine cell layer. The cones appear in the 5th month. They are a
protoplasmic extension of the outer neuroblastic layer (4–6). The rods appear in
the 6th month (5). The macula becomes thinner in the 7th month because the
cells of the different layers move laterally, and the foveal pit appears more
evident (Fig. 1.1) (7,8).

Figure 1.1 Diagram showing the development of the human fovea. A. Human fovea at birth. B. Human
fovea at 45 months. C. Human fovea at 72 years of age. Black lines mark the width of the rod-free foveola;
at birth, the foveola is wider (A) than at 45 months (B). Full foveal development is not complete until
sometime between 15 and 45 months of age.

The Retinal Pigment Epithelium

The outer layer of neuroectoderm (toward the sclera) becomes the retinal
pigment epithelium (RPE), the pigmented ciliary epithelium, and the iris dilator
muscle. The basement membrane of the RPE forms the innermost part of the
Bruch's membrane (9).
Pigmentation of the neuroectodermal cells occurs early and is completed
during embryonic life (10), whereas pigmentation of the uveal stroma, which is
derived from the neural crest, starts much later and is not complete until several
weeks after birth (9). The melanin granules of the neuroectoderm are larger and
chemically different from those of the uveal stroma (11). Moreover, melanin
content of the RPE is similar in all persons, regardless of race, in contrast to the
amount of uveal stromal pigmentation, which corresponds to the racial
pigmentation of the skin and hair (10).

Vascularization
Vascularization of the retina begins during the 16th week of gestation at the optic
nerve head (12) and normally reaches the ora serrata nasally by term. It is not
quite complete temporally at term because the distance from the optic nerve head
to the ora is greater.
There are differing theories as to how vascularization of the macula occurs.
Some have proposed that the capillary-free zone (CFZ) develops by regression
of previously formed capillaries, because the retina is thin enough in this area
that sufficient oxygen can diffuse inward from the choroidal circulation (12).
Others believe that the CFZ forms primarily, with the embryonic vessels
gradually encircling the center of the fovea and with no evidence of regression
(13).

The Choroid
In the first phase, at 4 weeks, the choroid begins to take form in the
undifferentiated mesenchyma surrounding the optic cup. At 5 to 6 weeks,
endothelial tubes near the pigment epithelium differentiate into capillaries.
Simultaneously, the Bruch's membrane and the vortex veins appear. With the
appearance of the posterior ciliary arteries at 8 weeks, the choriocapillaris is
fully established as a discrete layer.
During the growth phase in the 3rd month, the anterior capillaries arrange
themselves in a linear and radial pattern. The anterior supply of the
choriocapillaris is complete by 3 months, even though the ciliary body and iris
are not yet formed. The capillaries drain into the supra- and infraorbital venous
plexi.
The second phase of choroidal growth includes the formation of large
vessels (Haller's layer) at 4 months. The third phase begins around the 5th month
and is characterized by the formation of medium-sized vessels posterior to the
equator. Anterior to the equator, there are only two layers of vessels, the
choriocapillaris and medium-sized vessels.
Pigmentation of the choroidal melanocytes commences in the 5th month and
continues until after birth, beginning outward near the sclera and progressing
inward toward the Bruch's membrane. The peripapillary, intrascleral vascular
circle of Haller and Zinn develops between 3 and 6 months. Between the 6th and
10th month, the major arterial circle of the iris forms recurrent arterial branches
that supply the anterior choroid during its rapid growth phase (14).

TOPOGRAPHIC ANATOMY
The terms macula and fovea are used in different ways by the anatomist and the
clinician. Anatomically, the macula (macula lutea or central retina) is defined as
the portion of the posterior retina that contains xanthophyll and two or more
layers of ganglion cells. This region is about 5.5 mm in diameter and is centered
approximately 4 mm temporal and 0.8 mm inferior to the center of the optic disk
(15). It corresponds clinically to the posterior pole and is approximately bounded
by the superior and inferior temporal vascular arcades. On the basis of
microscopic anatomy, the macular area can be further subdivided into several
zones (Fig. 1.2).

Figure 1.2 Topographic anatomy of the normal macula. On the basis of microscopic anatomy, the macular
area can be further subdivided into several zones. I. Fovea containing the foveola (1). II. Parafovea. III.
Perifovea.

What is clinically referred to as the macula corresponds to the anatomic

fovea. The fovea (fovea centralis) is a depression in the inner retinal surface in
the center of the macula. It measures approximately 1.5 mm, or one disk
diameter in size, and it is more heavily pigmented than the surrounding retinal
tissue. The central floor of the fovea is called the foveola. The anatomic foveola,
often referred to clinically as the fovea, measures approximately 0.35 mm in
diameter. It lies within the CFZ, which measures approximately 0.5 mm in
diameter in most patients. A small depression in the center of the foveola is
called the umbo, where the retina is only 0.13 mm thick. A 0.5-mm-wide ring
zone where the ganglion cell layer, inner nuclear layer, and outer plexiform layer
of Henle are the thickest is called the parafoveal area. This zone is in turn
surrounded by a 1.5-mm zone referred to as the perifoveal area (16).

CLINICAL APPEARANCE
The anatomic subdivisions of the macula are ill-defined ophthalmoscopically.
The margins of either the 0.35-mm diameter foveola or the 1.5-mm diameter
fovea are difficult to define. A poorly defined zone of greater pigmentation (one-
fourth to one disk diameter in size) corresponds to the center of the macula,
which is maximum in the foveolar area. The foveal reflex is present in most
normal eyes, and it lies just in front of the center of the foveola (Fig. 1.3). We
can see the foveal depression with the narrow slit lamp beam.
Figure 1.3 Clinical appearance of the normal macula. A. Fundus in a young patient. B. Fundus in an adult
patient. C. Fundus in a moderately myopic patient. D. Tessellated fundus in an older patient. Large
choroidal vessels are visible in the macular area in (D) because of relative hypopigmentation of the RPE.
(B and D courtesy of Dario Fuenmayor-Rivera and Enrique Murcia.)

The central retinal artery supplies the inner half of the retina. It usually
divides into a superior and inferior trunk within the optic nerve head. These
trunks divide into a nasal branch and a temporal branch. The corresponding
retinal venous branches have much the same distribution as the arteries. They
give off arteriolar and venular branches that posteriorly occur primarily at right
angles to the parent vessel. They divide in a dichotomous way as they course
peripherally. The right-angle branches are referred to as first-order arterioles and
venules. One or more cilioretinal arteries derived from the ciliary circulation
supply the papillomacular area in approximately 20% of patients (17), and
occasionally the entire macula (Fig. 1.4). The blood vessel walls are normally
transparent (16,18).

Figure 1.4 FA shows a cilioretinal artery. (Courtesy of Dario Fuenmayor-Rivera and Enrique Murcia.)

GROSS ANATOMY
The retina loses its normal transparency within hours after death. Xanthophyll is
a yellow pigment made up of two carotenoids (zeaxanthin and lutein) (19,20). It
is apparent in the center of the macula and is highly concentrated in the foveolar
area. The maximal concentration of xanthophyll pigment is in the outer nuclear
and outer plexiform layers. However, we can find xanthophyll within the inner
plexiform layer inside the foveal area (19,21–23). The greatest concentration of
pigment is in the cones' central axons (23).
The entrance site for the short and long posterior ciliary arteries can be
visualized after removal of the choroid. There is no retinal circulation at the
foveola. The short posterior ciliary arteries are concentrated in the macular area
along the temporal margin of the fovea and the peripapillary area. The temporal
long posterior ciliary artery and ciliary nerve enter about one and one-half disk
diameters temporal to the center of the fovea (16).

HISTOLOGY
In the macula, we find the thickest portion of the retina surrounding the thinnest
portion, the foveolar area. At the umbo, the neural retina consists only of the
inner limiting membrane, the Henle's fiber layer, the outer nuclear layer, the
external limiting membrane, and the photoreceptors' outer and inner segments
(Fig. 1.5). The typical neural–retinal histology is established out of the foveola
(Fig. 1.6). The neural retina consists of the inner limiting membrane, the nerve
fiber layer (NFL), the ganglion cell layer, the inner plexiform layer (synaptic
processes between bipolar cells and ganglion cells), the inner nuclear layer
(nuclei of bipolar, horizontal, amacrine, and Müller cells), the outer plexiform
layer (synaptic processes between bipolar cells and photoreceptors), the outer
nuclear layer (nuclei of the rods and cones), the external limiting membrane
(formed by cell junctions between photoreceptors and the terminal optical
processes of Müller cells), and the photoreceptor layer (rod and cone cells). The
bipolar cells, ganglion cells, fibers of the outer plexiform layer (Henle's fiber
layer), and Müller cells are displaced circumferentially and show an oblique
orientation in the macula that causes thickening of the marginal zone and a
central thinning of the retina, forming the fovea.
Figure 1.5 Histology of the normal macula. CFZ, capillary-free zone; f, foveola; u, umbo.
(Microphotograph courtesy of Dario Savino-Zari.)
Figure 1.6 Schematic representation of the human retina illustrating its organization into discrete layers.
AC, amacrine cell; BC, bipolar cell; BM, Bruch's membrane; C, cone photoreceptor; ELM, external
limiting membrane; GC, ganglion cell; HC, horizontal cell; ILM, internal limiting membrane; M, Müller
cell; NFL, nerve fiber layer; R, rod photoreceptor; RPE, retinal pigment epithelium.

Müller cells are modified glial cells. They span the region from the internal
limiting membrane to the external limiting membrane, and they give support to
the neural elements of the retina. The internal limiting membrane consists of a
basement membrane, which is a surface modification of the vitreous body, and
the expanded vitreal processes of Müller cells. This membrane is relatively thick
in the macular region except in the area of the foveola. The internal limiting
membrane serves as an anchoring structure for the collagen framework of the
vitreous. The outer limiting membrane is formed by junctional complexes
between cell membranes of the Müller cells and inner segments of
photoreceptors. Müller cells are connected to the visual cells by a system of
terminal bars (24,25). These junctional complexes probably provide at least a
partial barrier to the passage of large molecules in either direction.
The retinal blood vessels supply the inner half of the retina. The major
branches of the retinal arterial system have the structure of small arteries,
persisting even beyond the equator (15). Retinal arteries do not have an internal
elastic lamina; however, they have a well-developed muscularis (five to seven
layers of smooth muscle cells posteriorly; one or two layers peripherally). Near
the optic disk, the retinal veins have three to four layers of smooth muscle cells,
and after a short distance, the muscle cells are replaced by fibroblasts. There is
controversy concerning the pattern of distribution of the capillary network in the
retina (diffuse arrangement or a two- or three-tier arrangement) (15,26,27). The
superficial network is predominantly postarteriolar and the deep network
prevenular. There is a distinct radial peripapillary capillary network; this
network richly interconnects with the inner retinal capillary layer (27). The large
arterioles and venules of the retinal circulation travel in the NFL and ganglion
cell layer. Close to the CFZ (0.4–0.5 mm in diameter), the capillaries form a
single layer, but elsewhere, the capillaries are present in two or more layers and
extend into the inner nuclear layer. The CFZ is normally vascularized during
prenatal development of the retina. This vascularization undergoes spontaneous
capillary obliteration just before or shortly after birth, forming the CFZ (28).
The foveola is composed entirely of cones; the central 100 μm of the foveola
contains only red and green cones (29). The peak foveal cone density averages
nearly 200,000 per mm2 and falls rapidly with increasing eccentricity, such that
cone density decreases nearly 10-fold within 1 mm of the umbo. Blue cone
density is highest in a zone between 100 and 300 μm from the center of the
fovea. The foveal cones take on a more rodlike shape; blue cones in the macula
tend to have inner segments 10% taller and a more cylindrical shape than their
red and green counterparts (29). Rod cells differ from cones, with their outer
segments consisting of stacks of flattened membrane disks that are separate from
the plasma membrane.
The RPE is a monolayer of hexagonal cells densely adherent to one another
by a system of tight cellular junctions or terminal bars that make up the outer
blood-retinal barrier, which maintains the subretinal space in a state of
deturgescence. In the fovea, there are 30 cones per RPE cell, while in the
periphery, there are 22 rods per RPE cell. Interdigitation of the RPE cells with
the rod and cone outer segments provides only a tenuous adhesion of the RPE to
the sensory retina. The RPE cells in the macular region are taller and contain
increased amounts of melanin pigment than elsewhere (30,31). There is an
inverse relationship between melanin and lipofuscin pigment concentration in
the RPE. Lipofuscin concentration increases initially during the first two decades
of life, and then again in the sixth decade. The concentration of lipofuscin in the
RPE is significantly greater in light- than dark-skinned persons, whereas the
concentration of melanin in the pigment epithelium is similar in light- and dark-
skinned persons. The melanin content of the pigment epithelium and choroidal
melanocytes decline with age (Fig. 1.3).
In young and middle-aged individuals, the RPE is tightly adherent to the
underlying Bruch's membrane by means of its own basement membrane. This
adherence decreases with advancing age. The Bruch's membrane consists of the
basement membrane of the RPE, the inner collagenous layer, an elastic layer, an
outer collagenous layer, and the basement membrane of the choriocapillaris.
Because of its porous structure, it probably plays a minimal role in regulating
movement of substances across it.
The choroid is the posterior aspect of the uveal tract; it is supplied by the
short ciliary arteries, and they are concentrated in the macula and peripapillary
region. These arteries form a rich anastomotic network that quickly empties
large quantities of blood into the choriocapillaris (sinusoidal network). The
choriocapillaris is fenestrated and arranged in a lobular pattern, with a feeding
arteriole in the center of each lobule and several venules peripherally (Fig. 1.7)
(32–40).

Figure 1.7 Schematic representation of the lobular pattern of the choriocapillaris. Each lobule is supplied
by an arteriole. BM, Bruch's membrane; CA, choroidal artery; CV, choroidal vein; RPE, retinal pigment
epithelium.

The prelaminar part of the optic nerve is supplied by the peripapillary

branches of the short posterior ciliary arteries. The choriocapillaris does not
communicate directly with the optic disk capillaries. The prelaminar capillaries
freely anastomose at the disk margin with those of the retina. Both capillary
systems drain into the venules leading to the central retinal vein (33,40).
The RPE is an integral part of the visual cycle. It is an important component
of photoreceptor renewal; new outer segment disks are continually added
proximal to the base, and the oldest disks at the distal end of the outer segments
are phagocytized by the RPE cells. The RPE also transports metabolic wastes
from the retina into the choriocapillaris. The melanin within the RPE cells
absorbs light that has not been captured by the photoreceptors, thus preventing
excessive light scattering within the eye. Melanin absorption may also confer
protection from photo-oxidative stress (41). The RPE cells are also thought to
secrete growth factors essential for proper differentiation of photoreceptors
during development (42).
The macula has the highest rate of blood flow of any tissue in the body. It
probably functions to stabilize the temperature environment of the retina (43)
because blood flow is greatly in excess of that needed to meet the nutritional
demands of the retina. The choriocapillaris supplies the RPE and outer retinal
layers. The choriocapillaris has an endothelium with a pore size sufficient to
allow some larger molecules, including proteins, to escape into the extravascular
space. There are no lymphatic channels in the eye; the perivascular and
perineural spaces in the sclera probably function as lymphatic channels. Thus,
the choriocapillaris endothelium controls the amount of extracellular fluid
normally present in the choroid.

BLOOD–RETINAL BARRIERS
Retinal perfusion is accomplished by a nonoverlapping, dual system of blood
circulation. For each system, there is a blood–retinal barrier analogous to the
blood–brain barrier. Both barriers confine even relatively small molecules,
because of a nonleaky tight junction between cells (44).
The outer blood–retinal barrier is constituted by the RPE; it blocks the
inward migration of small molecules from the choriocapillaris into the subretinal
space. Anatomically, these junctions include a zonula adherens and adjacent
zonula occludens of the RPE, both situated near the apex of the cell and
encircling it (45). The inner blood–retinal barrier is the retinal vascular
endothelium, including the capillary endothelium. The site of the barrier is the
specialized tight junctions (zonulae occludentes) between individual endothelial
cells (44,46).

NORMAL FLUORESCEIN
ANGIOGRAPHIC FINDINGS
Fluorescein angiography (FA) was developed in the 1950s as a means of
studying vascular flow. FA is still used for this purpose, but it provides much
additional information. Sodium fluorescein is excited by a blue light (465–490
nm), and it emits a fluorescent yellow-green light (peak wavelength of 520–530
nm). Because of its molecular weight (376 kDa), it diffuses freely out of all the
body capillaries except the retina. Approximately 80% of the dye is bound to
plasma proteins (albumin), and it is the unbound fluorescein that is detected
angiographically (16).
FA is performed using a fundus camera. Filters are used to produce the
exciting wavelength. The FA is photographed digitally or with black and white
film because of its superior resolution and speed. On the positive images,
fluorescein appears white and nonfluorescent areas appear black (46). The
normal FA shows the dual nature of the retinal circulation. The larger choroidal
vessels fill first, with almost immediate filling of the choriocapillaris. Rapid
perfusion of the choroid and leakage of the dye from the choriocapillaris give the
fairly uniform background fluorescence.
The tight junctions of the retinal pigment epithelial cells (the outer blood–
retinal barrier) block the fluorescein that leaks from the choriocapillaris and
diffuses through the Bruch's membrane. Normally, the fluorescein does not gain
access to the subsensory retinal space. The retinal vessels, including the
capillaries (the inner blood–retinal barrier), normally do not leak fluorescein.
Thus, the angiogram evaluates both of the blood–retinal barriers (44).
The normal macular region is hypofluorescent or has a barely visible
fluorescence because of the greater density of the RPE in this area and the
presence of xanthophyll in the outer retinal layers. In darkly pigmented
individuals, the increased density of choroidal melanocytes in the macula helps
obscure the background choroidal fluorescence. In addition, the capillaries
appear particularly distinct, because there is only one capillary layer surrounding
the foveal avascular zone (47). Only in the extramacular area, before the
arteriovenous phase, can details of perfusion of the larger choroidal vessels be
detected.
The FA consists of five phases (Fig. 1.8):
Figure 1.8 FA of normal fundus. A. Red-free photograph. B. Arterial phase. C. Early arteriovenous phase.
D. Intermediate arteriovenous phase. E. Venous phase. F. Recirculation phase. (Courtesy of Dario
Fuenmayor-Rivera and Enrique Murcia.)

1. Prearterial phase: The choroid and choriocapillaris fill with dye. A cilioretinal
vessel, if present, usually fills at the same time as the choroidal circulation,
before fluorescein is detectable in the other retinal vessels, approximately 1
 second before that of the proximal branches of the central retinal artery (Fig.
1.4).
2. Arterial phase: Lasts until the arteries are completely filled.
3. Arteriovenous phase: Characterized by complete filling of the arteries and
capillaries and the first evidence of laminar flow in the veins.
4. Venous phase: Begins as the arteries are emptying and persists until the veins
are filled with dye.
5. Recirculation phase: Follows the venous phase and represents the first return
of blood to the eye after fluorescein has passed through the kidneys. During
the recirculation phase, the outer edges of the major retinal vessels appear
relatively hyperfluorescent because of the greater amount of fluorescein in the
tangential section of the plasma cuff near the edge of the blood vessels.

All abnormalities in the FA can be understood as the presence of either too

much fluorescein (hyperfluorescence) or too little (hypofluorescence) in a
specific location (48).
In evaluating diseases of the macula, FA can be of value in detecting
alterations in blood flow, in permeability of the retinal blood vessels, in the
retinal vascular pattern, in the density of the pigment epithelium, and other
changes affecting the normal angiographic pattern in this area (16).

NORMAL INDOCYANINE GREEN

ANGIOGRAPHY FINDINGS
Indocyanine green (ICG) is a dye that was originally used in the photographic
industry. It was first used in ophthalmology by Flower and Hochheimer (49) in
the early 1970s to image the choroidal circulation. Although both experimental
and clinical investigations with ICG continued, it was not until the early 1990s
that it became an established method of investigation. This was because of the
increasing interest in the contribution of the choroid to retinal diseases and
improvements in technology. ICG, a tricarbocyanine dye, is injected
intravenously and is imaged as it passes through ocular vessels. An excitation
filter with a peak at 805 nm and a barrier filter with a transmission peak of 835
nm, corresponding to the maximum fluorescence emitted by the dye in whole
blood, are required. The standard technique is to inject 25 mg of ICG in 5 mL of
water slowly and to begin photographs 7 to 10 minutes after injection with late
photographs at 20 and 40 minutes. The circulating dye is rapidly excreted by the
biliary system. A preinjection infrared fundus photograph showing
pseudofluorescence or autofluorescence may help to avoid misinterpretation of
the angiograms (50).
Adverse reactions to ICG are more rare than those with intravenous
fluorescein. Mild reactions such as nausea, vomiting, sneezing, and transient
itching occur in 0.15% of cases (51). More severe reactions such as urticaria,
syncope, fainting, and pyrexia may also occur. Severe reactions such as
hypotensive shock (52), anaphylactic shock (53), and death have been reported.
Crossover allergy to iodine can occur in patients with seafood allergies, making
iodine and seafood allergy a contraindication to ICG angiography. Because the
liver primarily metabolizes ICG, it should be avoided in patients with hepatic
disease. Those undergoing hemodialysis are also at increased risk of
complications.
Photographic film with a conventional camera is not sensitive enough to
acquire ICG images—more sensitive digital systems are required (54–57). Low-
contrast individual images are digitally enhanced to allow better interpretation of
choroidal structures (58,59). ICG and FA can each be performed and the images
superimposed (60,61). New technologic developments now permit dynamic,
simultaneous FA and ICG using the confocal scanning laser ophthalmoscope
(62,63). The use of a wide-angle contact lens to visualize 160 degrees of the
fundus may help in the evaluation of flow characteristics and simultaneous
assessment of the periphery and posterior pole (64).
Interpretation of ICG angiography is difficult because multiple vascular
layers are displayed at one time. After choroidal arteries fill, the capillary phase
is characterized by a rather diffuse hyperfluorescence resulting from the additive
fluorescence effects of multiple crossing arterioles and venules. This
fluorescence is brighter in the foveolar area. However, because xanthophyll does
not efficiently absorb infrared light, the foveola cannot be located exactly during
ICG angiography (Fig. 1.9). The choriocapillaris layer itself can only be sensed
as a faint haze that becomes visible in the early venous phase. The images do not
change significantly during the next several minutes. With reduction in dye
fluorescence after about 5 minutes, discernibility of single choroidal vessels
decreases and there is a fairly uniform background fluorescence on which larger
retinal vessels are visible as brighter structures; after 10 to 15 minutes, however,
these vessels appear as darker structures. This rapid decrease in retinal vessel
fluorescence reflects the fast hepatic extraction of the dye from the blood. In
contrast, fluorescence in the choroid lasts much longer and could be interpreted
as exudation of the dye. However, a comparative examination of ICG
fluorescence demonstrates that plasma fluorescence of the dye is much brighter
than that of whole blood and lasts much longer (65,66). In fact, it decreases at
the same rate as background ICG fluorescence in the fundus. We therefore
believe that plasma gaps in choroidal capillaries must be sufficiently large to
become the main source of late choroidal fluorescence. As with fluorescein, ICG
hyperfluorescence cannot always be attributed to extravasation of the dye. Focal,
short-term hyperfluorescence is a common finding in the early venous phase and
probably is caused by a plasma vortex in venous loops or additive fluorescence
of crossing vessels. Though the pigment epithelium blocks only 10% of infrared
light, that is still enough to create a window-like hyperfluorescence at the site of
pigment epithelial atrophic defects (67).

Figure 1.9 Indocyanine green video angiography (ICG-V) of the normal fundus. (Courtesy of Dario
Fuenmayor-Rivera.)

NORMAL OPTICAL COHERENCE

TOMOGRAPHY FINDINGS
Time-domain optical coherence tomography (TD-OCT, Zeiss-Humphrey
Instruments, Dublin, CA) uses a continuously emitting, optically coherent laser
diode centered at 850 nm and focused at the retina. The superluminescence low-
coherence diode emits light with a 20 to 25 nm bandwidth. For thickness
measurements, the time delay of reflected or backscattered light is determined
using coherence interferometry. Reflectivity and distance information is
contained in the interference signal between a probe beam, reflected from
different structures within the investigated tissue, and light returning from a
variable-reference optical delay path. The length of one scan is adjustable for 43
nm working distance up to 3 mm in air (in vivo approximately 6 mm), and one
line can be measured within 1 second. The displayed image has a resolution of
500 × 500 pixels. The axial resolution of OCT is reported by the manufacturer to
be 10 to 20 μm (68).
The recent introduction of spectral-domain OCT (SD-OCT) allows imaging
of the macula with a much faster scan rate and at a higher scan resolution.
Compared with the commercially available time-domain OCT (Stratus OCT,
Carl Zeiss Meditec, Dublin, CA), the scan rate of SD-OCT is capable of
obtaining at least 18,000 axial measurements per second with an axial resolution
of 5 μm. The basic working principle of SD-OCT is similar to that of TD-OCT.
Both systems measure the echo time delay of backscattered light signals via an
interferometer. The light spectrum from the interferometer is detected by a
spectrometer. The interference spectrum data are then Fourier transformed to
generate axial measurements of the retina. SD-OCT has been shown to improve
visualization of the intraretinal structures, and the measurement repeatability is
even better despite the fact that three-dimensional SD-OCT generally has greater
macular thickness measurements compared with Stratus OCT (69).
The difference in foveal thickness measured by the two OCT instruments
would lie between 3.9 and 37.8 μm in 95% of pairs of observation. The normal
foveal thickness with SD-OCT is 216.4 ± 18 on the central fovea. The poor
agreement between three-dimensional SD-OCT and Stratus OCT may be
attributable to the different definitions of the posterior retinal boundary. In
Stratus OCT, the inner segment–outer segment (IS/OS) interface of the
photoreceptor layer is set as the posterior retinal boundary. Having a higher axial
resolution, delineation of the IS/OS photoreceptor interface and the RPE is
possible in three-dimensional SD-OCT, and the RPE is set as the posterior retinal
boundary (Fig. 1.10). The more posteriorly located reference line results in
greater values of macular measurement obtained by three-dimensional SD-OCT.
Measurement obtained from one system may not be used interchangeably with
the other.
Figure 1.10 Spectral-domain optical coherence tomography (SD-OCT) of the normal macula. ELM,
external limiting membrane; GCL, ganglion cell layer; ILM/NFL, internal limiting membrane/nerve fiber
layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform
layer of Henle; IS/OS PR, photoreceptor's inner and outer segments (ellipsoid zone); RPE, retinal pigment
epithelium.

Wakitani et al. (70) reported a study of macular thickness measurements in

203 healthy subjects with different axial lengths using TD-OCT. The thickness
values of three circular areas centered on the central fovea with diameters 350
μm, 1,850 μm, and 2,850 μm were 167 ± 21 μm, 212 ± 17 μm, and 231 ± 15 μm,
respectively. There was no significant difference in retinal thickness with age or
with increasing axial length of the eye. However, the average thickness of the
macula in females was significantly thinner than in males.
The contrast between different retinal layers is delineated by a narrow-field-
of-view image of a normal fovea. The anterior and posterior margins of the
retina are defined by highly reflective layers (the NFL and retinal pigment
epithelium/choriocapillaris). In the area of the fovea, the retinal pigment
epithelium/choriocapillaris appears distinct from the external segments of the
photoreceptors. The stratified structure of the retina is composed of alternating
layers of moderate and low reflectivity, and the photoreceptors appear minimally
reflective. Moderate backscattering is observed from the inner and outer
plexiform layers, which, like the NFL, consist of fibrous structures running
perpendicular to the incident beam. In contrast, the nuclear layers show minimal
backscattering because the cell bodies of the nuclear layers, like the
photoreceptors, are oriented parallel to the incident light. The increased
backscatter and the shadowing of the reflections from the RPE and
choriocapillaris identify the retinal blood vessels. The larger choroidal vessels
may also appear in the image and have minimally reflective dark lumens (71).
We can obtain information on three-dimensional structure, thanks to the
ability of OCT to acquire images of consecutive slices through the retina (Fig.
1.11). Characteristic features of the retina appear consistently in the serial
sections. The anterior and posterior surfaces of the neural retina are defined by
backscattering at the NFL and vitreoretinal interface, while the highly
backscattering red layer represents the RPE and choriocapillaris. The
development and resolution of the foveal depression, which reaches its
maximum depth at the fovea centralis, are revealed by the sequence of
tomograms. Retinal blood vessels derived from the superior and inferior
branches of the central retinal artery are evident in the tomograms from the
partial shadowing of the deep retinal structure beneath the vessels (71).
Figure 1.11 Spectral-domain optical coherence tomography of normal macula. A. Three-dimensional
image of the macula. B. Retinal thickness map. C. Normal fundus retinography.

NORMAL FUNDUS
AUTOFLUORESCENCE IMAGING
Autofluorescent imaging of the ocular fundus relies on the stimulated emission
of light from molecules, chiefly lipofuscin, in the RPE, in the photoreceptor
outer segments, and in the space between the photoreceptor outer segments and
the RPE (72). Currently, there are two ways to acquire fundus autofluorescence
imaging: one is using a fundus camera-based system (73) and the other is by
confocal scanning laser ophthalmoscopy (74).

Confocal Scanning Laser Ophthalmoscopy for Fundus

Autofluorescence Imaging
Confocal scanning laser ophthalmoscopy (SLO, Heidelberg Retina Angiograph
II, HRA II, Heidelberg Engineering, Heidelberg, Germany) has an argon laser
source that is able to emit a 488-nm excitation wavelength and a barrier filter to
detect emission from lipofuscin fluorophores over 500 nm. The illumination
beam is 3 mm in diameter and the full aperture of the dilated or undilated pupil
is used to collect the emitted fluorescence light from the posterior pole. The
confocal detection unit employs a 400-μm pinhole aperture to suppress light
from below or above the confocal plane. The size of the scanning field used is
30° × 30°. The frame grabber of the HRA can digitize image frames at a
programmable rate of up to 5 frames per second. Each frame contains 1,536
pixels vertically and 1,536 pixels horizontally in the high-resolution mode
(digital resolution 5 μm/pixel). All frames must be aligned and averaged to
create a final image. One of the difficulties encountered during fundus
autofluorescence imaging besides careful and standardized image acquisition is
the influence of media opacities, with cataract being the most prominent adverse
factor (74).

Fundus Camera–Based System

Ordinary fundus cameras can image autofluorescence of the fundus using the
same wavelengths. However, autofluorescence of the crystalline lens would also
be imaged, particularly in patients with nuclear sclerosis, creating fogging of the
image. To avoid this problem, a new filter set is used with a fundus camera to
image autofluorescence. A barrier filter of 695 nm (bandwidth 675–715 nm) is
selected (Spectrotech, Saugus, MA) to avoid imaging the autofluorescence of the
lens, which lies chiefly from 510 to 670 nm. SLOs use wavelengths to stimulate
fluorescence that are preferentially attenuated by increasing nuclear sclerotic
changes. Therefore, a bandpass filter for the excitation light of 580 nm
(bandwidth 500–610 nm) is selected to help reduce any variation in stimulating
light reaching the fundus caused by absorptive changes in the crystalline lens.
The gain for the digital camera is set at the maximum level through the
IMAGEnet software (Topcon, IMAGEnet, Oakland, NJ). The wavelengths
selected are well outside of that used for both fluorescein and ICG angiography.
The light exposure to the fundus is calculated to be orders of magnitude less than
that used for a typical fluorescein angiographic evaluation. The autofluorescence
photographs with confocal SLO imaging are noisier than the autofluorescence
photographs with the fundus camera–based system (73).

Interpretation of Fundus Autofluorescence

All autofluorescence values are adjusted using a histogram distribution for the
pixels in the image. A gaussian curve of values from 0 to 255 is obtained; 0
corresponds to black and 255 corresponds to pure white. A cumulative frequency
distribution is calculated, and the determination of the pixel value at the 1
percentile value is performed. Then, subtraction of this pixel value from the
values greater than 1 percentile is done. This process converts all values at or
lower than 1 percentile to black and shifts all other corresponding values in an
objective manner not influenced by operator interaction. Finally, these final pixel
values are evaluated to calculate the mean and standard deviation of the
brightness values. Fundus autofluorescence images usually are evaluated for the
presence of areas of decreased (hypoautofluorescence) or increased
(hyperautofluorescence) fundus autofluorescence. So, we consider that the RPE
cells are normal if the autofluorescence imaging shows some autofluorescence.
However, when autofluorescence imaging shows large autofluorescence, we
consider that the RPE is sick or stressed RPE cells, and the absence of
autofluorescence reflects the death of the RPE cells (73).

Normal Fundus Autofluorescence

A normal pattern of fundus autofluorescence is characterized by decreased
intensity at the fovea, under the retinal blood vessels, and in the peripapillary
region and further decrease over the optic nerve head, which appears dark (Fig.
1.12). The autofluorescence in the fovea is decreased because there is less
accumulation of lipofuscin in the fovea than nasally or temporally to the macula
(73). However, the autofluorescence in the fovea is minimally affected by the
absorption of the macula luteal pigments at a 580 nm excitation wavelength. In
addition, it has some effect of absorbance at a 488 nm excitation wavelength,
and it is not significantly affected by ocular pigmentation depending on the
choice of the wavelength excitation and barrier filters, because the higher the
wavelength, the less the absorption by melanin. There is a topographic
distribution of RPE lipofuscin content throughout the fundus, being higher at the
posterior pole, with a localized dip at the fovea, lower toward the peripheral
retina. This is consistent with histologic findings demonstrating a decay of
lipofuscin correlating to the density of photoreceptors toward the periphery. The
distribution by quadrants is highest temporally, then nasally, superiorly, and
lowest inferiorly. There are some facts that we must have in mind to understand
the normal autofluorescent pattern; one fact to be considered is the spatial
distribution of the photoreceptors. It is difficult to perform a correlation between
lipofuscin and cone photoreceptors because they decrease their density rapidly
between the fovea. At 3 or 3.5 degrees, the maximal density of the cones is
found at the fovea, and at 3 degrees from the fovea, we can find only 10% of
their maximal density. In the case of the rod photoreceptors, the distribution is
different: the rods' highest density is in a ring between 10 and 15 degrees, and
the highest loss of rods happens between 5 and 12 degrees (inside the highest
density ring of rods); after 15 degrees, the density of the rods starts to decrease
toward the periphery (73). It is important to address that the zone of minimal
fluorescence in the fovea overlaps the rod-free zone, and this fact can suggest
that the rate of lipofuscin formation in the cones may be slower than in the rods;
it would be confirmed if studies in humans correlated the study in rhesus
monkeys where the number of phagosomas in the RPE of the fovea (derived
from cones) is only one-third of the number of extrafoveal phagosomes in the
RPE (derived mainly from rods). Despite the fact that the greatest lipofuscin
accumulation overlaps the regions in which the rod loss is greatest, the
prediction of rod loss cannot be done by lipofuscin accumulation, because the
inferior retina had greater rod loss and lower fluorescence than the superior
retina.
FIGURE 1.12 A. Normal fundus retinography. B. Autofluorescence image of normal macula.

In the case of the Bruch's membrane, the fluorescence is only significant if

the excited wavelength used is in the blue light spectrum: the higher the
wavelength excitation, the lower the fluorescence of the Bruch's membrane. In
the red edge of the spectrum, the Bruch's membrane has little contribution to
autofluorescence.

ACKNOWLEDGMENTS
Supported in part by the Arevalo-Coutinho Foundation for Research in
Ophthalmology, Caracas, Venezuela.

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 2
ANIMAL MODELS FOR AGE-
RELATED MACULAR
DEGENERATION
MATÍAS E. CZERWONKO • MARIANA INGOLOTTI •
AMELIA NELSON JAMEL L. F. BROWN • D. VIRGIL
ALFARO III

INTRODUCTION
Age-related macular degeneration (AMD) is a late-onset, multifactorial,
neurodegenerative disease of the retina and the leading cause of irreversible
vision loss in the elderly in the Western world (1). It affects 30 to 50 million
individuals, and clinical hallmarks of AMD are observed in at least one-third of
persons over the age of 75 in industrialized countries (2). Animal models, in
particular, have contributed greatly to current knowledge regarding the etiology,
molecular mechanisms, and histologic features of this extremely prevalent
condition.
As we consider animal models of AMD, with the potential for preclinical
investigation, various pathologic and nonpathologic histologic elements must be
considered. In the particular case of AMD, we can roughly divide its typical
features into two types: histologic and functional alterations.
Among the histologic features that have been reported from the eyes of
patients with AMD are the following:

Thickening of the Bruch's membrane (BM)

Subretinal pigmented epithelium (RPE) basal laminar deposits and basal
 linear deposits (i.e., drusen)
Hyperplasia
Changes in the RPE including loss of the basal infoldings
Accumulation of immune cells (i.e., macrophages and microglia)
Atrophy
Photoreceptor atrophy
Deposition of activated complement proteins
Retinal or choroidal neovascularization (CNV)
Fibrosis
Accumulation of lipofuscin in RPE cells with increased levels of A2E and
corresponding increases in autofluorescence

Functional changes refer to decreased signals on electroretinograms (ERGs)

secondary to photoreceptor atrophy (3–7). Although AMD disease mechanisms
are still poorly understood, several pathogenic pathways have been proposed,
each associated with manifestations of AMD, including oxidative damage,
metabolic deregulation, and chronic inflammatory processes. These pathways
have been tested and confirmed in studies based on animal models, most of
which are explained in this chapter.
Mice have been widely used for generating models that simulate human
AMD features for investigating the pathogenesis, treatment, and prevention of
the disease. Focal atrophy of photoreceptors and RPE, lipofuscin accumulation,
and increased A2E can be present in aged mouse eyes, even though the mouse
does not have a macula. Unfortunately, drusen are rarely seen in mice because of
their simpler BM and different process of lipofuscin extrusion compared with
humans. Analyzing basal deposits at the ultrastructural level and understanding
the ultrastructural pathologic differences between various mouse AMD models
are therefore critical to comprehend the significance of research findings. More
recently, some other animal models have entered the world of AMD research.
While nonhuman primate models have gained popularity over the past few years
because of their resemblance to the ocular anatomic features of humans, rabbits
and pigs are less often used.
The purpose of this chapter is to provide a framework for better
understanding some of the existing animal models and the knowledge that has
been derived from their study regarding both dry and wet types of AMD. Some
of these models have encouraged the development of some promising lines of
investigation concerning novel therapeutic approaches. Nevertheless, it is
important to understand that most of these models are not necessarily etiologic
representations of human disease.

ETHICS OF ANIMAL USE

The use of animals in biomedical research dates back to the fifth century BCE.
Our ancient ancestors realized the homogenous relationship between humans
and other animals and the subsequent medical discoveries that come from
experimentation. Since then, nonhuman animals have proved themselves to be
appropriate models for scientific inquiries, contributing to medical science.
However, with the use of animals in research must come a regard for the ethical
treatment and general welfare of the animals used.
The first mention of animal usage in the medical field occurred around 500
BCE. Pythagoras, an ancient philosopher and mathematician, recognized a
common soul between animals and humans—a connection that could be used to
make certain comparisons. Hippocrates was the first to directly compare specific
organs. Vivisection was even practiced among certain scientists, including
Alcmaeon, Herophilus, and Erasistratus, at this time in history. Years later, the
use of vivisection was exercised even more so by one of the fathers of medical
experimentation, Galenus. The first true published experiment involving animals
did not occur until 1638 CE by English physician William Harvey. However, the
issue of ethics in these experiments was not mentioned until the 18th century.
British philosopher Jeremy Bentham published his ideas on the welfare of
animals, which led to the start of regulations and protection of animals. Bentham
is responsible for projecting the idea of utilitarianism, the ethical doctrine stating
if an action provides benefit to society, it is indeed morally right. He was the first
to question the actual suffering of animals in experiments, and laws regarding
such would be the result.
Britain was the first country to pass laws concerning animal welfare. The
first association to establish themselves as protectors of animals in medical
research was the Society for the Prevention of Cruelty to Animals, begun in the
year 1824 in England. The first law to pass was the British Cruelty to Animals
Act in 1876. The issue of animal rights did not die in England during the 1900s;
in 1969, FRAME (Fund for the Replacement of Animals in Medical
Experiments) was established.
In 1909, Americans started publishing on behalf of animal rights. The first
federal law passed in America regarding animal cruelty was created in 1966 and
called the U.S. Animal Welfare Act, serving to protect the welfare of laboratory
animals. Animal rights organizations such as PETA (People for the Ethical
Treatment of Animals) and the Johns Hopkins Center for Alternatives to Animal
Testing began forming in the 1980s. The three R's, replace, reduce, and refine,
became a sentiment for animal activists across the globe. Laws and regulations
on the ethics of animals in the laboratory are still fighting to be passed, and the
conflict lingers with a conclusion still remaining to be determined.
The first topic to be addressed when discussing animal research is the
obvious question: What is the purpose? Or, in other words, is there indeed a need
for animal experimentation? Some claim that animals and humans are simply too
different to make valid comparisons and that ultimately society does not benefit
from animal research. Of course, this is true if one was to compare completely
dissimilar organisms, such as two animals in entirely different animal kingdoms.
Nonetheless, properly executed animal experiments have led to many historical
medical breakthroughs, including the discovery and refinement of insulin in
dogs.
Another concern when evaluating the ethics of using animals in the
laboratory is the actual success rate of the research itself. How often does an
experiment involving animals actually lead to something valuable and how often
does it simply fail to achieve useful results? To answer this question, one must
determine what constitutes a low success rate, a term whose meaning is relative
to the person who is using it. Since a “low success rate” is not attached to a
tangible number, there is no way of knowing if animal experimentation would
qualify as such. The rebuttal to this argument points out that trial and error is one
of the characteristics of scientific research and explains that upon comprehension
of the scientific method should come a realization of the inevitable chance of
failure.
While there is still disagreement among people on opposing sides of the
issue, there is common ground being reached. Scientists have resorted to
alternative methods of experimentation. In many instances, in vitro techniques
can replace animal testing. Scientists take stem cells from humans to mimic real
human responses to testing. They can also use brain imaging, such as MRI or CT
scans, to study human brains instead of animal brains and even imitate real
human responses through computer simulation. Although the two different sides
of the issue conflict, alternative methods to animal testing serve as a satisfying
compromise.
Another point to be addressed is the inclusion of policies on ethical
standards in lab experiments and reports. One study shows that 40.7% of
published work involving animal testing was performed with a respect for
ethical standards. A different study, using articles from PubMed, shows that 53%
of the research had ethical policies. Both of these studies show that there is much
improvement to be done in this field. Inclusion of ethical standards in animal
research should hopefully reach 100%, proving that all research involving
animals displays ethical treatment to the laboratory animals. Not only does
reporting ethical standards in studies show that the science has taken a moral
standpoint, but it also implies scientific integrity and respect in the medical field
(8–12).

ANIMAL MODELS OF DRY MACULAR

DEGENERATION
Rodent Models for Dry AMD
The active role of oxidative stress, inflammation, and metabolic deregulation in
AMD pathogenesis has been well established (13). Pathologic hallmarks of the
dry type of AMD include drusen accumulation, BM thickening, RPE, and
photoreception degeneration followed by atrophy. Mice have been the most
commonly used models for retinal disease because their retina resembles the
human nasal and peripheral retina. The described murine models for dry AMD
have drusen-like lesions and RPE atrophy on funduscopy, ERG abnormalities,
lipofuscin accumulation, sub-RPE basal deposits, and BM thickening.
Undoubtedly, one of the most seductive advantages of murine models is the ease
to manipulate them genetically, surgically, and pharmacologically.
The aforementioned dry AMD murine models can be conveniently classified
into three categories:

Genetically engineered mice

Immunologically manipulated mice
Naturally occurring mouse strains

Most murine models are genetically engineered to manipulate genes that are
suspected to cause macular degeneration-like disorders in humans or might be
somehow associated to AMD pathogenesis. Inflammatory genes (Cfh, Ccl2,
Ccr2, and Cx3cr1), oxidative stress–associated genes (Sod1 and Sod2), and
metabolic pathway genes (mcd, Cp, Heph, ApoE, and ApoB100) have been
manipulated to create models with features that are typical of dry AMD. The
immunologically manipulated mouse model is immunized against
carboxyethylpyrrole (CEP), an endogenous biomarker of oxidative stress.
Finally, the naturally occurring mouse models include retinal degeneration
models with mice exposed to nocive and AMD-related conditions and the
senescence-accelerated mouse (SAM) models with systemic and retinal age-
related pathology, including BM thickening, basal deposits, and CNV.
In the following paragraphs, we explain the most important models of each
of the groups mentioned above. Overall, these models contrast with the murine
models of wet AMD that are mostly induced mechanically and are explained
later in this chapter.

LIPID AND GLUCOSE METABOLISM

MODELS
A growing body of the literature indicates the involvement of lipids,
lipoproteins, and glucose in the formation of extracellular lesions in aging BM,
basal deposits and drusen, and other AMD hallmarks. Observational studies have
indicated that maintaining adequate levels of fatty acids or a low glycemic index
(GI) diet may be particularly beneficial for early stages of the disease. From a
pathophysiologic perspective, lipids and cholesterol that concentrate within the
BM with aging are believed to restrict the metabolite transference between the
RPE and the choriocapillaris (14–18). From a structural point of view, lipids and
cholesterol deposits may promote the formation of both basal laminar and basal
linear deposits. We could fairly claim that there are three major metabolism-
related risk factors that have been associated to AMD pathogenesis:

Diet: High intakes of cholesterol and monounsaturated, polyunsaturated,

and saturated fats have been associated with the onset and progression of
AMD (19).
Cardiovascular diseases: An apparent correlation between the deposition of
cholesterol and lipids in atherosclerotic plaques and the material
accumulated in the BM was described. Nevertheless, numerous studies
have shown opposing results so that controversy still exists as to how tight
this association really is, if there is any (20–22).
Genetics: Polymorphisms in genes that code for lipid transport mediators
(apolipoproteins) showed a significant association with the risk of AMD
(23,24).
 The following sections are dedicated to mention and describe the most
important animal models that have been designed to assess the potential
connection between lipid and glucose metabolism and AMD pathogenesis.

Aging and a High-Fat Diet

To examine the histologic, histochemical, and ultrastructural changes in the BM
in C57BL/6 mice on a high-fat (HF) diet, Dithmar et al. compared 2-month-old
models on a normal diet and 8-month-old mice on a normal or HF diet.
Confirming the importance of aging, thickening of the BM was found in 8-
month-old mice on both normal and HF dietary regimens. However, 8-month-old
mice exposed to HF diets showed greater thickness compared to that of
nonexposed counterparts. Since the plasma cholesterol level was significantly
higher in mice on HF diets than the controls, the results were consistent with the
suspected role of lipids in the pathogenesis of AMD. A year later to Dithmar's
studies, Cousins et al. (25) compared 2-month-old and 16-month-old C57BL/6
mice fed normal and HF diets. In this case, basal laminar deposits were observed
in the 16-month group exclusively.

Aging and High Glycemic Index Diet

The GI indicates how fast blood glucose is raised after consuming a
carbohydrate-containing food. Human metabolic studies indicate that GI is
related to abnormal responses after meals. Recent epidemiologic evidence
indicates positive association between GI and risk for AMD in people without
diabetes. Therefore, it is reasonable to suspect low GI diets as protective factors
for the development of AMD (26–30).
Based on these assumptions, Uchiki et al. (31) compared mice that
consumed low and high GI diets, showing that the latter promote the apparition
of AMD-like lesions. Mice that consumed the lower GI diet displayed a
significantly reduced frequency and severity of age-related retinal lesions such
as basal deposits. Glycation-altered proteolysis is accounted for linking dietary
GI, aging, and age-related diseases like AMD. Not surprisingly, consuming
higher GI diets was associated with over threefold higher accumulation of
advanced glycation end products (AGEs) in the retina in the age-matched mice.
Both incidence and severity of retinal changes aggravated with age, as older
mice exhibited more thickening of the BM, more basal laminar deposits,
disorganization of RPE basal infolding, and increased photoreceptor atrophy
compared to 17-month-old counterparts (32).

Apolipoproteins
Cholesterol and its transporter, apolipoprotein E (ApoE), are major constituents
of sub-RPE deposits in AMD eyes. Based on these findings, the following
murine models were designed and studied:

ApoE knockout mice: ApoE is a constituent of very- low-density lipoprotein

(VLDL) synthesized by the liver and of a subclass of high-density
lipoprotein (HDL) involved in cholesterol transporting among cells. ApoE
mediates high-affinity binding of ApoE-containing lipoprotein particles to
the low-density lipoprotein (LDL) receptor and is thus responsible for the
cellular uptake of these particles. Mice with inactivated endogenous ApoE
gene exhibited very high levels of circulating cholesterol (33,34) and
revealed thickening of the BM combined with electrolucent particles and
membrane-bounded material at 8 months of age (35).
ApoEe2/ApoEe4 transgenic mice: With the purpose of studying the multiple
ApoE alleles seen in humans, transgenic mice were created replacing native
ApoE with human ApoE equivalents (36–38). Eyes of aged mice
expressing human ApoE2, ApoE3, or ApoE4 and maintained on a high fat
and cholesterol (HF-C) diet showed ApoE isoform–dependent pathologies
of diverse severity. They develop a constellation of changes that mimic the
pathology associated with human AMD such as
Diffuse subretinal pigment epithelial deposits
Drusenoid deposits
Thickened BM
Atrophy, hypopigmentation, and hyperpigmentation of the RPE

Interestingly, ApoE4 mice were the most severely affected, to the point of
having sometimes developed CNV. Transgenic ApoE4 mice fed HF-C diets have
also shown to accumulate amyloid b (Ab), a well-known constituent of human
drusen (39). What is more, systemic anti-Aβ immunotherapy was shown to
protect against loss of visual function and retinal damage, suggesting a role for
Ab in the AMD pathogenesis and functional manifestations (40). Ironically,
while the ApoEe4 allele is correlated with relative protection for AMD in
humans, in the mice, the opposite appears to be true (23,41–43). The reason for
this discrepancy between species still remains unclear.
 ApoE3-Leiden transgenic mice: ApoE3-Leiden is a dysfunctional form of
ApoE3 that is associated with a dominantly inherited form of familial
dysbetalipoproteinemia and early onset of atherosclerosis (44). With the
intention of investigating the ApoE3-Leiden mouse as an animal model for
retinal extracellular deposits, transgenic mice carrying the ApoE3-Leiden
gene were created. Eyes were obtained from ApoE3-Leiden transgenic mice
on a HF-C diet or on a normal mouse chow diet for 9 months. As controls,
eyes were collected from ApoE knockout mice on the same dietary
regimens. All eyes of the ApoE3-Leiden mice on a HF-C diet exhibited
basal laminar deposit, whereas ApoE3-Leiden mice on normal chow
displayed basal laminar deposits only occasionally. In both cases, however,
the ultrastructural features of these deposits were comparable with those
seen in human eyes, and they all showed immunoreaction with antihuman
ApoE antibodies. Importantly, no deposits were found in any of the control
mice. These results indicate that ApoE3-Leiden mice can be used as an
animal model for the pathogenesis of basal laminar deposits and that a HF-
C diet enhances the accumulation process. Furthermore, this study supports
the previously suggested involvement of dysfunctional ApoE in the
accumulation of extracellular deposits in the human disease (45,46).
ApoB100 transgenic mice: ApoB100 is the major apolipoprotein in LDL
cholesterol. Lipoprotein particles in the BM have been shown to contain
ApoB100. To study how ApoB100 might contribute to the formation of
lipoproteinaceous deposits in AMD, several groups have examined mice
that express human ApoB100. Young ApoB100 mice developed basal
laminar deposits when fed with a HF diet and exposed to oxidative blue-
green light (47). At older ages, ApoB100 mice exposed to normal diets
exhibited BM thickening, loss of the basal infoldings of the RPE, and basal
laminar deposits (48,49). When these animals were additionally fed HF
diets, more basal linear deposits were observed.

Lipoprotein Receptors
LDL receptor (LDLR) knockout mice are incapable of importing cholesterol,
leading to increased plasma cholesterol levels and development of
atheromatosis. Since atherosclerosis is a well-established risk factor for
developing AMD, a murine knockout model with LDLR deficiency was used to
evaluate changes in the BM. As it was expected, mice lacking LDLR exhibited a
degeneration of the BM with accumulation of lipid particles, which was further
increased after fat intake due to elevated blood lipid levels.
 Alternatively, alterations in the gene that codes for VLDL receptor (VLDLR)
have also shown some impact on the onset and progression of retinopathies.
Mice with both alleles mutated do not show signs of dyslipidemia but still
develop retinal neovascularization as early as 2 weeks of age (50–52). These
vessels grow toward the subretinal space, and can form choroidal anastomosis or
cause retinal hemorrhages between 30 and 60 days. Deeper analysis of this
model revealed the development of CNV, elevated levels of vascular endothelial
growth factor (VEGF), and dysregulation of the Wnt pathway all of which
suggested VLDLR as a negative regulator of CNV (53). Therefore, modulation
of the VLDLR may have a great potential from a therapeutic perspective.
More recently, polymorphisms in CD36 have also been shown to be
protective factors in AMD (54). CD36 is a scavenger receptor that binds
oxidized LDL (OxLDL) and is mostly expressed in the apical and basolateral
membrane of the RPE (55–57). Mice deficient for CD36 accumulate subretinal
OxLDL even when fed a regular diet (58). Even though lipids in the RPE come
mostly from photoreceptor outer segments and are exocytosed through the base
of the RPE to be eliminated via the choroid, RPE cells can also take up oxidized
lipids from their cell base, suggesting they may be involved in clearing the
subretinal space from such deposits. Laminar deposits in the BM have been
found to contain OxLDL among other compounds. Consistently, CD36
deficiency in mice resulted in both age-associated accumulation of oxidized
lipids and BM thickening. Coherently, treatment of HF-C-fed ApoE null mice
with a CD36 agonist was shown to diminish thickening of the BM notably as
well as to partially lessen photoreceptor atrophy and preserve function (58).

Cathepsin D
Cathepsin D is an aspartic protease that is highly expressed in the RPE and plays
a decisive role in processing photoreceptor outer segments (59,60). Cathepsin D
is secreted as a proenzyme, which accumulation has been linked to RPE
dysfunction (61). To study the mechanism, transgenic mice with a mutant form
of cathepsin have been developed (62). Mice homozygous for the transgene
demonstrated areas of RPE atrophy as early as 9 months of age (63). Between 10
and 18 months old, these mice exhibited areas of RPE hypertrophy,
photoreceptor atrophy, and reduced ERGs. Remarkably, they also displayed
small yellowish spots in the fundus that were similar to basal laminar and basal
linear spots.
OXIDATIVE DAMAGE MODELS
Clinical and experimental evidence supports that chronic oxidative stress is a
primary contributing factor to numerous retinal degenerative diseases, specially
AMD (64–66). Oxidative damage is normally minimized by the presence of a
wide range of antioxidant and efficient repair systems. Unfortunately, aging
intensifies oxidative damage while decreasing antioxidant capacity, thereby
perturbing the oxidative homeostasis. Postmortem eye examinations from AMD
patients have shown signs in line with extensive free radical damage (67).
Consistently, antioxidant-enriched diets have been shown to reduce the
progression from dry to wet AMD (68).
Several mouse models of chronic oxidative stress displayed many of the
most characteristic hallmarks of AMD. These models can be classified into two
groups: those that lack intrinsic antioxidant mechanisms and those where
additional oxidative stress is applied. However, whether oxidative stress is an
etiologic component or if it is just involved in disease progression is still
controversial.

Carboxyethylpyrrole-Adducted Products
Recent evidence has implicated AMD as an immunologically mediated disease
(69). The protein adduct CEP is present in AMD eye tissue and in the blood of
AMD patients at higher levels than found in age-matched non-AMD tissues.
Autoantibodies to CEP are also higher in AMD blood samples than in controls.
It has been suggested that a signal from the outer retina initiates an immune
response in AMD. CEP acts as a reliable biomarker of oxidative stress and is
currently suspected to be one of those signals (70,71).
To test such hypothesis, Hollyfield et al. (72,73) created a mouse model by
immunizing animals with CEP-adducted mouse serum albumin. Essentially, two
groups were compared: a short-term group that received a strong immunologic
challenge over 3 months and a long-term group that was inoculated with a
weaker challenge but over a year. Both groups successfully developed antibodies
to CEP-adducted albumin. At 3 months, the short-term group showed deposition
of complement component C3d in BM, RPE swelling and lysis, sub-RPE
deposits, pyknosis of overlying photoreceptors, and invasion of macrophages.
Long-term counterparts, on the other hand, exhibited a three- to fivefold
thickening of BM. There existed a solid correlation between the levels of CEP-
specific antibodies and the severity of pathologic alterations. Importantly,
although CEP is also proangiogenic via a VEGF-independent pathway (74),
neither the short-term nor long-term group exhibited signs of CNV, confirming
that the utility of this model is limited for dry AMD only.

SOD Models
Superoxide dismutases (SODs) are one of the major antioxidant defense
systems, which consist of three isoforms in mammals: the cytoplasmic SOD
(SOD1), the mitochondrial SOD (SOD2), and the extracellular SOD (SOD3).
All of these isoforms require catalytic metal (Cu or Mn) for their activation (75).
To investigate the role of oxidative stress in AMD pathogenesis, transgenic mice
targeting the SOD oxidative stress recovery pathways have been created and
examined. With some significant variances, knockout mice for different SOD
genes showed typical features of AMD.
SOD1 levels are particularly high in the retina, and its protective role against
oxidative damage is unquestionable. Imamura et al. showed that SOD1 knockout
mice display age-related pathologic changes in the retina reminiscent of AMD as
early as 7 months of age. Among the most frequent findings were the presence
of drusen, the thickening of BM, and the development of CNV (being thereby
suitable for simulating wet forms of AMD as well) (76). However, prior to the 7-
month period, retinas from these animals were absolutely indistinguishable from
age-matched wild-type animals (76). As with many other models, lesions
increased in number and severity with age: At 10 months of age, 86% of mice
had drusen, compared to very few drusen in age-matched wild-type mice (76),
and at a year of age, RPE vacuolization and degenerative changes were noted.
Remarkably, yellowish drusen-like deposits between the RPE and BM stained
for some of the most typical drusen biomarkers such as vitronectin,
carboxymethyl lysine, and tissue inhibitor metalloproteinase 3 (TIMP3) (76,77).
Senescent SOD1 knockout mice demonstrated evidence of necrotic death of cells
in the inner nuclear layer (INL) and reduced ERGs (78).
SOD2 knockout mice have been used as models for dry AMD as well, but
not without some difficulties. Contrary to SOD1, the antioxidant enzyme
manganese superoxide dismutase (MnSOD), encoded by SOD2, is in the
mitochondrial matrix, and polymorphisms in its encoded gene have been
strongly associated with the development of AMD (79). Unfortunately, most
SOD2 knockout mice died of dilated cardiomyopathy by the early age of 10
days, limiting their use as a model for maculopathies (80). Nevertheless, retinal
histology from some of the few that survived over 3 weeks demonstrated
significant central thinning of all retinal layers (81). Justilien et al. (82) managed
to overcome the limitations of this model by transfecting mouse retina and RPE
with an adenovirus that expresses a ribozyme that degrades SOD2, thereby
reducing its levels only locally and diminishing major systemic consequences.
Thickening of BM, degeneration of the RPE, increased autofluorescence,
elevation of A2E levels, and atrophy of the photoreceptors were among the most
remarkable histologic findings.

Ceruloplasmin and Hephaestin

Iron is an essential cofactor for many enzymes, but ferrous iron (Fe2 +) can cause
oxidative damage via the Fenton reaction. Such oxidative stress increases in the
body with aging and may contribute to AMD pathogenesis when affecting the
retina (83). Indeed, compared to normal eyes, AMD eyes show a statistically
significant increase in total iron in the RPE and BM (84). Since ceruloplasmin is
believed to facilitate iron export from cells, it is reasonable to expect humans
without functional ceruloplasmin to develop drusen and retinal pigmentary
changes later in life (85–87). Nevertheless, mice lacking functional
ceruloplasmin only showed a trivial iron overload (88) with mild retinal
alterations. The most satisfactory explanation for such discrepancies lies in the
presence of a second ferroxidase named hephaestin (Heph), which may
compensate, at least partially, the impact caused by the ceruloplasmin loss
(88–90). As a result, it was necessary to create mice with combined deficiencies
in Cp and Heph so as to assess the potential influence of iron overload in AMD
(91). By 5 months of age, these double knockout models had fruitfully increased
iron levels and displayed subsequent iron-laden electron-dense vesicles inside
the RPE cells. Focal areas of RPE hypertrophy and hypopigmentation in the
midperipheral retina, subretinal deposits, photoreceptor atrophy, and subretinal
neovascularization start appearing at the age of 6 to 9 months, and after a year,
these models showed infiltration of macrophages, focal areas of
hyperautofluorescence, and complement disposition (92). It is of interest to note
that the RPE hypertrophy in this model was greater than what had been observed
in AMD retinas (91,92). Unfortunately, most double knockout mice die from a
movement disorder at a very young age, thus hindering possibility to study long-
term effects.

Exogenous Oxidative Conditions: Cigarette Smoke,

Hydroquinone, High-Fat Diet, and Blue Light
It is unanimously accepted that smoking constitutes the major preventable risk
factor for AMD (93–97). Cigarette smoke contains over 4,000 potentially
damaging toxins, many of which are pro-oxidants including carbon monoxide,
nitric oxide, and hydroquinone (98). The association between AMD
pathogenesis and oxidative harm caused by environmental factors has been
studied in multiple animal models. Espinosa-Heidmann et al. described the
histologic alterations of mice exposed to combinations of HF diets, blue light,
and whole cigarette smoke versus oral hydroquinone. Animal exposed to these
conditions exhibited changes to the BM and had increased basal laminar deposits
(25,99). Plus, several mice exhibit invasion of the choriocapillaris into the BM
suggestive of early AMD. Wild-type mice treated with hydroquinone in their
drinking water develop decreased expression of Ccl-2 in the RPE and choroid
and demonstrated altered ratios of proangiogenic VEGF versus antiangiogenic
pigment epithelium-derived factor (PEDF) (100). Therefore, it was hypothesized
that hydroquinone may raise the risk of CNV by disturbing the normal balance
between angiogenic factors. Nevertheless, some studies have reached different
results regarding hydroquinone activity so that it remains a matter of debate.

OXY Rat
The term OXY rats refer to an inbred strain of Wistar rats that were chosen for
susceptibility or resistance to the early development of cataracts when fed with
diets rich in galactose (101). The susceptible line (OXYS) displays high levels of
hydroxyl radical formation and lipid peroxidation causing mitochondrial
oxidative damage and a senescence-accelerated model. As a result, these rodents
show premature aging and suffer age-related conditions such as cataracts,
emphysema, scoliosis, tumors, and myocardiopathy relatively early in life
(101–104). As for the retina, funduscopic analysis on these rats revealed
choriocapillaris and atrophic areas in the RPE at the age of 45 days (105).
Eventually, rats also exhibited thickening of BM, drusen and RPE detachments,
and, by the age of a year, photoreceptor atrophy, decreased ERGs, destruction of
the choriocapillaris with signs of fibrosis, and even hemorrhagic detachment of
the retina secondary to neovascularization (101,105,106).

COMPLEMENT PATHWAY AND

INFLAMMATION MEDIATORS
Chronic inflammation contributes to the onset and progression of multiple age-
related degenerative diseases including AMD (107,108). The pathology of AMD
lesions reveals signs of chronic persistent inflammatory damage such as
macrophage infiltration and microglial accumulation. Plus, several acute-phase
reactant proteins, in particular elements of the complement cascade, have been
identified as molecular components of drusen shaft (69,109–112).
Interestingly, underneath the AMD-related inflammatory processes, there is
a strong genetic connotation. Polymorphisms in many of the genes that have
been accounted for AMD encourage a proinflammatory state in the eye denoted
by increased C-reactive protein and decreased inhibitory complement factors
(113,114). Such polymorphisms include genes that code for factor H (CFH),
complement component 2 (C2), complement component 3 (C3), complement
factor B (FB), and complement factor I (FI) (115–119). Suffice to mention the
particular case of the Y402H polymorphism in CFH to understand the great role,
these genetic variations may have within AMD pathogenesis. Not only does such
polymorphism increase the risk of AMD five- up to sevenfold, but it has also
been found in half of AMD cases. Additional polymorphisms related to toll-like
receptor (TLR) (e.g., TLR-3 and TLR-4) and chemokines (e.g., CX3CR1 and
CCR3) have been also linked to the development and progression of AMD
(120–124). In fact, the association between chronic inflammation and AMD is
strong, and murine models that target specific aspects of the inflammatory
process have been created to further elucidate this connection. These models are
as varied as interesting and are discussed in detail in the following sections.

COMPLEMENT CASCADE
CFH Models
CFH negatively regulates the complement system by inhibiting the alternative
pathway either by promoting factor I–mediated inactivation of C3b or by
displacing factor Bb from the C3bBb complex and blocking the formation of C3
convertase (125,126). CFH dysfunction may lead to excessive inflammation and
tissue damage, which may contribute to the pathogenesis of AMD in the retina
(127). Patients and mice lacking functional CFH develop macular drusen similar
to those seen in AMD (128–131). At the age of 2 years, these genetically
engineered mice suffered decreased visual acuity, reduction in rod-driven ERG
a- and b-wave responses, increased subretinal autofluorescence, complement
deposition in the retina, and disorganization of photoreceptor outer segments.
However, though there is an association between CFH and AMD, the CFH
knockout mice do not display many of the hallmark AMD features. Contrary to
other models of AMD, almost 30% of these animals showed a substantial
thinning of their BM. Plus, since CFH itself is a main component of drusen, the
loss of this protein may reduce the volume of sub-RPE deposits.

Transgenic CFH Y402H Mice

Recent studies have shown an increase risk of AMD in individuals with the
Y402H polymorphism in the complement factor H gene (132). This
polymorphism has been located to a region of CFH that binds heparin and C-
reactive protein (124). In order to understand the role that such particular CFH
mutation have in AMD pathogenesis, Ufret-Vincenty et al. (133) created
transgenic mouse lines expressing the Y402H polymorphism under control of
the human ApoE.
At the age of 1 year, transfected mice showed larger amounts of drusen-like
deposits than those observed in either wild-type mice or even CFH knockout
mice. While immunohistochemistry has revealed an increased amount of both
microglial and macrophages in the subretinal space, the electron microscopy
showed thickening of the BM and deposits of C3d in the basement membrane.
Contrary to the CFH knockout mice, transgenic CFH Y402H models did not
show signals of photoreceptor atrophy. This occurrence might be explained
either by the younger age at which these animals were examined (1 year vs. 2
years) or by the existence of some functional activity of CFH protein that
disallowed a more severe dysregulation of the alternative pathway.

Transgenic Mice Overexpressing C3

C3 plays a major role in the complement activation and generation of immune
responses by integrating signals from every complement-activating pathway.
Since C3 can activate complement system and the complement system is a major
risk factor for developing AMD, it is reasonable to expect that overexpression of
this protein might lead to accelerated development of retinal pathology. To put
this theory in practice, Cashman et al. (134) injected mice in the subretinal space
with a recombinant adenovirus-expressing murine C3. Such delivery of C3
induced significant functional and anatomic changes that reproduce not only
many of the features of AMD but also those of other retinal diseases.
Examination of transfected eyes revealed more retinal changes (significantly
increased vascular permeability, endothelial cell proliferation and migration,
RPE atrophy, loss of photoreceptor outer segments, reactive gliosis, retinal
detachment, and reduced retinal function) relative to those injected with a
control adenovirus. Deposition of the membrane attack complex was observed
on endothelial cells and photoreceptor outer segments.
This novel model may be useful in assessing the role of complement in
retinal pathology and in developing anticomplement therapies for retinal
diseases associated with complement activation. However, as for its accuracy to
emulate AMD, there are some limitations that could not be ignored. In particular,
the fact that C3-overexpressing animals demonstrated an unexpectedly high
incidence of retinal detachments (a feature not shared with AMD) raises
question about its similarity to the actual disease. Plus, it is unknown whether or
not the mere activity of the transfected adenovirus could contribute to the
pathologic features, thereby biasing the results.

C3a and C5a Receptor

Both C3 and C5 have been found in drusen (39,135). Although these proteins are
best known for their role in opsonization and formation the membrane attack
complex, their activity is multifaceted. In fact, mice lacking the receptors for
these components showed smaller lesions in the laser-induced CNV model,
decreased levels of VEGF expression, and impaired leukocyte recruitment (136).
Such data support the hypothesis that activation of the C3a and C5a receptors
contributes in the appearance of CNV in AMD.

Chemokines and Chemokine Receptors

Chemotactic cytokines, also named chemokines, are a family of small cytokines,
which name is derived from their ability to induce directed chemotaxis in nearby
responsive cells (137). Some chemokines are considered proinflammatory and
can be induced during an immune response to recruit cells of the immune system
to a site of infection, while others are considered homeostatic and are involved in
controlling the migration of cells during normal processes of tissue maintenance
or development. From a structural perspective, however, chemokines are
frequently divided into four families: CXC, CX3C, CC, and C (138,139).
As for AMD, chemokines appear to be crucial in the subretinal
microglia/macrophage accumulation observed and may also participate directly
in the genesis of several abnormal processes such as retinal degeneration and
CNV (108,140,141). The CCL2/CCR2 and CX3CL1/CX3CR1 ligand/receptor
pairs excel among all the chemokines studied (142). Particularly, the T280 M
allele of CX3CR1 gene has been shown to be associated with AMD in diverse
patient populations (143–146). There are several reports using knockout mice for
these ligand/receptor pairs in an attempt to decipher the inflammatory
mechanisms of AMD. These models have successfully displayed many of the
most distinctive AMD characteristics.

Ccl2 and Ccr2 Single Knockout Mice

Ccl2, also known as monocyte chemoattractant protein 1 (MCP-1), enables the
binding of monocytes to the endothelium for subsequent tissue extravasation
(147,148). RPE cells under oxidative stress can upregulate Ccl2 (149), so that
increased intraocular levels have been reported both in exudative AMD (150)
and in mouse models of CNV (151). Macrophages extracted from eyes
undergoing the laser-induced CNV model, which are primarily responsible for
the angiogenic reaction (152), managed to get in the eye by moving toward this
higher concentration of the chemical in question. Bearing this in mind, it is easy
to understand why the recruitment of macrophages/microglia to the injury site
and the subsequent CNV happened to be diminished in both Ccr2 and Ccl2
knockout mice (153). Nevertheless, Ambati et al. (154) reported the spontaneous
appearance of yellowish drusen-like subretinal deposits in these mice after 9
months of age, combined with other AMD features including thickening of BM,
an increase in autofluorescence and lipofuscin granules, photoreceptor
malfunction, and the occurrence of CNV. It had been hypothesized that the
deficiency in macrophages/microglia recruitment through a CCL2-/CCR2-
dependent pathway could have prevented the clearance of accumulating debris in
BM, which would have eventually taken to drusen formation. Nevertheless,
further studies from Ccl2 knockout mice have rebutted such initial interpretation
(153), since drusen-like subretinal deposits initially happened to be
accumulations of swollen macrophages. Finally, Luhmann et al. found no
difference between thickening of BM, RPE and photoreceptor atrophy, and ERG
amplitudes compared to age-matched wild-type animals. Consequently, the
actual influence of the Ccl2/Ccr2 pathway to AMD pathogenesis still remains
unclear.

CX3CR1 Single Knockout Mice

CX3CR1 is a chemokine receptor that has been found in microglial cells in the
retina (144,155,156). T280M allele of the CX3CR1 gene leads to a dysfunctional
monocyte migration associated with AMD. Studies of two independently
generated CX3CR1-deficient mice (157,158) have exposed alterations in line
with AMD (144). By the year of age, these animals displayed drusen-like
deposits that were not apparent in age-matched wild-type counterparts. However,
these lesions were lipid-bloated subretinal microglia and not sub-RPE
extracellular deposits. By the 18 months of age, CX3CR1 knockout mice also
experienced significant retinal thinning. It is generally believed that deficiency
of functional CX3CL1 on retinal microglia may reduce egress of these cells from
the retina, causing in term the formation of subretinal deposits.

Ccl2/Cx3cr1 Double Knockout Mice

Ccl2 and CX3CR single knockout mice only exhibit signs of maculopathy at
older ages (16 and 18 months, respectively), and the phenotype was not fully
penetrant. In an attempt to accelerate the development of AMD-like features,
Tuo et al. (145) created double knockout (DKO) mice. The model was a success:
DKO mice displayed retinal alterations early in life (with the earliest occurrence
at 6 weeks) making a seductive alternative to single knockout animals. By 9
weeks of age, all DKO mice had developed subretinal drusen, which worsen
with aging. Immunostaining revealed increased complement in BM, RPE, and
choroidal capillaries (159). DKO mice had focal thickening of BM, increased
levels of A2E, infiltration of microglial, and photoreceptor atrophy. In older
animals, areas of atrophy and chorioretinal scars were observed. Up to 15% of
eyes displayed CNV as early as at 3 months. These data support DKO mice as
convenient models for both dry and wet AMD.
Nonetheless, the validity of this model has been recently rebutted by Raoul
et al. (142), whose independently developed lines of DKO mice did not display
well-defined features and were not phenotypically different from single
knockout counterparts. According to the authors, differences in the results exist
because of a bias in the previous selection regarding the breeding of animals,
since those worst drusen may have been unintentionally chosen for AMD factors
independent of CCL2 or CXCR1. Besides, it has been also reported that such
lines may have been adulterated with concomitant unrelated mutations, which
may had an impact in the ultimate phenotypes as well (160,161). Therefore, the
conclusions concerning this murine model should be taken with caution.

Nonhuman Primates for Dry AMD

Macular degeneration occurs in rhesus and cynomolgus macaques. In both,
retinal lesions are characterized by drusen accumulation in the central retina.
However, onset of fundus changes and inheritance patterns differ in these two
primate species. Macular degeneration in the cynomolgus macaque appears to be
a model for early-onset maculopathies rather than AMD. In contrast, adult-onset
macular degeneration in rhesus macaques resembles human AMD. Genetic
studies in rhesus macaques may resolve, which of two closely linked adjacent
genes on human (HTRA1 and LOC387715/ARMS2) are responsible for the
disease.

Early-Onset Macular Degeneration in Cynomolgus

Monkeys (Macaca fascicularis)
At the Tsukuba Primate Center, an early-onset macular degeneration with
autosomal dominant inheritance occurred in a large pedigree of cynomolgus
macaques (Macaca fascicularis) (162–165). A similar syndrome has been
identified in the Japanese macaque (Macaca fuscata) (161). Symptoms appeared
as early as 2 years of age and exacerbate gradually with aging. While
funduscopy revealed fine, yellowish white dots within the macula of affected
animals, drusen from these animals have been confirmed by histopathology to
closely resemble human equivalents and have been shown by
immunohistochemistry and proteomic analysis to contain many human
hallmarks, such as:

Apolipoprotein E
Amyloid P component
Various-sized complement component C5
The terminal C5b-9 complement complex
Vitronectin, membrane cofactor protein
Annexins
Crystallins
Immunoglobulins

Young age of onset and inheritance pattern in this model are reminiscent of a
number of early-onset maculopathies in humans, rather than AMD. Umeda et al.
assessed the contribution of early-onset human maculopathy susceptibility genes
in this cynomolgus colony. They found no association between the monkey
phenotype and 13 genes that are known to underlie early macular degeneration
syndromes in humans (162). The dominant inheritance and early onset of this
disease could facilitate production of animals for preclinical therapy
development.
Adult-Onset Macular Degeneration in Rhesus
Monkeys (Macaca mulatta)
Even though natural developments of both dry and wet forms of advanced AMD
are only occasionally found in monkeys, many groups have been capable of
documenting the signs of early to intermediate AMD in older rhesus macaques
(Macaca mulatta) (164,166–175). Funduscopic examination of a closed colony
of rhesus macaques found in Cayo Santiago revealed macular drusen in
approximately half of animals older than 9 years and all specimens older than 25
years (168,176–178). Both histologic and ultrastructural examination set the
presence of small and large drusen within the BM as well as beneath the RPE. In
some cases, RPE cells overlying drusen were atrophic. Among the composites
found in the drusen of cynomolgus macaques, we can remark the following:

The apolipoprotein E
Amyloid P component
Complement component C5
The terminal C5b-9 complement complex
Vitronectin, membrane cofactor protein
Annexins
Crystallins
Immunoglobulins

For further studying, a breeding colony derived from these macaques was
established at the University of Florida in 1994, and equivalent features were
displayed (179). In spite of the much higher occurrence of macular drusen in
young adult to middle-age macaques compared to that observed in humans, the
disease seems to progress more slowly and only occasionally lead to geographic
atrophy or CNV (167,176,178). Contrary to early-onset macular degeneration in
cynomolgus macaques, rhesus macaques appear to have a nonmendelian
inheritance pattern. Francis et al. (169) genotyped a population of rhesus
macaques and found that a susceptibility locus in the rhesus homologue of
human 10q26 was associated with macular phenotype. These genes contain
sequence variants significantly associated with affected status in humans and
macaques. As expected, the LOC387715/ARMS2 gene is present in simians
only, thus corresponding with development of the macula. In contrast to human
studies, this study indicated that only the promoter polymorphism in HTRA1
appears to be significantly associated with the disease phenotype.
ANIMAL MODELS OF WET MACULAR
DEGENERATION
Only about 10% of patients suffering from AMD have the wet type. This form,
known also as exudative or neovascular, is characterized by the formation of a
pathologic CNV responsible for most cases of severe blindness (180).
Bleeding, leaking, and scarring from these abnormal blood vessels
eventually cause irreversible damage to the photoreceptors and rapid vision loss
if left untreated. Yet, studies based on animal models have opened the door
toward a new era as far as treatment, prognosis, and morbidity in wet AMD are
concerned. In fact, it was the better understanding of the molecular basis for this
condition that allowed the replacement of aggressive ablative therapies with
more sophisticated and seductive alternatives (181). Agents that delay
neovascular growth and moderate subretinal hemorrhages are one of the most
hopeful breakthroughs in the field of therapeutics for advance AMD and are
currently under evaluation.
The majority of present models for wet AMD rely on either laser or direct
mechanical injury to the RPE/BM complex or alteration of the RPE and
surrounding environment by external interventions (such as exogenous
compounds injected in the subretinal space). Nonetheless, genetically altered
mice have been gaining popularity among experts, who use these “internal”
interventions to explore angiogenic homeostasis within the RPE–choroidal
interface, specially the equilibrium between proangiogenic VEGF and
antiangiogenic PEDF. The interface of these two approaches has brought several
antiangiogenic agents into clinical use. Even though these studies currently focus
on inhibiting VEGF (182), additional use of PEDF via gene therapy constitutes a
realistic idea and may occur in the future (183).
Even though these exudative AMD models recapitulate many of the clinical
and histologic manifestations of CNV in humans, the time to develop, course of
progression, size, and appearance of the lesion vary significantly among them
(184). These models and the differences between them are going to be discussed
in detail in the following sections.

LASER-INDUCED MODELS OF CNV

Laser Injury in Rodents
Laser-induced neovascularization can be achieved in both mice (136,185,186)
and rats (187,188). Although this laser trauma model was first tried in nonhuman
primates (189), rodent adaptations are nowadays more popular mainly because
they offer attractive advantages in terms of cost, number of CNV lesions for
statistical analysis, and time course. In fact, both mice and rats showed an early-
phase CMV over the first week, displaying mature membranes after only 10 days
(184,190). Laser trauma models basically consist of using high-powered,
focused laser energy in order to induce several breaks in BM. The power
employed to generate CNV must be fixed carefully, as burns without enough
intensity to break the BM may not result in CNV and extremely intense burns
may result in extensive choroidal hemorrhage followed by avascular scarring
without CNV.
There is no ideal method to detect laser-induced neovascularization; each
approach has their own strengths and weaknesses and often complements each
other. Neovascular development can be monitored in anesthetized rodents with
high-resolution angiography with fluorescein isothiocyanate–dextran to assess
vessel leakage, although quantifying is sometimes challenging as mature CNV
complexes may not leak at all (190). Plus, tissues can be scanned postmortem
with paraffin histology. Since these studies have an important quantitative
component, care must be taken during processing so that pathology of each laser
site can be correlated with its in vivo angiographic progression. Histochemical
visualization and immunochemical methods using antibodies may contribute in
detecting endothelial cells and thereby neoformed vessels (187,191). However,
histologic examinations are overall demanding and time-consuming processes,
which may be also limited by the technical difficulty to achieve adequate
sampling, as was previously mentioned. Finally, using image analysis software is
an appealing alternative to investigate the histologic area of CNV in these
models (192). Sclera–choroid–RPE flat mounts may be created and combined
with fluoresceinated high molecular weight dextran angiography to allow
computer-assisted, quantitative imaging (184). However, there are two major
problems regarding this method: First, there is no certainty that the vascular
tissue imaged in this technique is choroidal in origin (187), and second,
nonperfused vessels sometimes are not detected (193). Recently, Campos et al.
have proposed another method of assessment in which rat eye cups were
fluorescently labeled with markers for nuclei, endothelial cells, microglia, and
filamentous actin and then flat-mounted specimens were evaluated with confocal
microscope. Computer software generated three-dimensional reconstructions for
qualitative and quantitative analysis of confocal image stacks. This technique
provides excellent morphologic detail and facilitates the study of critical early
events in CNV, including the rupture of the BM and the formation of endothelial
clusters before vessel formation.
Laser-induced CNV models have several differences with the actual wet
AMD that must be taken into consideration. First, laser use may ominously harm
the overlying neural retina to a larger extent than what is normally observed in
eyes of AMD patients. Second, laser trauma models fail to emulate the complex
sequence of events that leads ultimately to CNV in the actual maculopathy. Such
functional disparity is a consequence inherent to the model fabrication process:
Trauma-induced CNV occurs secondary to an acute injury and inflammation
response and not to long-standing senescent degeneration and chronic
inflammation as happens in the genuine disease.
Regardless of the drawbacks mentioned above, rodent laser trauma models
have contributed greatly toward our present understanding of the pathogenesis of
CNV, including

The role of angiogenic cascade (80,194–202)

The role of intracellular kinase signaling of growth factors (192,203)
The participation of numerous cell types involved in CNV formation
(194,204,205) and the role of RPE (206)
The role of the complement cascade in experimental CNV (207)
The proof of concept for pharmacotherapy of CNV and gene therapy
(192,208–223)

Laser Injury in Nonhuman Primates

Just as in rodents, laser photocoagulation is used to break the BM and induce a
subsequent fibrovascular proliferative response in the choroid. This response is
the basis for modeling CNV in late-stage AMD regardless of the animal
employed. The primate model of experimental CNV, created by Ryan et al.
(224,225), was termed “subretinal neovascularization” at the time and was the
first animal model of CNV more than 30 years ago. Although this procedure was
originally tried in rhesus and cynomolgus macaques, the technique has been
recently adapted for use in the squirrel monkey (226). Ryan (189) used an argon
laser to induce small spots of photocoagulation in a reproducible grid pattern in
the temporal retina, avoiding the capillary-free zone of the fovea. This method
continues to be in use. Typical argon laser exposure parameters for the induction
of CNV have included a 50-μm spot size, 100-ms duration, and powers ranging
from 300 to 700 mW but in some studies as high as 1,500 mW. Spots are
funduscopically visible as bubbles at the time of photocoagulation.
Photocoagulation is sometimes repeated to produce a break in the BM and
bubble formation.
Photocoagulation induced thrombosis of choroidal vessels followed by
reendothelialization 48 hours later and growth of new vessels into the subretinal
space by a week (227). As explained above, newly formed vessels are more
permeable so that neovascular development can be monitored with fluorescein
angiography to assess vessel leakage. As a matter of fact, outcome measures of
efficacy include the extent of growth of laser-induced new vessels, the total area
of blood vessel leakage, and the percentage of laser lesions resulting in clinically
significant vessel leakage, as visualized by fluorescein angiography and in some
cases confirmed by histopathology. By these criteria, CNV peaks at
approximately 2 to 4 weeks after photocoagulation. Similar to the CNV that
occurs in human ocular disease, laser-induced CNV follows predictable stages of
development: early membrane formation, establishment of a mature
fibrovascular network, and involution (228). Experimental agents are typically
given at approximately 1 month after injury, before vessels begin to resolve
(226,229). Spontaneous neovascular involution (indicated by decreased
fluorescein leakage) commences at approximately 3 to 7 weeks and then
gradually progresses (over a period of approximately 2 to 13 months) until
leakage is no longer apparent at the site (230). The extent of new vessel growth
compared to poorly vascularized scarring can be variable in all models and is
influenced by species, location of injury in the retina, and intensity of the laser
beam. In some cases, laser injury can result in anastomotic vessels between
choroidal and retinal circulations (229). This lesion, known as retinal
angiomatous proliferation, can be present as part of advanced AMD in humans,
and it holds a poor prognosis.
Advantages of the primate model include the close approximation of the
monkey retina and macula to the human, size of the primate eye for drug
delivery studies, and utility of the model for development of human clinical
trials. Disadvantages include the expense of the animals, animal care and
husbandry, length of experiments, and ethical issues regarding use of primates
when rodents (mice and rats) are a reliable model. The primary drawback of the
model is that not all eyes respond with new vessel formation, with an incidence
of CNV as low as 30% (225,231). Latest studies have managed to optimize laser
parameters to produce advanced lesions more frequently (232). Still, group sizes
of 10 monkeys are typically required to document significant effects, and in
some studies, smaller group sizes have generated inconclusive results (233,234).

Laser Injury in Rabbits and Pigs

Rabbit
elDirini et al. (235) used rabbits in an attempt to find an intermediate laser-
induced CNV model between rodents and primates, thereby avoiding the
expense and ethical issues concerning the latter. Nonetheless, rabbits exhibit
significant anatomical discrepancies, as they do not have a macula and their
retina vascular supply is different. The model utilized subretinal
endophotocoagulation to create histologically identified CNV.

Pig
Another intermediate laser-induced CNV model is the pig. Saishin et al. (219)
used a laser to create defects in the BM in the pig eye and establish histologic
evidence of CNV in 100% of lesions. Image analysis of histologic sections was
used to evaluate drug delivery for the treatment of the CNV. Kiilgaard et al.
studied xenon laser versus diode laser versus mechanical disruption of the BM in
a pig model of CNV. The authors found histopathologic evidence of CNV in
54%, 83%, and 100% of the animals, respectively (236). The pig eye is
approximately the same size as the human eye, and the retina vascularization is
similar to the human. Like the rabbit, the pig circumvents the cost and ethical
issues of the primate. The utility of the pig is for drug delivery experiments.
However, the pig lacks the advantages of high-throughput, short-duration
experiments as available in rat and mouse models of laser-induced CNV.

Surgically BM Rupture
Trauma to the BM can cause localized fundus areas of hemorrhage and
infarction with subsequent neovascular connections between the retina and
choroid (237). Pioneer studies undertaken by Kiilgaard et al. (236) compared
eyes in pigs that underwent retinal photocoagulation with a xenon lamp and
retinal laser photocoagulation with those exposed to mechanical ruptures of the
BM following surgical debridement of the RPE without damage to the
neuroretina. The results were conclusive: Not only did the surgical method show
higher success rates of CNV induction (100% vs. 83% and 54%), but it has also
produced CNV membranes that were morphologically more similar to those
present in human exudative AMD. Contrary to CNV obtained by using laser or
xenon burns, morphology of surgically induced lesions was not dominated by
retinal gliosis and retinal neovascularization, probably due to a preservation of
the neuroretina.
Also, Lassota et al. (238) found that the original surgical technique could be
optimized by avoiding RPE removal prior to the BM perforation. Compared to
those obtained with the original surgical intervention, CNV membranes induced
by using this method were significantly thicker and had a higher cellular content,
were more richly vascularized, and also exhibited the highest propensity to leak
in fluorescence angiograms. It is important to note that even when the surgical
model offers benefits of reduced neuroretinal damage, the technique is limited
by higher cost and the necessity of a three-port vitrectomy.

SUBRETINAL INJECTION MODELS

CNV has been immunologically and mechanically induced in animal models,
primarily by injection of synthetic peptides, viral vectors containing VEGF,
cells, and inert synthetic materials. The proximity to the choroidal vasculature
along with the perturbation of the RPE caused by the injection itself, when
combined with proper angiogenic signals, appears to be sufficient stimuli to
encourage invasion of the subretinal space by CNV complexes (196).

Subretinal Matrigel Injection

Although the pathogenesis of CNV in AMD is not entirely clear, it has been
strongly associated with the presence of abnormal extracellular deposits in the
space between the RPE and BM (4,6,239,240). Supporting this notion, recent
studies have shown that artificially created sub-RPE deposits are sufficient to
induce the development of CNV in mice (241) and in rabbits (242).
Subretinal/RPE injection of Matrigel, a basement membrane extract (243) that
solidifies after implantation in tissue, was reported to induce CNV in both ccl2-
deficient and wild-type rodents, possibly by encouraging the release of
angiogenic factors and recruit host cells (242). As mentioned in previous
sections, the normal functioning of CCL2/CCR2 pair confers protection against
age-related maculopathies. Consistently, data revealed that CCL2 knockout mice
developed more severe disease than wild-type counterparts. These findings
support Matrigel subretinal injection as a useful generator of AMD-like
pathologic changes while confirmed the importance of CCL2 in AMD
pathogenesis. More recently, studies showed that rabbits exposed to subretinal
injection of Matrigel alone or Matrigel with VEGF also responded positively,
developing CNV in 100% of the lesions (242). Zhao et al. used Matrigel
injection to assess the necessity of a proper relationship between the RPE and
BM so as to prevent abnormal angiogenesis. Matrigel was injected to the
subretinal space of rats to create an amorphous deposit, causing RPE cells to
migrate toward photoreceptors and then form a new layer between the deposit
and photoreceptors, resulting in RPE translocation. The BM devoid of RPE
attachment becomes susceptible to invasion by new blood vessels from the
choroid, thereby resulting in CNV (241).

Subretinal Lipid Injection

Lipid and oxidized lipid depositions are found in eyes of AMD patients as well
as non-AMD aged BM (244). Rabbits and rats exposed to subretinal injections
of an oxidized lipid (HpODE) have displayed CNV (245,246). Since animals in
which subretinal injections caused breaks in the BM were excluded from the
study, the authors were in position to blame the lipid injection for the histologic
changes.

Subretinal Complement Compounds Injection

The role of the complement cascade in the formation of drusen and CNV is
unquestionable and has been already discussed (122,136,247). Lyzogubov et al.
(248) employed different doses of subretinal PEG-8 to create a new model of
CNV. PEG-8 is an activator of both the classical and alternative pathways of the
complement system. In this model, PEG-8 injection induced activation of the
complement cascade and dose-dependent CNV production.

Macrophages
Macrophages are believed to have a pivotal role in the development of CNV
(249,250). Subretinal injection of macrophages on wild-type or Ccl2 knockout
mice (251) caused CNV with concomitant fibrosis. Nevertheless, since the
protocol included rupture of the BM with a laser at the site of injection, the
meaning of the findings is indefinite.
GENETICALLY ENGINEERED ANIMAL
MODELS OF CNV
Subretinal VEGF Gene Therapy
VEGF is the quintessential promoter of vasculogenesis and angiogenesis
processes in both CNV models and human wet AMD (252,253). In effect, CNV
membranes removed from AMD patients showed VEGF remarkable
immunoreactivity (254,255). It has been hypothesized, therefore, that
overexpression of VEGF would result in concomitant angiogenesis within the
eye.
Numerous groups have sought to put into practice this theory by giving
subretinal injections of adenovirus vectors expressing VEGF to rodents.
Transgenic expression of VEGF in RPE cells resulted in choroidal vascular
permeability, leukocyte adhesion, and intrachoroidal neovascularization, as
measured by angiography and histology. If the VEGF transgene is expressed in
the photoreceptor layer, new vessels originate from the inner retinal vasculature
within the retina and extend into the subretinal space, where they form clusters
of vessels surrounded by RPE cells (256). Adeno-associated virus–mediated
delivery of PEDF to the subretinal or vitreous space results in significant
reduction of neovascularization resulting from laser injury in mice (223). Plus,
pharmacologic blocking of its signaling has shown results as promising as
consistent by successfully inhibiting laser-induced CNV in experimental animal
models (219,257).
The advantage of these models is the ability to study various biologic
components of CNV by comparison with controls and crossbreeding
experiments. Disadvantages relate to the length of time for the CNV to develop,
the relatively small percentages of eyes that develop CNV, and the small size of
the CNV. These models have supported the concept that the best models of CNV
are those that incorporate physical disruption of the BM into the model.

SOD1 Knockout Mice

Senescent Cu–Zn SOD–deficient mice displayed fundus and histologic evidence
of CNV in 8.3% and 10%, respectively (76). A marker of oxidative damage to
DNA (I-OHdG) was detected in the RPE of senescent Sod 1 knockout mice but
not in controls. The CNV appeared to connect with retinal vessels in 16-month-
old compared with year-old mice.

Ccr2-/Ccl2-Deficient Mice
The Ccr2-/Ccl2-deficient mouse model of AMD has received considerable
attention (154). In this model, transgenic mice deficient in either Ccl2 or Ccr2
fail to recruit macrophages to the area of the RPE and BM. This allows for
accumulation of C5a and IgG, both of which induce VEGF production.
Thorough examination of these transgenic mice showed a CNV incidence of
25% (154). These findings have contributed to the understanding of the
pathophysiology of CNV, specifically regarding to macrophage recruitment.
Although the areas of CNV are very small, recent work has shown that CCR3 is
specifically expressed in choroidal neovascular endothelial cells in either of
these knockout mice (258).

Ccl2/Cx3cr1 DKO Mice

RPE changes and drusen-like lesions exhibited by these mice have been already
mentioned. However, approximately 15% of these animals eventually exhibit
histologic evidence of CNV. The fact that lesions in these mice take only 6
weeks to appear makes it a very appealing model for studying spontaneous
development of neovascularization. However, further work is required to assess
the molecular mechanisms underneath this neovascularization.

VLDLR-Targeted Mutant
Homozygous strains of mice with targeted mutations for VLDLR gene
developed new blood vessels in the area of the outer plexiform layer of the retina
and choroidal anastomoses by 3 months (51).

ApoE Overexpression
Eyes in hypercholesterolemic mice displayed AMD-like changes in the BM/RPE
complex (35). ApoE4 overexpressing transgenic mice fed with HF-C diet
developed CNV in addition to drusen-like and basal laminar deposits as early as
65 weeks of age (36). The CNV was observed in 19% of male and 18% of
female mice and was demonstrated by several methods.

Cp Heph Knockout Mice

Iron overload, observed in these transgenic mice, causes AMD-like alterations.
The mice developed funduscopically evident lesions at the level of the RPE, and
100% of the mice developed histologically identified subretinal
neovascularization, although it is not apparent if the neovascularization arose
from the retina or choroid (91). Furthermore, the mice do not appear to develop
the drusen-like or basal laminar deposit-like lesions as seen in AMD.

Spontaneous Bst Chromosome 16 Mutant

An angiogenic phenotype has been described in Bst/+ mice that is age related
and clinically evident and resembles human CNV. This represents a spontaneous,
genetically determined model of CNV. Bst/+ mice offer the possibility of
exploring the molecular mechanisms of CNV without the need for exogenous
agents. The Bst mutation demonstrates a broad range of phenotypic expression
that includes CNV, focal retinal detachment, retinal dysplasia, persistence of the
hyaloid vascular system, patchy absence of retinal differentiation, and
colobomas. Almost 90% of the mice exhibited subretinal neovascularization
identified with histologic examinations. The CNV was associated with RPE
abnormalities, retinal hamartoma-like lesions, and connections of the
neovascularization with the retina and choroid through defects in BM. Although
the CNV was age related, there were neither drusen-like nor basal laminar
deposit-like lesions in this model.

ANIMAL MODELS FOR ANTI-VEGF

THERAPY
Anti-VEGF therapies are widely used to treat wet AMD. Bevacizumab (BVZ), a
full-length antibody originally developed to treat advanced colon
adenocarcinoma, found its way through ophthalmology, and it is chosen for
diseases as relevant as exudative AMD, diabetic retinopathy (DR), retinal vein
occlusions, and retinopathy of prematurity. Likewise, pegaptanib, an aptamer to
VEGF165, and ranibizumab (RVZ), the Fab fragment of the VEGF, were
developed exclusively for intravitreal usage (259). We must note that both in
vitro and in vivo preclinical studies were performed in order to determine its
efficacy and safety (260).
Animal models used to test these therapies were mostly rabbits, rats, mice,
and nonhuman primates. Rabbits were used primarily to describe the
pharmacokinetics of intravitreal RVZ and BVZ. These studies showed that BVZ
had longer half-life but was detected in low concentrations in serum and humor
vitreous of the fellow uninjected eye (261,262). Pharmacokinetics and serum
bioavailability were studied in cynomolgus monkeys as well. Six to twenty-four
hours after intravitreal injection, retina levels of the anti-VEGF drug were one-
third that in the vitreous, and its half-life was 3 days with a parallel clearance
from all ocular compartments. Consequently, it would be suitable for clinical use
in wet AMD by an intravitreal injection once a month (263).
Rats were chosen to investigate the toxicity of all three anti-VEGF therapies
using two different doses in both healthy and N-methyl-D-aspartate (NMDA)–
induced retinal ganglion cell (RGC)–damaged animals. No toxic effects of BVZ
and RVZ were found in either healthy or damage rat models (264).
Mouse models of subretinal neovascularization and exudative retinal
detachment were employed in order to compare the effects of intravitreal
injections of RBZ and BVZ. For this purpose, Miki et al. selected two models:
transgenic mice, in which the rhodopsin promoter derived expression of human
VEGF in photoreceptors (rho/VEGF mice), and double transgenic mice, which
had doxycycline-inducible expression of VEGF in the retina (Tet/opsin/VEGF).
This last one represented an aggressive model of proliferative retinopathy due to
high VEGF expression. The gathered data suggest that BVZ is not only as useful
as RBZ for treatment of subretinal neovascularization in mice but also superior
in severe models (265).
New antiangiogenic approaches are being studied including small RNA
(ribonucleic acid) interfering molecules and multikinase inhibitors. Two small
interfering RNAs that selectively silence messenger RNA encoding for VEGF
are under investigation:

Bevasiranib (bevasiranib, OPKO Health, Miami, FL) targets against VEGF

—an mRNA.
SIRNA-027 (AGN211745 or SIRNA-027, Allergan, Irvine, CA) targets
VEGF receptor 1.

Tyrosine kinase inhibitors, which block the phosphorylation of all known

VEGF receptors such as Vatalanib® (formerly PTK-787, Novartis International
AG, Basel, Switzerland), have shown efficacy in mice animal models of retinal
and CNV. Maier et al. investigated the effect of this drug in a mouse model of
ischemia-induced retinopathy. Mice were exposed to 75% oxygen on postnatal
day 7, and on day 12, they injected the drug intravitreally. The authors concluded
that a single intravitreal injection was capable of significantly reducing
angioproliferative retinopathy (266).

ANIMAL MODELS OF OTHER RETINAL

DEGENERATIONS
Retinal degenerations reunite a group of entities characterized by the progressive
loss of retinal function due to cell death. These conditions comprise different
phenotypes that can be present at birth and develop during infancy or even
during adulthood. Plus, the spectrum of vision impairment is also wide and can
range from night blindness and loss of peripheral or central vision to total loss of
vision.
Retinal degenerations are usually classified in hereditable and
nonhereditable. In the hereditable retinal degeneration group, we can include
retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and Stargardt
disease. On the other hand, AMD, glaucoma, and DR belong to the
nonhereditable group, which affect a larger number of people worldwide (267).

Retinitis Pigmentosa
RP is an inherited disorder that leads to blindness because of the loss of
photoreceptor cells. It is caused by the mutation of multiple genes that encode
for proteins responsible for the maintenance and function of cones and rods. Its
prevalence is around 1 case per 3,000 to 5,000 individuals (268), and the
inheritance can be sporadic, X-linked, and autosomal recessive or dominant,
being the latest accounted for 25% to 30% of all cases of RP (269).
Since neither successful treatment nor cure has been discovered, exploiting
animal experimentation to develop new strategies may be crucial. The greatest
number of known mutations that cause RP occurs in 32 genes, specially in the
rhodopsin (RHO), retinitis pigmentosa 1 (RP1), and retinitis pigmentosa GTPase
regulator (RPGR) genes. However, no single mutation accounts for more than
10% in related patients (270).
RP pathogenesis and therapy have been studied in a wide range of animals
including rodents, dogs, cats, and pigs. Of those, two animal models described in
literature stand out for their utility, and they differ in their Mendelian
inheritance. RPGR mutations are the most common cause of X-linked retinitis
pigmentosa (XLRP). Most of these mutations are present in ORF15, the purine-
rich terminal exon of the predominant splice variant expressed in retina. A
naturally occurring mouse animal model of XLRP has been recently defined.
The retinal degeneration 9 mice carry a 32-base-pair duplication at ORF15 that
leads to a shift in the reading frame causing a nonsense mutation. Therefore, no
protein is translated, and thus, mice exhibit pigment loss and thinning of the
outer nuclear layer (271).
The second model corresponds to an autosomal dominant retinitis
pigmentosa (adRP) miniature pig that carries the most common human mutation
Pro23His (P23H) RHO. Miniature pigs were chosen instead of domestic pigs
because its smaller size makes it easier to manipulate. Mini pigs with the
Pro23His (P23H) RHO mutation successfully developed the clinical phenotype
of human RP: a dramatic reduction in the scotopic b-wave amplitude in the full-
field ERG, reflecting rod photoreceptor dysfunction, and a subsequent, albeit
delayed, reduction in the photopic b-wave amplitude, reflecting cone
photoreceptor dysfunction along with extensive degeneration of the outer
nuclear and reduced thickness of the INLs. Authors supported this model over
rodents on the grounds of convenient eye size, better access to the subretinal
space improving surgical interventions, and a more appropriate biologic model
(since approximately 14% of the photoreceptors in the pig are cones, compared
to only 1% in mice (272)). Cat and dog models of inherited RP are currently
used in research as well (273,274). However, its restricted genetic information,
the presence of major and minor histocompatibility differences using cell-based
therapies, the societal concern over experimentation in them, and the limited
access to retinal progenitor and stem cells in these species constitute major
limitations that frequently impede their use.

Diabetic Retinopathy
DR is a major microvascular complication of diabetes mellitus and the leading
cause of preventable blindness among the working-aged population with a
significant impact on the world's health systems. DR's most accepted
classification includes a nonproliferative (NPDR) and a proliferative form (PDR)
(275). NPDR, previously called background retinopathy, is determined by the
presence of microaneurysms and dot and blot hemorrhages. According to these
microscopic findings, NPDR can be further subdivided into three stages:

Mild: microaneurysms only.

Moderate: more than just microaneurysms but less than in the severe
nonproliferative form.
Severe: more than 20 intraretinal hemorrhages in each of four quadrants or
 definite venous beading in two quadrants or prominent intraretinal
microvascular abnormalities (IRMAs) in one quadrant. No signs of
proliferation must be found.

On the other hand, PDR is characterized by the presence of neovessels that

conduct to vitreous/preretinal hemorrhages, fibrosis, and tractional retinal
detachments.
Animal models have been extensively used in diabetes research. To date,
species such as mice, rats, cats, dogs, pigs, and nonhuman primates are being
used to understand the pathogenic mechanisms underneath DR. Models of DR
are complex and can be achieved by genetic alteration, pharmacologic induction,
feeding a galactose diet, and spontaneous selection inbreeding. Recently, protein
molecular biologic techniques have produced a large number of new animal
models for the study of diabetes, including knockin, generalized knockout, and
tissue-specific knockout mice.
Rodents, especially mice, are the most widely studied animal DR models due
to their short generation time, easy manipulation, and the inherited
hyperglycemia that affect certain strains. They are suitable for reproducing early
stages of the disease but less useful in demonstrating all neural and vascular
complications typical of the advanced phases, so they do not exactly mirror the
human condition (276). Consequently, researchers have chosen nondiabetic
animals to study proliferative retinopathy where VEGF and IGF-1 factors can be
manipulated independently.
Rodent DR models can be either spontaneous or chemically induced.
Currently, diabetes can be provoked by destroying the pancreatic β-cells with
alloxan or streptozotocin, generating a type 1 diabetic model. These animals
maintained a hyperglycemic state for up to 24 months with small amounts of
insulin and develop early-stage DR findings. This method is frequently
performed in rats because mice can be difficult to maintained alive once diabetic.
Interestingly, variations in the retinal response to diabetes in between rat species
have been reported (277). By contrast, spontaneously diabetic rodents, for both
type 1 and type 2 diabetes, have been broadly described in literature (Table 2.1).

Table 2.1 DIABETIC RETINOPATHY RODENTS ANIMAL MODELS

 Larger animals, including dog, cats, and pigs, have been used in DR research
with relative success, but not exempt from limitations. All of these models,
although morphologically similar to human DR, tend to be expensive and
complex because of the lack of specific antibodies and molecular biology
reagents. To add more, techniques for manipulation and genetic characterization
are somewhat experimental and therefore less precise than in small animal
models. Indeed, nonhuman primates stand out among other models discussed
above because of the presence of a macula. Cynomolgus monkeys (Macaca
fascicularis) that were pancreatectomized or treated with streptozotocin and
obese rhesus monkeys (Macaca mulatta) that spontaneously develop diabetes at
middle age have been used in DR studies. As in humans, DR develops fairly
slowly in these models. Johnson et al. reported ophthalmoscopical changes in
aged monkeys with spontaneous diabetes. Their findings showed intraretinal
hemorrhages and large areas of retinal capillary nonperfusion along with
macular edema (287). These models can almost perfectly mimic every aspect of
the human condition. However, the longer gestational periods, the lack of
reagents for experimentation, the high cost of maintenance, and the ethical issues
involved represent disadvantages that cannot be ignored.

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 3
PATHOGENESIS AND
PATHOPHYSIOLOGY OF AGE-
RELATED MACULAR
DEGENERATION ANA
ANA MACHÍN MAHAVE • ERNESTO ROMERA REDONDO
JOHN BARNWELL KERRISON

INTRODUCTION
Age-related macular degeneration (AMD) is the leading cause of severe visual
acuity loss in the United States among people older than 60 years, representing
54% of legal blindness (1). In the general population, the number of people in
this group is increasing. As a result, vision loss from macular degeneration is a
growing problem. Ninety percent of AMD patients have dry AMD, and 10% of
AMD patients have wet AMD. Currently, an estimated 8 million Americans are
affected with early AMD, and over 1 million will develop advanced AMD
within the next 5 years (2).
Many changes in the macula could be due to normal aging. The focus of
study has been which aging processes are implicated in the pathogenesis of
senile macular degeneration or at what stage they become pathologic (3). The
critical question to be answered is “Is AMD a normal process of aging or is it a
disease by itself?” (4–7). Most of the authors defend the theory in which AMD is
an advanced stage or perturbation of the normal process of eye senescence
(3,4,8,9).
The pathophysiology of AMD is incompletely understood, but genetic tools
offer new insights into the development and progress of AMD (10).

RISK FACTORS
AMD is a multifactorial disease in which multiple genetic and environmental
factors are involved.
Age: Age is the largest risk factor for AMD (11–17).
Smoking: The mechanism by which smoking might affect the retina is
unknown; however, there exists a direct association between the risk of
developing advanced AMD with the number of cigarettes smoked (18,19).
Sunlight exposure: Individuals who wore sunglasses regularly were less
likely to develop soft drusen (20). Results from the Beaver Dam Study suggest
that people who spent leisure time outdoors were at an increased risk of
developing early AMD (21). On the other hand, some studies have shown little
or no association between sunlight exposure and the risk of AMD (22–24).
White individuals: It is postulated that increased levels of melanin could
increase the free radical scavenging potential of the retinal pigment epithelium
(RPE) and Bruch's membrane (BrM), thereby protecting against the risk of
advanced AMD (25–28).
Female sex: Female gender might be a risk factor in individuals older than
75 years (29), with double the risk of developing wet AMD in comparison with
age-matched men. However, nonstatistically significant differences have been
demonstrated (30).
Cholesterol levels, obesity, hypertension, cardiovascular disease, and
increased dietary fat: These factors seem to be related to the risk of developing
AMD, but different studies do not agree (31–37).
Genetics: Several studies show a genetic factor in the pathogenesis of the
disease (38–41). The gene for apolipoprotein E (APOE) is the first identified
susceptibility gene for AMD and has been also associated with other diseases of
aging including Alzheimer's disease. APOE epsilon 2 allele is associated with a
50% increased risk of AMD (42); however, APOE epsilon 4 allele is associated
with 57% reduction in risk of wet AMD (43). Several loci have been also
associated with AMD, including two major loci in the complement factor H
(CFH) gene on 1q32 and the ARMS2/HTRA1 locus on the 10q26 gene cluster
(44,45).
In short, genes influence many biologic pathways, but genetic susceptibility
can be modified by environmental factors. Together, they are greatly predictive
of onset and progression of disease (46,47).

PATHOPHYSIOLOGY AND PATHOLOGY

The pathology of AMD is characterized by degenerative changes affecting outer
retina (photoreceptors), RPE, BrM, and choriocapillaris. These structures,
collectively called Ruysch's complex (4,48), provide an optimal environment for
retinal function—high-resolution and color vision (cones), and peripheral vision
and vision at dusk (rods).
AMD can be classified according to its onset, distinguishing early and late
AMD. Early pathologic changes in AMD involve basal deposits (laminar and
linear) (49) in BrM, which cannot be distinguished by clinical evaluation. Late-
stage AMD shows loss of RPE, decreased choriocapillaris density, and decreased
lumen diameter of the choriocapillaris. There is no neovascularization in
atrophic or dry AMD. In wet AMD, neovascularization, exudative change, and
disciform scar formation are observed (50).

THE RETINAL PIGMENT EPITHELIUM

The RPE is a central element in the pathogenesis of AMD. It is a postmitotic,
cuboidal monolayer of pigmented cells, which improves visual resolution and
neutralizes photo-oxidative stress. The RPE has a very high metabolic rate and is
rich in mitochondria. It is located between the neural retina and choroid. Because
of its neuroectodermal origin, the RPE is considered part of the retina. The inner
boundary (apical membrane) interdigitates with the outer segments of
photoreceptors. The outer boundary (basolateral membrane) faces BrM forming
the outer blood–retinal barrier (BRB). Functions of the RPE include regeneration
of bleached visual pigments; formation and maintenance of two extracellular
matrixes, the interphotoreceptor matrix and BrM; transport of nutrients, ions,
and water between photoreceptors and the choriocapillaris; and phagocytosis of
membranous discs of the outer segments of photoreceptors (51). Another pivotal
function of the RPE is light absorption and protection against photo-oxidation.
The RPE is also involved in the immune privilege of the eye through the
secretion of immunosuppressant factors (52).

TRANSPORT
Transport through the RPE is bidirectional. From the subretinal space to choroid,
the RPE transports electrolytes and water. From the blood to the photoreceptors,
the RPE transports glucose and other nutrients.
Transport from blood to the photoreceptors encompasses glucose and other
vital nutrients. The RPE absorbs glucose, retinol, ascorbic acid, and fatty acids in
the blood and delivers them to the photoreceptor. For the transport of glucose,
the RPE has high amounts of glucose transporter (GLUT) in both their apical
and basolateral membranes. Another notable function of the RPE is transport of
retinyl to ensure its supply to the retinal photoreceptors. In this process, many
complex intermediate steps take place. A critical step involves an RPE enzyme
that isomerizes all-trans-retinyl esters into 11-cis-retinal, which is essential for
rod and cone function (33,53).
The delivery of the fatty acids such as docosahexaenoic acid and
eicosapentaenoic acid to the photoreceptor is another important RPE function
(54). These omega-3 fatty acids cannot be synthesized by the nervous tissue but
are an essential component of the membranes of neurons and photoreceptors.
They are synthesized in the liver from the precursor, linolenic acid, and are
carried in the blood by plasma lipoproteins (33). In addition, docosahexaenoic
acid is the precursor of the D1 neuroprotectin that protects RPE from oxidative
stress (25,55). The main sources of omega-3 fatty acid are fish products. The
Blue Mountain Eye Study and some case–control studies have found evidence of
reduced risk of advanced AMD and in those who eat fish regularly (34,56,57).
Transport from photoreceptors to blood is important for ions and water. The
RPE transports ions and water from the subretinal space (apical side) into the
blood of the choroid (basolateral side). This process requires energy, and the Na
(+)/K (+)-ATPase, on the apical membrane of the RPE, provides the energy (58).
Tight junctions between RPE cells result in a barrier between the subretinal
space and the choriocapillaris. Notably, paracellular resistance of this barrier is
10 times the transcellular resistance. For this reason, water cannot pass through
the paracellular RPE pathway and passes mainly through the transcellular
pathway via aquaporin-1 (59,60).

LIGHT ABSORPTION AND PROTECTION

AGAINST OXIDATIVE STRESS
The retina is the only neural tissue in the body that is exposed to direct sunlight,
which favors the oxidation of lipids and is toxic for the photoreceptors. Retina
consumes proportionally more oxygen than other tissues, generating free radicals
that are toxic to the cells of the retina. The RPE is essential to countering
oxidative stress in the retina. The RPE cells contain pigments, such as melanin
and lipofuscin, which absorb and filter the light. The RPE also produces
antioxidant molecules including lutein, zeaxanthin, or ascorbate as well as
enzymes, such as superoxide dismutase and catalase, that reduce reactive species
(61).

PHAGOCYTOSIS
The RPE is a phagocytic system that is essential for the renewal of
photoreceptors. Each photoreceptor has an inner and outer segment, the outer
segment of each cone has a membrane folded 700 times, and the outer segment
of every rod has about 1,000 discs (62).
The photoreceptors are often exposed to constant high light levels, which
leads to accumulation of oxidized proteins and lipids. The concentration of
photo-oxidized substances increases within the photoreceptors daily. The
transduction of the light depends on the photoreceptor by appropriate
functioning and structure of the proteins, retinyl, and cell membranes. Therefore,
to maintain the excitability of the photoreceptor outer segments, they are
undergoing constant renewal, reconstructed from its base. The tips of the outer
segments of the photoreceptors, containing the highest concentration of free
radicals, proteins, and photo-oxidized lipids, detach from the photoreceptors.
Outer segments maintain a constant length through the coordinated release of its
extremities and the formation of new discs. The discs detach from the outer
segments and are phagocytosed by the RPE (63), which digest and deliver the
essential molecules, such as docosahexaenoic acid and retinyl, back to the
photoreceptors to rebuild their light-sensitive outer segments (32,64). Sometimes
the content of the phagolysosomes are incompletely degraded, forming the
residual bodies that are the substrates for lipofuscin formation. The residual
bodies in the RPE accumulate with age, beginning as early as 16 months (65,66).
The limited autophagocytic capacity of the RPE eventually results in an
accumulation of metabolic waste in RPE cells throughout life. Dry AMD often
begins in the parafoveal ring, where lipofuscin concentration is highest.
However, lipofuscin disappears with cell death and a reduction in
autofluorescent lipofuscin is a sign of progression toward late AMD.
SECRETION
RPE cells produce and secrete growth factors as well as essential factors for
maintaining structural integrity of the retina and choroid. These molecules favor
the photoreceptors' survival and ensure a structural integrity for optimal
circulation and nutrient supply. RPE secretes pigment epithelium–derived factor
(PEDF), vascular endothelial growth factor (VEGF), fibroblast growth factors
(FGF-1, FGF-2, and FGF-5), transforming growth factor–β, insulin-like growth
factor I, neuronal growth factor (NGF), growth factor derived from the brain,
neurotrophin-3, ciliary neurotrophic factor, several interleukins, chemokines,
tumor necrosis factor–α, colony-stimulating factors, and various types of
metalloprotease tissue inhibitors (67,68).
The most important factors with regard to the pathophysiology of AMD are
PEDF and VEGF. PEDF is a neuroprotection and antiangiogenic factor. PEDF
inhibits endothelial cell proliferation and stabilizes choriocapillaris endothelium.
It is important in embryonic eye development. Mice lacking PEDF develop more
aggressive retinal vascularization in ischemia (69). VEGF is secreted in low
concentrations during physiologic conditions, thereby preventing endothelial cell
apoptosis. In addition, VEGF regulates vascular permeability, thus stabilizing
endothelium fenestrations (70).

IMMUNE PRIVILEGE
The eye is an immune-privileged site, and the RPE cells have an important role
in the maintenance of this immune privilege (71,72). In 1993, Liversidge et al.
indicated how RPE cells could inhibit lymphocyte proliferation (73). Many
pathways have been suggested to explain RPE immunosuppression (74). On the
one hand, RPE inhibits T-cell proliferation through secretion of prostaglandins
and immunosuppressive cytokines. On the other hand, apoptosis and
phagocytosis properties have demonstrated both directly (decreasing T-cell
number and clearing apoptotic lymphocytes) and indirectly (down-regulating
proinflammatory cytokine secretion, such as IL-1b) (75,76).
Ambati et al. (77) in 2003 postulated in a study of mice that there is an
accumulation of complement fragments that might damage RPE and induce
VEGF production by RPE cells, resulting in the development of choroidal
neovascularization (CNV). This study provides insight into the role of
macrophages and complement in the pathophysiology of AMD (78).
BRUCH'S MEMBRANE
BrM is a complex pentalaminar extracellular matrix (ECM), located beneath the
RPE, and includes three layers: a central elastic layer sandwiched between two
collagenous layers. BrM is lined by the basement membranes of the RPE and the
choriocapillaris (basal laminae). Apart from its structural support function, BrM
is also highly permeable to fluid and small molecules like oxygen, glucose, and
other molecules necessary to maintain retinal health and function (79).
Proteoglycans play an important role in BrM (80). Their negative charge impairs
the passage of positively charged macromolecules that are essential for
maintenance of RPE.
Changes in BrM begin in the third or fourth decade of life (5,81,82). These
changes include the accumulation of membranous debris on both sides of the
elastic lamina, drusen between RPE basal lamina and inner collagenous layer of
BrM, and basal laminar deposits that may occupy the same space as drusen or
develop between the RPE cell and its basal lamina (79) and appears as an
amorphous material. How drusen develop and why they can vary in location and
features are unknown.
Drusen usually contain fragments of RPE cells (9), a core of glycoproteins,
an outer dome of crystallins (83,84), chaperone proteins, APOE, vitronectin, and
proteins related to inflammation (amyloid P, C5, and C5b–9 complement
complex) (85). Drusen are classified as either hard or soft. Hard drusen look like
small, yellow nodules and are not associated with AMD (86). Soft drusen appear
as large, white or light yellow, dome-shaped elevations that can resemble
localized serous RPE detachments.
BrM doubles in thickness and declines in elasticity between the ages of 10
and 90 years. It contains no lipids during the first 30 years of life, but lipid
concentrations rise in later years (4). In addition to the structural alterations that
take place in BrM during aging, its fluid permeability becomes substantially
reduced. BrM acts as a scaffold for the choriocapillaris and RPE cells, regulating
their survival. When this function is diminished, anoikis occurs. Anoikis is an
apoptosis phenomenon resulting from incorrect cell adhesion, and this process
takes place in photoreceptors, RPE cells, and possibly endothelial cells (4) of the
choriocapillaris. The development of either excessive material in BrM or erosion
of BrM has important consequences. It may lead to choroidal endothelial cell
loss, instigate chronic local inflammation, and produce ischemic stress on the
oxygen hungry photoreceptor cells.
CHORIOCAPILLARIS
Choriocapillaris is a plexus of capillaries with fenestrations on the side facing
BrM and is part of the innermost layer in the choroid. The capillary network is
designed in a lobular pattern for rapid blood flow to supply the high metabolic
demand of the photoreceptors and RPE (82). In aging eyes, the lumina of the
choriocapillaris and the choroidal thickness are reduced by half. The resulting
hypoxia stabilizes hypoxia-inducible factor 1α, which activates genes encoding
proteins such as erythropoietin that protect the photoreceptors (87). Hypoxia also
increases the secretion of growth factors such as VEGF A within Ruysch's
complex on the basal side of the RPE cells, causing development of choroidal
neovascular membranes (4,88).

PATHOPHYSIOLOGY OF LATE AMD

Late-stage AMD shows the presence of RPE loss (cell death) or choriocapillaris
attenuation without neovascularization in dry or atrophic AMD and exudative
changes, neovascularization, or disciform scar in wet or neovascular AMD.

GEOGRAPHIC ATROPHY
Deposits of cellular debris in BrM alter the pathway of nutrient exchange
between the RPE and choriocapillaris and lead to the downturn of RPE cells. At
the same time, RPE cells overlying drusen are usually inflated and depigmented,
and the adjacent photoreceptors are often damaged or dead (8). When the RPE
degeneration is more advanced, thick basal laminar deposits take place in the
BrM and obstruct nutrient exchange. These deposits may also decrease the
adhesion of the RPE to the inner collagenous layer of BrM facilitating serous
detachment of the RPE and retina (89,90).
Throughout their history, drusen reflect the state of the adjacent RPE cells.
The greater the number of drusen and the larger they are, the greater the
probability that the RPE cells will be damaged (8). Soft drusen easily became
confluent and can reach a size that produces serous detachments of the RPE (91).
Drusen sometimes disappear. Macrophages, pericytes, and choriocapillaris
contribute to the removal, leading to their replacement by fibrous tissue.
By this time, RPE deterioration is critically advanced, and some cells may
proliferate into small clumps, detach from BrM, and migrate into the retina. The
final consequence of this process is geographic atrophy. Depigmentation exists
in atrophic areas; however, at the edge of the lesions, pigmentation develops due
to proliferation, hypertrophy, or phagocytosis of liberated melanin and lipofuscin
(8,92). During the process of geographic atrophy, neural retina and damaged
RPE cells disappear and the surviving rods and cones become shorter.

DISCIFORM SCAR
The main site of disturbance in neovascular AMD is BrM. However, the changes
in BrM that predispose to neovascularization are unclear. With aging and as a
consequence of membrane calcification, small fractures or gaps develop in BrM.
Abnormal secretion from the deteriorating RPE cells might serve as attractant
for endothelial cells, phagocytic cells, and lymphocytes, resulting in
neovascularization. Lipofuscin accumulation, RPE membrane thickening, and
intracellular residual body increase help stimulate new vessel formation.
Therefore, new vessels occur as a consequence of an imbalance in the
stimulating and inhibiting influences of growth factors, and any disruption in the
diffusion through BrM to choroid could alter this balance. Once established, the
new vessels have tendency to leak and bleed. When this occurs, the fluid extends
laterally beneath the RPE in the same plane where basal laminar deposit and
soften drusen are located.
There are three types of pigment epithelial detachment (PED) associated
with AMD: drusenoid, serous, and fibrovascular. Drusenoid PED is a confluence
of several soft drusen. Fibrovascular PED is a form of occult CNV. Serous PED
is not exactly a breakdown in the outer BRB, it seems to develop from a
dysfunction in the complex RPE cells–BrM. Fluid moves from the retina into the
BrM as a result of active movement of ions by the RPE cells. If BrM became
hydrophobic, resistance to water flow could cause fluid to collect between RPE
and BrM (74). Serous exudates and blood may enter the retinal space. The
retinal detachment may extend beyond the zone of RPE detachment and result in
a rapid destruction of the RPE and photoreceptors over an area much larger than
the region of vascular invasion, so the loss of vision is more severe in
neovascular AMD than it is in geographic atrophy. Hemorrhage under the RPE
or retina stimulates proliferation of fibrous tissue that produces a disciform scar,
increasing the size of the area of blindness.

CONCLUSION
The main features of the AMD are the formation of drusen; thickening and other
signs of damage of BrM; depigmentation and RPE hyperplasia; and
accumulation of basal laminar deposits beneath the RPE. However, these are not
unique or pathognomonic of this disease as we often observe them in most eyes
of elderly individuals (93–96). In summary, the main signs of AMD increase
with age, but only in some persons, they progress to the stage of cell death and
functional loss. According to this point of view, AMD is an advanced stage of a
process that occurs in all eyes. It is part of a common and continuing process in
which the transition between age and disease is marked by loss of vision (9).
In 1972, Hogan (97) described the role RPE plays in the macula and
postulated the existence of a gradual decrease in the activity of RPE, which
resulted in accumulation of metabolic waste in BrM and loss of permeability.
Zarbin (10) summarized five key concepts:

1. AMD includes disorders of aging and pathologic events.

2. Oxidative stress plays an important role in the damage of the RPE and BrM.
3. The RPE damage results in chronic inflammation of BrM and the choroid.
4. Choriocapillaris and RPE injury lead to the formation of an abnormal ECM
that produces an alteration in the diffusion of nutrients between the retina and
the RPE, which in turn causes more damage.
5. RPE and choriocapillaris atrophy occurs as well as CNV growth.

It is hoped that in the future, better understanding of the pathophysiology

and histopathology will lead to new treatments engaging different phases of the
disease. Hopefully, vision loss can be delayed and/or even avoided in AMD.

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Shuang Ying, ed. Degeneration, age related macular degeneration—the recent advances in basic research
and clinical care. InTech; 2012. http://www.intechopen.com/books/age-related-macular-degeneration-the-
recent-advances-in-basic-researchand-clinical-care/bruch-s-membrane-the-critical-boundary-in-amd. ISBN:
978-953-307-864-9.
80. Kliffen M, de Long PT, Luider TM. Protein analysis of human macula in relation to age-related
maculopathy. Lab Invest. 1995;73:262–272.
81. Sarks SH. New vessels formation beneath the retinal pigment epithelium in senile eyes. Br J
Ophthalmol. 1973;57:951–965.
82. Handa JT. Pathophysiology of the retinal pigmented epithelium and Choroid. In: Quiroz-Mercado H,
ed. Macular surgery. 2nd Ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2011:10–16.
83. Crabb JW, Miyagi M, Gu X, et al. Drusen proteome analysis: an approach to the etiology of age-related
macular degeneration. Proc Natl Acad Sci U S A. 2002;99:14682–14687.
84. Hageman GS, Luther PJ, Victor Chong NH, et al. An integrated hypothesis that considers drusen as
biomarkers of immunomediated processes at the RPE-Bruch's membrane interface in aging and age-related
macular degeneration. Prog Retin Eye Res. 2001;20:705–732.
85. Umeda S, Suzuki MT, Okamoto H, et al. Molecular composition of drusen and possible involvement of
anti-retinalautoimmunity in two different forms of macular degeneration in cynomolgus monkey (Macaca
fascicularis). FASEB J. 2005;19:2088.
86. Geen WR, Enger C. Age-related macular degeneration histopathologic studies. The 1992 Lorenz E.
Zimmerman lecture. Ophthalmology. 1993;100:1519–1535.
87. Grimm C, Wenzel A, Groszer M, et al. HIF-1-induced erythropoietin in the hypoxic retina protects
against light induced retinal degeneration. Nat Med. 2002;8:718–724.
88. Dámore PA. Mechanisms of retinal and choroidal neovascularization. Invest Ophthalmol Vis Sci.
1994;35:3974–3979.
89. Braunstein RA, Gass JDM. Serous detachments of the retinal pigment epithelium in patients with senile
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90. Gass JDM: Pathogenesis of disciform detachment of the neuroepithelium. III. Senile disciform macular
degeneration. Am J Ophthalmol. 1967;63:617–644.
91. Green WR, Mc Donnell PJ, Yeo JH. Pathologic features of senile macular degeneration.
Ophthalmology. 1985;92:615–627.
92. Burns RP, Feeney-Burns L. Clinico-morphologic correlations of drusen of Bruch´s membrane. Trans
Am Ophthalmol Soc. 1980;78:206-223.
93. Feeney-Burns L, Eldred GE. Age-related changes in the ultrastructure of Bruch´s membrane. Am J
Ophthalmol. 1985;100:686–697.
94. Green WR. Clinicopathologic studies of senile macular degeneration. In: Nicholson DH, ed. Ocular
pathology update. Chicago, IL: Year Book Medical Publishers; 1982:115–144.
95. Hoshino M, Mizuno K, Ichikawa H. Aging alterations of retina and choroid of Japanese: light
microscopic study of macular region of 176 eyes. Jpn J Ophthalmol. 1984;28:89–102.
96. Ring HG, Fugino T. Observation on the anatomy and pathology of the choroidal vasculature. Arch
Ophthalmol. 1967;78:431–444.
97. Hogan MJ. Role of the retinal pigment epithelium in macular disease. Trans Am Acad Ophthalmol
Otolaryngol. 1972;76(1):64–80.
 4
ETIOLOGY OF LATE AGERELATED
MACULAR DISEASE
MAXIMILIANO OLIVERA

INTRODUCTION
Age-related macular disease (AMD) is a chronic, progressive disease of the
retina, specifically the macula. AMD is an important cause of vision loss in the
United States and all over the world. AMD, in all of its forms, has an overall
prevalence of 6.5%, but some demographics, such as non-Hispanic White
people, have a higher incidence (7.3%). Non-Hispanic Black people have a
lower incidence rate (2.4%). The incidence of AMD increases with age. The
incidence in individuals over 60 years old is 13.4% compared to an incidence in
the 40- to 59-year-old group of 2.8% (1).
Like many other chronic diseases, it is important to understand its complex
pathophysiology in order to develop screening and treatment strategies.
Although we do not have a complete understanding of the complex
pathophysiology of AMD, several studies over the last 20 years have identified
many environmental and genetic risk factors. Identification of risk factors allows
for the development of screening strategies in order to make a diagnosis as early
as possible, improving the chances of a better visual outcome. For example, the
understanding of pathophysiologic mechanisms involved in the development and
progression of neovascular AMD, such as the role of the vascular endothelial
growth factor (VEGF), permitted the design and development of anti-VEGF
therapy for AMD. This has resulted in a radical change in the expectations for
patients and physicians (2).
CLINICAL CLASSIFICATION OF AMD
AMD is not only about changes in the eye fundus examination. To diagnose
AMD and determine what kind of AMD the patient has, it is important to
consider the patient's symptoms.
In addition, it is important to understand that the aging eye is associated with
certain changes (3). Most of these changes are almost clinically undetectable,
and patients have no visual symptoms at all. These changes include the
following:

A decrease in density and distribution of photoreceptors

Loss of melanin granules, lipofuscin accumulation, and residual body
accumulation in the retinal pigment epithelium (RPE)
Accumulation of basal lamellar deposits, formed by a lipid-rich granular
material and collagen fibers, between the lamina basalis (plasma
membrane) of the RPE cells and inner face of the basal membrane of RPE
Changes in choriocapillaris

The characteristic physical sign of AMD is the presence of drusen. Drusen

are small, rounded, yellowish lesions located beneath the RPE, inner to the
Bruch's membrane. Drusen are classified by size: small (diameter less than 64
μm), intermediate (diameter between 64 and 124 μm), and big (diameter 125 μm
or above). They may also be classified by the discreteness of their boundaries:
hard (well defined), soft (not well defined), and confluent (contiguous limits
between drusen).
In addition to drusen, other physical signs such as RPE hyper- and
hypopigmentation, RPE atrophy, RPE detachments, choroidal neovascular
membranes (CNVM), and hemorrhages give us information about the clinical
status of the patient with AMD. Clinically, it is useful to classify AMD into
early/late or dry/wet.

Early AMD
Early AMD is usually asymptomatic and clinically presents with drusen and
RPE changes consisting of focal hyper- or hypopigmentation.

Late AMD
The main characteristic of late AMD is severe loss of vision.
The late stages of AMD have two different forms (dry and wet), although
both forms may occur simultaneously in the same eye. Over time, dry AMD may
develop to wet AMD.

Dry AMD
Patients with dry AMD have poor visual acuity, hyperpigmented RPE spots
(usually juxtafoveal), and an area with visible choroidal vessels. Choroidal
vessels are visible because the RPE cells over them have become atrophic. As
these areas become confluent, they take on an appearance referred to as
geographic atrophy. No neovascularization is present.

Wet AMD
Wet AMD is characterized by the presence of CNVM. Patients with wet AMD
may experience a sudden onset of decreased visual acuity, metamorphopsia, and
paracentral scotoma. Clinical findings include subretinal fluid, subretinal or sub-
RPE hemorrhages, subretinal or intraretinal lipids, pigmentary subretinal ring,
RPE irregularities and detachment, gray-white subretinal lesions, and cystoid
macular edema (3).

EPIDEMIOLOGY
Incidence of AMD
According to the 2005–2008 NHANES (National Health and Nutrition
Examination Survey) (1), the most AMD-compatible lesions were found in
persons aged 60 or older in all the three ethnic groups studied (non-Hispanic
Black, non-Hispanic White, and Mexican American). The incidence of early
AMD is similar for non-Hispanic White persons aged 40 to 59 years (3%) and
Mexican American persons aged 40 to 59 years (2.7%). The incidence of early
AMD is lowest in non-Hispanic Black persons. Late AMD was more prevalent
in non-Hispanic White persons.
The estimated total prevalence of any AMD in the US population aged 40 or
older was 6.5%. Of a total of 7.2 million persons having any kind of AMD, 0.89
million (95% CI, 552,000–1.2 million) were estimated to have late AMD (see
Ref. (1) for more details).

Risk Factors for AMD

The most important risk factor for AMD is age, but over the years, many factors,
including genetic, demographic, behavioral, dietary, and other factors, have been
studied as risk or protective factors for AMD. The AREDS (Age-Related Eye
Disease Study) was a clinical trial sponsored by the National Eye Institute, one
of the National Institutes of Health in the United States. The trial was designed
to investigate the natural history and risk factors for AMD and cataracts and to
evaluate the effect of high doses of antioxidants and zinc on the progression in
patients with AMD. This study describes the demographic, behavioral, medical,
and nonretinal factors associated with progression to neovascular AMD and
central geographic atrophy (CGA).
Of all the studied factors, only a few were statistically associated with AMD.
Subjects with advanced AMD, relative to the early AMD group, tend to be older,
have fewer years of formal education, smoke more, have higher body mass index
(BMI), have higher blood pressure, be myopic, have a history of angina, be more
likely to have a lens opacity, and be less likely with hormone replacement
therapy. Neovascular AMD was associated with white race and smoking more
than 10 pack-years. CGA was associated with fewer years of formal education,
being obese (higher BMI), smoking more than 10 pack-years, and not using
antiacids (4).
There were also some other weak associations, such as diabetes, use of
nonsteroidal anti-inflammatory agents, and hormone replacement therapy, that
were reported in other studies (5–14) but not fully demonstrated on AREDS. Of
note, hormone replacement therapy was reported to be a protective factor in
some patients with specific polymorphisms in ARMS2 (HtrA serine peptidase 1)
(15).
In contrast to risk factors for progression to neovascular AMD and CGA,
black race (16,17); increased intake of docosahexanoic acid (18),
monounsaturated fatty acids (19), fish (20–22), and dark green leafy vegetables
(23); and higher levels of serum carotenoids (24) were associated with a lower
risk of progression.

GENETICS OF AMD
Patients with a positive familiar history of AMD have a higher risk of
development of this disease (8,25,26). Late AMD is a polygenic disease. No
single-gene defect accounts for disease. There are variations and defects in genes
functionally related to complement and immune processes, high-density
lipoprotein (HDL) cholesterol, and mechanisms involving collagen formation,
extracellular matrix production, and angiogenesis considered to be associated
with the onset, progression, and bilateral involvement of early, intermediate, and
advanced states of AMD (see Table 4.1 and Refs. (27–34) for more details) (35).
Several pharmacogenetic studies found that variants in some genes are related to
different treatment outcomes (Table 4.2) (36).

Table 4.1 GENETIC LOCI ASSOCIATED TO AMD

Adapted from Lim LS, Mitchell P, Seddon JM, et al. Age-related macular degeneration. Lancet.
2012;379:1728–1738.
Table 4.2 PHARMACOGENETICS AND AMD

Adapted from Chen Y, Bedell M, Zhang K. Age-related macular degeneration: genetic and environmental
factors of disease. Mol Interv. 2010;10(5):271–281.

Genetic variations in genes encoding the complement proteins and regulators

have been identified as protective or risk factors for AMD. Many of them are
single nucleotide polymorphisms (SNPs), causing a change of a single amino
acid in the polypeptide chain that could affect the binding affinity of the
complement protein to its substrates. This suggests that certain individuals with
some of these variations may be genetically predisposed to AMD. Variations of
the complement system likely interact with environmental risk factors to
determine overall risk.

Complement Factor H
Complement factor H (CFH), an important regulator of the alternative pathway,
was the first complement protein to be implicated in the pathogenesis of AMD.
Physiologically, CFH binds C3b and accelerates the decay of the alternative C3
convertase (C3bBb) and acts as cofactor for the inactivation of C3b by
complement factor I (CFI) (37). CFH is produced locally in the eye by the RPE
cells (38) and accumulates in the drusen, sub-RPE space, RPE,
interphotoreceptor matrix, and choroid (38,39). Environmental factors vary CFH
production by the RPE cells. In vitro studies show an increase in RPE CFH
production by interferon gamma (40) and reduction in conditions of oxidative
stress (38).
The CFH gene is located at chromosome position 1q23. Mutations of this
gene manifest as dominant mendelian disorders (41). In 2005, three groups
simultaneously (27,42,43) reported a nonsynonymous SNP in the CFH gene
(rs1061170), resulting in a substitution of tyrosine by histidine at position 402 of
the polypeptide (Y402H), which was important in the development of AMD.
This change alters the ligand binding site of CRP (C-reactive protein), heparin,
M protein, and glycosaminoglycans, probably leading to a reduced binding to
cell surfaces and therefore impaired regulation of the alternative C3 convertase
(44). A meta-analysis (45) suggested that this variant (Y402H) is a contributing
factor in over half of all cases of AMD. Other SNPs throughout the CFH gene,
including the SNP 162 V (38), resulting in substitution of isoleucine with valine
residue within the C3b binding site, have been associated with AMD
(32,38,41,46–50) and AMD progression (51–55).

Variations in VEGF Gene

Some authors (56–58) proposed that the SNP A/A in the allele rs3024997 and
G/G re2010963 in the VEGF gene are associated with a better response to
antiangiogenic (bevacizumab) therapy with regard to visual acuity outcomes. In
a more recent study (59), using a multivariate data analysis and a higher number
of patients, this SNP was not found to be associated with a different response to
antiangiogenic therapy.

WHAT HAPPENS IN THE EYE WITH

AMD?
In order to understand AMD, it is important to understand the physiologic aging
of the eye. The age-related changes in the retina that predispose a person to
AMD occur in the outer retina: the photoreceptors (PRs), the RPE, the Bruch's
membrane, and the choriocapillaris. Most of these changes are not clinically
detectable until a late stage, when they start to affect the visual function of the
patients.
The Photoreceptors
PR cells translate light into electric activity that can be understood by the brain
and central nervous system. Anatomically, PR cells have four different regions:
the outer segment (OS), the inner segment, the cell body, and the synaptic
terminal. The OS of the PR is composed of membranous disks, which have a
high concentration of visual pigment. Rhodopsin, the visual pigment of rods, is a
G protein–coupled receptor that when stimulated by a photon of light undergoes
a conformational change initiating a series of biochemical steps leading to the
onset of the electric activity (3). Numerous studies of the human and animal
retina show that excessive light exposure leads to photochemical injury causing
damage to the outer segment of the PR. Excessive light exposure is considered to
be an environmental factor associated with AMD (60), but the magnitude of this
risk is hard to evaluate and is controversial. Even under normal light conditions,
the PR incurs significant oxidative stress, due to the great energy requirement for
visual phototransduction (61). This stimulation increases the oxygen-reactive
species, damaging DNA and other macromolecular complexes important for the
PR survival (62,63). For a long-term survival of the PR cells, it is important to
have a healthy RPE participating in the visual cycle, renewing the PR OSs and
producing the interphotoreceptor cell matrix (3).

The Retinal Pigment Epithelium

The RPE is a postmitotic, cuboidal monolayer of cells located between the
neural retina and the Bruch's membrane. Physiologically, it has a very high
metabolic rate and performs several important functions for the retina. Of all the
functions of the RPE, the most important for understanding AMD (64) are the
following:

Regeneration of bleached visual pigments (opsins)

Formation and maintenance of the interphotoreceptor matrix and Bruch's
membrane
Transport of fluids and ions between PRs and choriocapillaris
Phagocytosis

The reconstitution of the visual pigment rhodopsin occurs mainly in the RPE
cells, through many intermediate steps. This mechanism has a key role for
normal function of both cones and rods, transforming the all-trans-retinyl esters
into 11-cis-retinal (65). As a phagocytic system, the RPE is essential for the
renewal of PRs (66). Each PR has hundreds of disks in its outer segment. These
disks are formed by plasma membrane, containing transmembrane protein
rhodopsin, which is positioned in combination with four phospholipids and
docosahexanoic acid. The OS disks of the PR cells are engulfed by the RPE. In
the RPE, the disks fuse with lysosomes, forming phagolysosomes, where the
contents are degraded. In young, healthy individuals, most of the disks are fully
degraded, and lipofuscin accumulation is minimal, but over time, the self-limited
phagocytic and degradative capacity of the RPE cells becomes more and more
overloaded. This incompletely degraded membrane material accumulates in the
form of lipofuscin in secondary lysosomes or residual bodies (67).
Lipofuscin is a yellow-brown, autofluorescent molecule that accumulates in
all postmitotic cells, particularly the RPE (68–71). The presence of lipofuscin
may act as a cellular aging indicator, and its quantity in tissues may be estimated
by amounts of autofluorescence present. The autofluorescence from the eye
fundus, mostly derived from lipofuscin, can be clinically and noninvasively
quantified, allowing for an estimate of the aging degenerative process of the eye
and a diagnostic and follow-up method for patients with AMD (72). Some
factors increase (vitamin E deficiency) while others decrease (oxygen-free
conditions and vitamin A deficiency) lipofuscin pigment formation.
The retinoid A2E is the major fluorophore of lipofuscin (73). Once it is
synthesized, it cannot be eliminated by the RPE. Precursors of A2E, all-trans-
retinal and ethanolamine, are formed within the PRs (74), but the fully
synthesized A2E molecule arises from the phagolysosomal compartment of the
RPE cells. When it reaches a critical concentration, the metabolism of
phagocytized OS lipids by the RPE is impaired. Phagocytized and oxidized OS
membranes are extruded by the RPE into the Bruch's membrane, contributing to
drusen formation and membrane thickening.
A2E is toxic for the RPE. It inhibits the proton pump of lysosomes (75),
causing leakage of the contents of the lysosomes into the cytoplasm of RPE
cells. It inhibits phagolysosomal degradation of PR phospholipids (76) and can
also damage the DNA of RPE cells. A2E also accumulates in the mitochondrial
membranes, decreasing mitochondrial activity and enabling the translocation of
cytochrome C and AIF (apoptosis-inducing factor) to the cytosol and nucleus,
respectively. Functionally, the release of cytochrome C from the inner
mitochondrial membrane generates oxidative stress and decreased electron flow,
leading to impaired ATP synthesis. Both mechanisms are highly relevant for
apoptosis by causing leakiness of the inner mitochondrial membrane and release
of the propapoptotic proteins, activating the caspase cascade. AIF is a pro-
apoptotic protein, which is strictly located in the mitochondria. Its translocation
to the nucleus induces apoptosis, functionally independent from caspases (77).
A2E also inhibits the normal activity of the enzyme RPE65 isomerohydrolase, a
key enzyme of the visual cycle, which is responsible for the isomerization of all-
trans-retinyl ester to 11-cis-retinol, the precursor of 11-cis-retinal (78). This
inhibition could cause a disruption of 11-cis-retinal supply to the retina and
result in PR dysfunction. A2E is known to perturb the cholesterol metabolism in
the RPE cells, causing cholesterol accumulation within the late
endosome/lysosome, inhibiting acid lipase and resulting in a feed-forward cycle
of cholesterol accumulation in the RPE. This interferes with rhodopsin activation
and PR membrane remodeling (79). A2E is also thought to be photosensitizing
due to its broad light spectrum–absorbing capacities, particularly in the visible
ranges, and thus generating reactive species of oxygen (80,81).

The Bruch's Membrane

The Bruch's membrane lies beneath the RPE and has three layers: two
collagenous layers on the top and bottom and a central elastic layer (82).
Proteoglycans are an important constituent of the Bruch's membrane (83). Their
negative charges hinder the passage of the positively charged macromolecules
necessary for RPE maintenance. Changes in the Bruch's membrane begin prior
to the development of clinically significant AMD. As soon as the third decade of
life, basal lamellar deposits and membranous debris, precursors of AMD, begin
to appear (84,85).
Hard drusen appear between the basement membrane of the RPE and the
inner collagenous layer of the Bruch's membrane in the third decade of life (86).
It is not well understood how drusen develop. Drusen have a complex structure.
They have a core of glycoproteins and an outer dome containing crystallins,
chaperones, apolipoprotein E, vitronectin, inflammation-related proteins (as
amyloid P, C5, C5b-9 complement complex), and fragments of RPE cells
(87–91). Basal laminar deposits (BLD) contain granular electron-dense material,
coated membrane bodies, and wide- or long-spaced fibrous collagen (92–96).
Vesicles and membranous debris, referred to as basal linear deposits,
accumulate beneath the RPE basement membrane and correlate
histopathologically to soft drusen (93,97).
Features of the normal aging of the Bruch's membrane include increased
lipid concentration (99–104), calcification (98), and decreased concentration of
metalloproteinases. The most common lipid in the aged Bruch's membrane
deposits is the phospholipids, derived from local cells rather than blood (101).
There is an age-related decrease in adhesion molecules, laminin and fibronectin,
that is inversely correlated with the lipid content of the Bruch's membrane (100).
In contrast, another study concluded that the main lipid component of these
deposits was esterified cholesterol, similar to depositions in other membranes
throughout the body that occur with age, suggesting a blood origin of these
rather than cellular (105).
These changes contribute to the thickening of the Bruch's membrane,
interfering in normal function of this membrane by decreasing the hydraulic
conductivity. Thus, fluids pumped out by the RPE cells, toward the choroid,
encounter the resistance provided by the thickened membrane, leading to the
formation of serous RPE detachments. Such morphologic changes lead to
alterations in cellular adhesion, which could cause the triggering of an apoptosis
(106,107).

Choriocapillaris
The choriocapillaris is the vascular layer of the eye. Between the retina and the
sclera, it is formed by an extensive fenestrated vascular network derived from
the perforating ciliary arteries, branches of the ophthalmic artery. Its main role is
to provide oxygen and macromolecules to the outer retina. Over the years, the
choriocapillaris becomes less fenestrated and the lumina are reduced, causing
less delivery of oxygen and macromolecules to the retina (98,108,109). This
change results in hypoxia and activation in the transcription of genes such as
erythropoietin (for PR protection) (110) and vascular endothelial growth factor A
(VEGF-A) (111,112), causing the development of CNVM.

Choroidal Neovascular Membranes

CNVMs are fibrovascular complexes that arise from the choroid, penetrating the
Bruch's membrane and proliferating in the sub-RPE and subretinal space. This
complex is formed by two major components: neovascular sprouts and
fibroblasts. Proliferation of this fibrovascular complex leads to disturbances in
the normal architecture of the choriocapillaris, Bruch's membrane, RPE,
photoreceptors, and retina.
Since the Macular Photocoagulation Study in the 1970s, CNVM was
classified based on its localization relative to the fovea (extrafoveal, juxtafoveal,
and parafoveal) and the pattern of dye leakage on fluorescein angiography (FA)
(classic and occult). Advances in diagnostic techniques for CNVM, particularly
indocyanine green angiography (ICGA) and optical coherence tomography
(OCT), allowed determination of the retinal layer in which CNVMs are located.
A new classification for CNVM based on FA, OCT, and ICGA patterns
includes type 1 (CNVM beneath RPE), type 2 (subretinal CNV), and type 3
(retinal angiomatous proliferation) (113).

UNDERSTANDING HOW ALL THESE

CHANGES LEAD TO AMD
The aging process of the eye affects the PRs, the RPE, the Bruch's membrane,
and the choriocapillaris. In these tissues, accumulation of substances, functional
changes, and apoptosis may occur, and important pathophysiologic mechanisms
are activated by these changes.

Angiogenesis
Angiogenesis, leading to the formation of CNVM, is one of the most important
pathophysiologic features of late AMD. In angiogenesis, blood vessels grow in
adult tissue from sprouts on preexisting vessels. This process involves several
steps: cell migration, proliferation, and survival; vascular maturation; wall
remodeling; and degradation of the extracellular matrix (114–116). Angiogenesis
has a complex molecular regulation, representing a balance between the
angiogenic and the antiangiogenic factors, affected by different pathologic states
such as hypoxia, ischemia, or inflammation (2).

VEGF-A, Hypoxia, Inflammation, and Oxidative

Stress
VEGF-A is a remarkable angiogenic factor. This homodimeric glycoprotein with
a molecular weight of 45 kDa (117) has four major isoforms, distinguished by
their molecular weight, acidity, and whether they are bounded to heparin.
VEGF-A is synthesized by RPE cells constitutively and is elevated in early
forms of AMD and hypoxia. VEGF-A is highly selective for endothelial cells. It
has diffusion characteristics that allow it to reach its target. It has several
regulation mechanisms and affects the angiogenesis mechanism in different
stages, including endothelial cell proliferation, survival, and migration, and also
promotes the vascular hyperpermeability (118). VEGF receptors, VEGFR-1 (Flt-
1), VEGFR-2 (Flk-1/KDR), and VEGFR-3 (Flt-4), are expressed predominantly
in endothelial cells and to a lesser extent on monocytes and macrophages (119).
Normal human choriocapillaris expresses all VEGF receptors in the
choriocapillaris endothelium next to the RPE layer, suggesting a paracrine
relation between RPE cells and the choriocapillaris (120). VEGF has also been
shown to have survival function in the retina (121) and has a role in the
maintenance of the adult choriocapillaris via stimulating the formation of
fenestrations (122).

Hypoxia
The role of hypoxia on the pathogenesis of AMD is controversial. While some
studies (123) showed that patients with AMD have decreased blood flow in the
choriocapillaris, others (124) could not confirm a decrease in blood flow but
found a decrease in pulse amplitude. It also has been theorized that the
previously described age-related changes in the Bruch's membrane may limit the
diffusion of oxygen and create an ischemic environment. Physiologically, pO2
decreases linearly with distance from the choriocapillaris to the inner segment of
the PRs. Under normal conditions, little of the O2 in the choriocapillaris blood is
extracted. Some studies have shown that as perfusion decreases, the oxygen
extraction from the choriocapillaris increases (125), showing that there is
significant reserve. This suggests that hypoxia is not the most important factor in
causing an imbalance between angiogenic and antiangiogenic factors.

Inflammation
Studies of excised choroidal membranes and the growth patterns of CNVM
suggest more is involved than just ischemia in driving neovascularization.
Histologic studies of the CNVM show vascular endothelium as well as a variety
of inflammatory cells, such as lymphocytes, macrophages, and foreign body
giant cells (126,127), similar to granulation tissue on a wound-repair response
(128). Activated macrophages secrete proteolytic enzymes, collagenase, and
elastase, which can erode an attenuated Bruch's membrane and thereby assist the
migration of choroidal capillaries (129,130). Also, inflammatory cells produce
cytokines such as interleukin-1, which induce VEGF, as shown in cultured
choroidal fibroblasts (131). In one study (132), the amount of VEGF was found
to be proportional to the number of macrophages in the specimen, suggesting
that inflammation is important for the regulation of angiogenesis. Therefore,
upregulation of VEGF may be the link between inflammation and promotion of
new blood vessels in an eye with AMD.
Before the formation of the CNVM, there are signs of inflammation in an
eye with AMD. Drusen deposits between the Bruch's membrane and RPE may
contain bioactive fragments of complements (C3a and C5a) (133). These
substances induce VEGF expression and have significant chemotactic activity
that further invites inflammatory cells to the macular region (134). Another key
actor of the complement system in AMD is the CFH. CFH binds and inactivates
C3b deposited on intact host cells, allowing destruction of foreign or damaged
cells (135). This is achieved through an important locus, domain 7 on the
molecule (136–139). Domain 7 binds to heparin or sialic acid on host cells.
Therefore, it is possible that alterations in domain 7 or the heparin/or sialic acid
binding region of factor H could lead to or augment the destruction of normal or
injured ocular cells as well as other foreign cells in an appropriate environment
(44).
Extracellular drusen also contain advanced glycation end products (AGE),
high levels of oxidized low-density lipoproteins (ox-LDL), and oxysterols
(87,140,141). The receptor for AGE (RAGE), normally not markedly expressed
in the retina, although highly accumulated in RPE cells, PRs, and
choriocapillaris in advanced AMD (142,143), activates the nuclear factor kappa
B (NFκB), master regulator of both innate and adaptive immunity. NFκB is a
central regulator of IL-6, a cytokine that has been shown to be an important
regulator of choroidal neovascularization (144–146), and VEGF. Damaged RPE
and oxidized proteins and lipids in the Bruch's membrane have been postulated
to activate and recruit dendritic cells from the choroid, enhancing the immune
response in this area (88,147).
In a laser model of CNV, CD18 and ICAM-1 are expressed during
development of CNV. Targeted disruption of either of these inhibits the
development of CNVM. Animals models of CNV have been developed that
mimic many aspects of CNVM in AMD. Mice expressing monocyte
chemoattractant protein-1 (MCP-1) or its cognate, CC chemokine receptor-2,
developed drusen, lipofuscin accumulation, geographic atrophy, and CNV (148).
Depletion of the monocyte cell lines inhibits experimental CNV (149–151).
With time, the neovascularization appears to “burn out,” leaving a cicatricial
mass almost completely devoid of vessels. If ischemia is the only cause of
neovascularization, one would expect these vessels to regress, which would
increase the amount of ischemia present.
The Complement System
The complement system plays a fundamental role in host defense against
pathogens, elimination of immune complexes and apoptotic cells, and adaptive
immune responses. It consists of over 40 proteins and regulators found in the
systemic circulation and some tissues. Three different activation pathways exist,
each one with different triggers: the classical pathway (triggered by an antibody–
antigen complex), the alternative pathway (triggered by binding to a host cell
pathogen surface), and the lectin pathway (triggered by polysaccharides on
microbial surfaces). These three pathways converge on the terminal complement
complex (TCC), a multiprotein complex, formed from the binding of C5b to the
plasma complement proteins C6, C7, C8, and C9 (152).
Components of the classic, alternative complement pathways and TCC have
been found to be present in drusen (153), especially in older eyes with AMD.
Alterations of the complement system or its regulators can lead to damage of
the tissues and is known to be implicated in a wide spectrum of diseases. Several
studies suggest that complement plays a key role in the development of AMD
(154).

Other Growth Factors Involved in Angiogenesis

Several other growth factors are involved in the regulation of angiogenesis in the
eye. These mechanisms help us to understand why conventional therapies do not
succeed in some cases and spur the development of new therapeutic strategies.

Pigment epithelium–derived factor (PEDF), produced by the RPE, is a

neurotrophic PR growth factor and generally is thought to inhibit
angiogenesis. It is the main antiangiogenic cytokine in the eye. Under
hypoxic conditions and wet AMD, secretion of PEDF is decreased, thereby
allowing the endothelial mitogenic activity of VEGF to go unchecked
(154–156). Some paradoxical effects have been identified (155,157).
Platelet-derived growth factor (PDGF) is involved in the development and
differentiation of vessel walls. It is required to recruit pericytes and to
promote maturation of the microvasculature. It is a potent chemoattractant,
dedifferentiater, and mitogen for both fibroblasts and RPE cells (158–160).
The potential therapeutic effect of anti-PDGF therapy still needs to be
studied (161).
Basic fibroblast growth factor (bFGF) is a potent angiogenic molecule in
 vivo and is produced by a variety of cell types including vascular
endothelial cells of the choriocapillaris, fibroblasts, astrocytes, and RPE
cells (162,163). bFGF induces the secretion of VEGF and HGF by Müller
glial cells and stimulates cell proliferation (163–165). One study comparing
the antiangiogenic effect of bevacizumab (anti-VEGF) alone and combined
with anti-bFGF antibodies observed that targeting bFGF in addition to
VEGF could show synergistic effects in CNV treatment (166).
Angiopoietin-2, detected in CNVM lesions (167), is upregulated by hypoxia
and VEGF. It may facilitate hypoxia and VEGF-induced neovascularization
by destabilizing existing vasculature (168,169).
Metalloproteinases are enzymes that remodel the extracellular matrix in
neovascular processes. They can be found in AMD CNVM lesions, and
they are upregulated by the VEGF (170,171).

Oxidative Stress
Oxidative stress refers to a progressive cellular damage caused by reactive
oxygen species (ROS) contributing to protein misfolding and evoking functional
abnormalities during RPE cellular senescence (172). Light can induce the
formation of reactive species, which in turn can lead to the formation of
dysfunctional or toxic molecules of lipids and proteins or damage the DNA.
Oxidized molecules are formed in the PR OSs as a normal part of daily life.
Over the years, the amount of these molecules increases because the mechanisms
needed to clear the reactive species out of the cells become insufficient (173).
Oxidative stress also interferes with the capacity of the heat shock proteins and
the ubiquitin–proteasome way to repair or degrade damaged proteins (174–176).
Aging primarily influences the oxidative balance between oxidizing factors
and antioxidants. Other factors, such as cigarette smoking and alcohol
consumption, aggravate oxidative imbalance and DNA damage (177).
Antioxidant genes (178) and variants of the CFH gene influence the role of
oxidative stress in AMD (179).
A wide variety of oxidative lesions to DNA have been described, including
single- and double-stranded DNA breaks and the development of cross-linked
lesions (180,181). The cell responds to genomic damage through repair
processes employing a large number of proteins. The cell may turn off growth
and replication until the repair process is complete (182). Responding to the
Samurai law of biology (better dead than wrong), those cells unable to repair
their DNA damage undergo apoptosis.
 To help protect against inappropriate oxidation, there are basically three
levels of protection: molecular, cellular, and tissue. On the molecular level, the
cell has antioxidant vitamins and enzymes. These include vitamins C and E,
superoxide dismutase, catalase, glutathione transferase (183), glutathione
reductase, and glutathione peroxidase. The antioxidants may limit inappropriate
oxidation in the first place or may terminate propagation reactions. On a cellular
level, two main responses may occur. The cell may try to adapt to the oxidative
stress by increased activation of transcription factors and proteins (184) that
helps control gene expression of antioxidant enzymes. The cell could also start
the apoptosis mechanism. Tissue mechanisms for responding or protecting
against oxidative stress include complex intercellular signaling pathways as
discussed below.
Scavenger receptors (185,186), in particular CD36, are present on the RPE
cell and participate in phagocytosis of spent OSs. It is possible that ordinary
everyday exposure of CD36 receptor to oxidized lipids in the PR OS helps
maintain the constitutive secretion of VEGF by RPE cells. Excessive secretion
of VEGF by RPE cells is supposed to be one factor responsible for the initiation
of CNV. This raises the possibility that excessive exposure to oxidative damage
may lead the RPE cells to secrete excessive VEGF. Armstrong et al. (187) have
shown that injection of lipid peroxide derivate into the vitreous cavity caused
retinal neovascularization that persisted for 4 weeks. After the injection, there
was a cascade of cytokines secreted, including VEGF. The Bruch's membrane
has an exponential increase in lipids with age. The lipids preferentially
accumulate in the same region where the neovascularization grows. The Bruch's
membrane has no known intrinsic mechanism to protect against oxidative
damage to lipids, which accumulate over time. This mechanism could be
important in the formation of CNVM in AMD.
Lipid peroxides not only accumulate in the Bruch's membranes but also
accumulate in atherosclerotic plaques. In atherosclerotic vessels, the body
mounts an aggressive cell-mediated mechanism to contain the oxidized material
(188,189), principally using vascular endothelial cells and macrophages also
stimulating the production of VEGF by these cells. The body has a number of
defined strategies and methods for dealing with degenerating cells and tissue.
Many of the same strategies and methods that are used in atherosclerosis of
vessel walls are also used in the eye. Injection of these lipids has led to ocular
neovascularization in the rabbit (190–192).
As discussed, the accumulation of oxidative damage may be a cause of
neovascularization, but the same oxidative damage may also induce a senescent
aging phenotype (193), with possible apoptosis. Oxidative damage can lead to an
increased accumulation of lipofuscin within RPE cells, a finding linked to the
development of geographic atrophy.

CONCLUSION
In this chapter, we discussed the etiology of age-related macular disease.
Epidemiologic and genetic factors, clinical classification, anatomical changes,
and how these changes affected the development and evolution of the AMD
were discussed. While a complete understanding of AMD and its
pathophysiology is still lacking, new evidence and understanding are rapidly
accumulating. There is not a single gene defect or a single molecular disturbance
that causes AMD. It is a complex, multifactorial disease that may require a
variety of treatment strategies to preserve vision for our patients.

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 5
ETIOLOGY OF LATE AGERELATED
MACULAR DISEASE
MARIANA INGOLOTTI • ERIC P. JABLON JOHN
BARNWELL KERRISON • HUGO QUIROZ-MERCADO D.
VIRGIL ALFARO III

INTRODUCTION
Age-related macular degeneration (AMD) is the leading cause of central vision
loss in the over-65 population in developed countries. It is a bilateral,
degenerative, and chronic disease limited to the macula so that the peripheral
retinal function is relatively preserved. Progressive histopathologic changes (Fig.
5.1) in the retinal pigment epithelium (RPE), retina photoreceptor cell layer,
Bruch's membrane (BM), and choriocapillaris are seen in these patients.
Figure 5.1 Histopathological changes in the two main forms of AMD. This diagram is showing the
structural and functional changes of a normal RPE cell when developing dry or wet AMD. A. The RPE in a
young retina has a homogenous distribution of the melanin granules, a BM without deposits, and a
preserved function. B. In dry AMD, the RPE melanin granules are localized basally, and lipofuscin
granules increase in number while the efficacy of the disks digestion reduces. Note the formation of drusen
between the basement membrane of the RPE and the inner collagen layer of the BM and the thickened BM.
C. In wet AMD, CNV develops, and an inflammatory response develops with an influx of lymphocytes
and macrophages to the scene. These fragile new vessels leak and lead to subretinal hemorrhage.

The most common histologic findings in nonneovascular or dry AMD are

drusen; a thickened and basophilic BM; and geographic atrophy, hypertrophy,
and hyperplasia of the RPE. Choroidal neovascularization (CNV), pigment
epithelial detachment (PED), and disciform scar are important features of
neovascular or wet AMD. The purpose of this chapter is to give a detailed
description of the histopathologic findings encountered in AMD with emphasis
on the ultrastructure, composition, and formation of different lesions.

NORMAL AGING OF THE RETINA

In discussing the histopathology of AMD, one must consider normal aging
changes in the outer retina, a major risk factor for the development of this
disease. The photoreceptor layer, RPE, BM, and choriocapillaris constitute the
Ruysch's complex (1). This complex suffers age-associated changes that
commence at approximately the age of 20.
Certainly, the RPE shows ultrastructural changes seen in electron
microscopy. Its melanin granules migrate to the basal portion of the cell, and the
amount of lipofuscin granules increases as a result of the phagocytosis of the rod
and cone outer segments. With age, the efficacy of rod and cone outer segment
digestion reduces, and they can be extruded into the BM and in the space
between the basement membrane of the RPE and its cellular membrane where
they accumulate. Lipids from the digestion of organelles like mitochondria and
endoplasmic reticulum can also deposit here (2).
Lipofuscin is an autofluorescent pigment formed through the oxidation of
unsaturated fatty acids. In short, it is an undigestable residue of cytoplasmic
catabolism. Studies showed that content of lipofuscin in the cells increases with
age. On the contrary, melanin content decreases showing an inverse relation
between these two compounds not only in their final concentration but also in
their topographical distribution. This interesting finding may suggest a protective
mechanism in the formation of lipofuscin (3).
Thickened BM is another alteration that occurs over time. Histologically,
there is an increase in basophilia with hematoxylin and eosin (H&E) stain
because of progressive calcium salt and lipid deposition in both inner and outer
collagenous zones (4). Moreover, studies have shown that a large portion of the
lipids found in this tissue are lipoprotein-like particles that increase with
advancing age. These deposits were found to increase in the inner collagenous
and elastic layers, but not in the outer collagenous layer. Authors have related
these findings to the formation of a hydrophobic barrier within the BM that
altered the filtration capacity through the area (5). These deposits may also be
related to small granules, another major deposit found at the BM by Huang et al.
They suggested there could be an interaction between this two inclusion types,
but the identity of these small granules could not be determined (6). In addition,
the accumulation of cholesterol esters with advancing age can be compared to
accumulation of cholesterol esters in the intima of large arteries in
atherosclerosis (7). Finally, there is an increase in collagen cross-linking in the
inner portion of this membrane causing a negative effect on its permeability.
This cross-linkage provides strength and density to the collagen network
together with the loss of flexibility, elasticity, and filtration properties. Therefore,
the RPE collagenases become less effective in the removal of other BM
components considering this tight network is blocking their way (8).
The photoreceptor layer manifests a different response to aging.
Immunohistochemical analysis concludes that the cone photoreceptor population
shows more anomalies compared with rod photoreceptor population in non-
AMD retinas. Nevertheless, the signs of degeneration have an early onset in
cones, but rods appear to die more promptly. Cones can prolapse into the outer
plexiform layer and subretinal space and lose their synaptic contact without
succumbing to cell death as do rods (9).
Some authors consider AMD to be an accentuation of normal aging changes.
Age is a key factor in its pathogenesis. Nevertheless, many people maintain an
excellent vision in spite of advancing age and its consequences.

DRY/NONNEOVASCULAR AMD
Drusen
Drusen are deposits of extracellular matrix localized between the basement
membrane of the RPE and the inner collagen layer of the BM. They can extend
to the outer collagenous zone if there is discontinuity in the central band of
elastic fibers (10,11). They are often seen in the aging retina, as a hereditary
condition (dominantly inherited drusen) or secondary to a variety of intraocular
processes including inflammation, trauma, and chronic retinal detachment (2).
However, an increase in number, size, and confluence was established as a risk
factor for the development of AMD (12). Because of this, it is important to
distinguish which yellow deposits will lead to atrophy of the RPE or CNV. The
American Academy of Ophthalmology proposed the following classification
according to the size of the drusen (13):

Small (less than 64 μm of diameter)

Intermediate (64–124 μm of diameter)
Large (≥125 μm of diameter)
 Immunohistochemical and proteomic studies of drusen composition have
revealed different proteins other than RPE remnants (lipofuscin) like
immunoglobulins, class II antigens, acute-phase proteins (e.g., fibrinogen, C-
reactive protein, and vitronectin), components of the complement cascade and its
inhibitors, apolipoproteins B and E, and lipids (e.g., cerebrosides), among others
(8,14,15). These deposits are collectively known as drusen, but they are not all
alike. Drusen main types are hard, soft, and diffuse or confluent, although
authors have also described several others subtypes such as basal, nodular,
mixed, and calcified regressing drusen (11).
Hard drusen are pinpoint-sized, sharp-edged yellowish retinal lesions easily
seen on ophthalmoscopic exam. They are hyperfluorescent in the early stages of
the fluorescein angiography and stain late without leakage. Histopathologically,
hard drusen are hyaline-like, dense, rounded, homogenous bodies restricted to
the basement membrane of the RPE and BM. They consist of a uniform periodic
acid-Schiff-positive material and are eosinophilic on the H&E stain (Fig. 5.2A
and B) until they start accumulating calcium and consequently become
basophilic (16). They also stain lightly positive with special staining technique
for lipid (2). On the electron microscopy, they are finely granular or amorphous
nodules with similar electrodensity to the basal membrane of the RPE. They may
also contain pale vesicles, tubular structures, curly membranes, and wide-banded
collagen (15). The overlying RPE is thinned due to degenerative changes as
described in Ulshafer et al. (17) electron microscopy studies. There are several
hypotheses concerning the formation of hard drusen. Some authors have shown
that the main initiating event in their development is the shedding of portions of
RPE cells into the BM that are then hyalinized during the apoptosis process
(18,19). Others have demonstrated that they are formed by lipidization and
degeneration of single cells (20,21). This matter is still uncertain.
Figure 5.2 Histopathologic section of drusen (asterisk) showing atrophy of the overlying pigment
epithelium and photoreceptors. A. Masson trichrome digital stain 40×. B. H&E stain 20× (Courtesy Zárate
JO, Alvarado M© 2011. Unpublished data.)

In contrast, soft drusen have less well-defined boundaries and tend to

become confluent. Histologically, they are composed of a pale-staining
amorphous more irregular and granular material located in the thickened inner
layer of the BM. Green et al. (16) pointed out that they represent a serous
detachment of the thickened inner aspect of the BM along with the RPE. On
electron microscopy, this thickened area consists of vesicles, membranous
debris, and wide-spaced collagen associated with the basal laminar deposits (22).
The basal deposits represent one of the earliest morphologic changes in the
AMD. The two main types of basal deposits are basal laminar deposits
(BLamDs) and basal linear deposits (BLinDs) (23). BLamDs are seen as soft
granular eosinophilic material or extensive plaques that elevate the atrophic RPE
from the inner surface of BM. On electron microscopy, these deposits are found
to be limited to the plasma membrane and the basement membrane of the RPE
and are comprised of extracellular matrix material with wide-spaced collagen
(24). These deposits contain laminin, collagen type IV, vitronectin, extracellular
matrix–modulating metalloproteinases, activated complement, glycoproteins,
heparan sulfate, cholesterol, carbohydrates, and apolipoproteins B and E (8,25).
In contrast, BLinDs are located in the thickened inner layer of the BM and
appear to be electron dense rich in lipid material. These deposits represent what
many authors have described as “diffuse drusen” (26). With progressive death of
pigment epithelial cells, drusen start to fade by decreasing in size and becoming
what some authors called calcified drusen (27). In fact, RPE atrophy, represented
by hypopigmentary changes on clinical exam, is seen concomitantly with
calcified or refractile drusen in histopathologic studies (28).
In summary, soft drusen along with basal linear deposits reveal an RPE
dysfunction that influences the development of CNV (29,30). It has been
reported that the thickening due to these deposits predisposes the BM to splitting
and consequent complications such as retinal PED, neovascularization, and
scarring (31).

Geographic Atrophy
Geographic or areolar atrophy also called advanced “dry” AMD involves an
extensive degeneration (150 μm or larger) of the RPE and the overlying
receptors that are metabolically dependent upon the RPE. It is labeled as
“geographic” because the areas of atrophic RPE are well demarcated and not
related to any specific anatomy structure (32). Some authors describe a
nongeographic atrophy in cases where atrophic areas are noncontiguous or
manifest as speckled depigmented areas (13). On funduscopic examination,
geographic atrophy is seen as a sharply demarcated round or oval
hypopigmented spot, frequently found either juxtafoveal or parafoveal, that
allows an enhanced view of the underlying large choroidal vessels (1,33). On
fluorescein angiography, geographic atrophy demonstrates an early
hyperfluorescent area due to the window defect in the RPE. The loss of
epithelium transmits fluorescein brightly, but the extent of the lesion does not
change in the late phases.
 On microscopy, areas of geographic atrophy lack photoreceptors and RPE
cells, and the outer plexiform layer is adherent to the BM (Fig. 5.3A and B) (4).
This is seen as hypopigmented or atrophic areas overlying a diffusive thickened
inner aspect of the BM (26). Calcified or retractile drusen, as previously
described, are also observed (28). The RPE demonstrates other changes such as
hypertrophy and hyperplasia, which are clinically seen as focal
hyperpigmentation (23). Clumps of pigmented cells in the subretinal space and
the outer retinal layers may also be observed histologically (26).

Figure 5.3 Geographic atrophy. A. Retinal and choroidal tissue. Photoreceptor lesion with fibrosis (arrow)
H&E stain 20×. B. Macular retinal atrophy and fibrosis (arrow). Masson trichrome digital stain 60×.
(Courtesy Zárate JO, Alvarado M© 2011. Unpublished data.)

Studies performed by Schatz et al. demonstrated that in addition to RPE

atrophy, one may also observe atrophy of the choriocapillaris. During fluorescein
angiography, the choriocapillaris fills more slowly than normal indicating an
ultrastructural damage (32). It is not understood if the choriocapillaris atrophy is
secondary to RPE deterioration or vice versa. In fact, there is direct in vitro
evidence that RPE cells may support the survival of the choriocapillaris
endothelial cells by both insoluble molecules and growth factors such as basic
fibroblast growth factor (bFGF). In other words, without them, the cells become
apoptotic (34). Therefore, the absence of RPE or anything that can reduce its
availability, like deposits in BM, produces choriocapillaris atrophy. An
alternative theory postulated by Ciulla et al. (35) supporting the vascular
pathogenesis of AMD hypothesizes that there is a primary vascular alteration in
the choroid, which eventually causes a collateral effect on the RPE. This issue
requires further research.
Even though at one time it was assumed patients with geographic atrophy
were not at risk of developing exudative complications, it is now well known
that neovascular changes can occur (16). It has been established that this new
vessel formation depends on the viability of the RPE so that it can only grow in
the edges of the lesion where the pigment epithelium remains intact or
hypertrophic (27). Furthermore, Kliffen et al. illustrated this event with a light
microscopic image and demonstrated that the junctional zone was comprised of
hypertrophic RPE cells and basal laminar deposits. They assumed that if
neovascularization was present, this was where it was supposed to be found in
association with macrophages and giant cells (36). Also, geographic atrophy can
coexist with subretinal neovascularization or be followed by serous detachment
of the RPE (11).

Pigment Epithelial Detachment

After a while, basal laminar deposits enlarge and coalesce providing a larger
cleavage plane in BM, which is no longer seen as soft drusen but as a serous
PED. Indeed, this appears to be a key event in the evolution to the development
of subretinal neovascularization (16).
The inner aspect of the BM also suffers from rip or tears as a common
complication of PEDs. The clinical and fluorescein angiographic aspects of these
lesions were first described by Hoskin et al. in 1981. They determined that the
tear always took place at the edge of the PED and that the remaining detached
pigment epithelium retracted, leaving an area of nude BM. The retracted
epithelium was folded parallel to the edge of the rip and, in some cases,
reattached afterward to the BM at a new site. There was no modification on the
overlying retina. Moreover, in the fluorescein angiogram, the nude BM made the
choroidal vessels be seen in the first frame prior to hyperfluorescence within 2
seconds. There was no leakage into the subretinal fluid, but some late dye
accumulation was identified subretinally as well as dark hypofluorescent folds of
residual detached pigment epithelium (37). Tears may also develop after laser
photocoagulation or antiangiogenic therapy in patients with neovascular AMD
(38,39).

WET/EXUDATIVE/NEOVASCULAR AMD
Choroidal Neovascularization
Choroidal also known as subretinal neovascularization (CNV) is one of the main
features of wet AMD. The new vessel formation extends from the choroid into
the thickened portion of the BM or from the margins of the optic nerve head
(40). Studies have confirmed that a prior break in the BM is not necessary for
vessel proliferation because the new vessels can make their way through it by
themselves (41). CNV is often seen in retinas in which softening and confluence
of drusen have taken place (42). In histologic sections, Albert et al. (11)
described the following findings: (a) breaks in BM; (b) a granulomatous
inflammatory pattern with macrophages, lymphocytes, and fibroblasts; (c) basal
laminar deposits and soft drusen; and (d) RPE depigmentation, hypertrophy,
hyperplasia, and folding. On electron microscopy, both mature and newly
formed capillaries have fenestrations similar to their choriocapillaris precursors.
They also have high permeability to fluorescein and may cause leakage or
fluorescein blockage (43). Even though they are initially very small, are
nondetectable, and have capillary characteristics, they acquire arterial and
venous features with time. Ophthalmoscopically, they can either be seen through
the atrophic RPE or hidden underneath fibrous grayish tissue or RPE
hypertrophy. CNV can be localized to the macula, peripapillary retina, or
peripheral retina. CNV can be restricted to the BM (Fig. 5.4A and B) or trespass
the RPE and grow into the subsensory space associated with fibroblastic RPE
metaplasia and migration of macrophages (33). Anastomosis between choroidal
and retinal vessels can develop. This characteristic is often seen in late stages of
AMD when the disciform scar is well established (2). The neovascular
membranes can be recognized in the fluorescein angiogram as a fine network of
vessels with early filling, associated with exudation and subretinal hemorrhage.
These specific elements are toxic for the RPE and photoreceptors, and they may
lead to RPE detachment, proliferation of fibrous tissue, cystoid macular edema,
and occasional circinate retinopathy (33,44,45). In this setting, fibroblasts can be
identified invading the hematoma. Also, the surviving RPE cells are transformed
into elongated spindly cells while the overlying photoreceptor atrophy resulting
in a submacular nodule, plaque, or fibroglial scar tissue that contracts and
distorts the retina (40).
Figure 5.4 Wet AMD. A. Histologic section shows CNV at the BM. Retinal pigment epithelium (RPE),
choroid (C), and sclera (S). H&E stain 40×. B. Magnified view of the choroidal neovessels (asterisk). H&E
stain 60× (Courtesy Zárate JO, Alvarado M© 2011. Unpublished data.)

Sometimes, the boundaries of subretinal membranes may appear obscure in

the fluorescein angiography but can be better defined using indocyanine green
angiography (ICG). Viewing specific characteristics of the membranes is crucial
while making treatment decisions. There are two main types of membranes:
classic (well-defined) membranes and occult (poorly defined) ones. Classic
membranes are well-demarcated, hyperfluorescent areas in early phases of the
angiogram with blurry boundaries in late phases. They are located in the
subretinal space where they are observed as an inverted layer of refractive RPE
on the external aspect of the membrane (46). In contrast to classic membranes,
occult membranes are poorly defined because they grow within sub-RPE
deposits where there is not enough space for the pooling of the dye causing the
so-called angiographic term late fluorescence of undetermined source (33). They
are firmly adherent to the RPE and BM and have no potential plane of cleavage
that allows surgical removal without destroying the overlying retina (46).
The pathogenesis of this process remains unclear. Although it has been
proposed that vascular proliferation is a response to outer retinal ischemia, the
lack of retinal neovascularization, typical outer ischemic retina atrophy, and
absence of choriocapillaris occlusion render this hypothesis weak (2).

Retinal Angiomatous Proliferation

Retinal angiomatous proliferation (RAP) is a distinct form of CNV in the
paramacular area in exudative AMD. The concept of RAP was first established
by Yannuzzi et al. (47) in 2001 due to three elements: (a) early appearance of
neovascularization, (b) a contiguous telangiectasia in the retina, and (c) the
variable or late onset of retinal–choroidal anastomosis. It had already been
described, despite some inconsistencies, by several authors (48–50).
In RAP, new retinal vessels expand toward the inner retina and below the
RPE. Three stages of the disease have been established based on clinical
biomicroscopic examination, fluorescein angiography, ICG, and optical
coherence tomography. Its histopathologic correlation has been obviated (51).
Immunohistochemical studies performed in neovascular membranes obtained by
surgical excision demonstrated hypoxia inducible factor one (HIF-1) alpha and
HIF-2 alpha expression in vascular endothelial cells in the neovascularization.
This suggests that RAP tissue is in a hypoxic state predisposing it to VEGF
expression. Valuable data regarding RAP pathogenesis were provided by this
study. Migration of CD68-positive macrophages expressing hypoxia factors in
the area of neovascularization was observed, suggesting that ischemic and
inflammatory factors are associated with the development and progression of
RAP (52).

Disciform Scar
The formation of a disciform scar represents the end stage of AMD. As
previously described, the CNV fragile endothelium is associated with leakage
and bleeding, leading to RPE detachments. In the process of reorganizing the
damaged tissue, a fibrovascular disciform scar is generated.
The major histopathologic components of this scar are mounds of dense
collagenous connective tissue on the inner surface of the BM (24). It is generally
associated with the loss of neural tissue (photoreceptors) in the outer retina. As a
general rule, the thinner and smaller the scar's diameter, the greater the
photoreceptor survival (33). In spite of this, morphometric studies confirmed that
the ganglion cell and inner nuclear layers are relatively preserved (53). Cystoid
degeneration and lamellar and full-thickness macular holes are other examples of
possible degenerative changes (4). RPE proliferation, hyperplasia, and fibrous
metaplasia have been reported. These epithelial cells are responsible for the
production of large quantities of extracellular matrix material. The disciform
lesions can be partially pigmented because of melanin and hemosiderin presence
and may be difficult to differentiate from nevi, malignant melanoma, or pigment
epithelium tumors (45). Vascular supply is provided by choroidal vessel in most
cases, rarely by retinal vessels (4).
 A rare but devastating complication of this condition is a massive subretinal
hemorrhage. It has been documented by Wood et al. (54) that neovascular tissue
present within the macular disciform lesions was the source of such bleedings
after a complete retinal detachment occurred. Other studies suggest that
anticoagulant medication, widely used among the AMD age group, can be a risk
factor for developing this unfortunate outcome. Therefore, the authors
recommend that anticoagulant therapy should be prescribed in cases only when
it is indicated (55). Moreover, another complication known as senile Coats'
response can also develop, resulting in extensive exudation in and under the
retina with lipid and cholesterol deposition (16).

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SECTION II

Classification of Age-Related Macular Degeneration

 6
CLASSIFICATION OF EXUDATIVE
AGE-RELATED MACULAR
DEGENERATION
MICHELLE L. CROUSE • WISSAM CHARAFEDDIN
GERARDO GARCIA-AGUIRRE • GABRIELA E. GRANELLA
D. VIRGIL ALFARO III • JOHN BARNWELL KERRISON
ERIC P. JABLON

INTRODUCTION
Age-related macular degeneration (AMD) has been subject to many divisions,
namings, and classification schemes since it was first described by Haab in 1885
(1). The disease has since been subdivided into two major forms: nonexudative
(dry AMD) and exudative (wet AMD). In nonexudative AMD, visual loss
develops as the result of geographic atrophy involving the foveal center (2,3). In
exudative AMD, visual loss develops secondary to choroidal neovascularization
(CNV), which leads to bleeding, exudation, and eventual scar formation (4).
Exudative AMD is distinguished from nonexudative AMD when the integrity of
the Bruch's membrane–retinal pigment epithelium (RPE) complex separates and
forms a pigment epithelial detachment (PED). While nonexudative AMD has not
been subject to subdivisions, exudative AMD has several subclassifications. In
the past, exudative AMD has been most commonly divided into occult and
classic types, based on characteristic lesion formation. In classic exudative
AMD, lesion boundaries were well demarcated on fluorescein angiography (FA),
while occult lesions had irregular boundaries and stippled hyperfluorescence.
Other classifications have been based on the grading systems of various studies
and clinical trials (5). In 1995, based on postoperative results in patients with
CNV, Gass (6,7) suggested a shift in the classification of exudative AMD using
the anatomic localization rather than the appearance on FA as was proposed in
the Macular Photocoagulation Study (MPS) (8,9). Such classification was
limited by the lack of imaging available at the time but can now be used due to
multimodal imaging, employing the use of FA, optical coherence tomography
(OCT), and indocyanine green (ICG) angiography.
A new classification scheme proposed by Freund et al. (10) builds on the
Gass model, dividing neovascularization into four types depending on location:
type 1, located under the RPE; type 2, above the RPE, in the subretinal space;
type 3, characterized by intraretinal neovascularization (IRN), also known as
retinal angiomatous proliferation (RAP); and type 4, polypoidal choroidal
vasculopathy (PCV), considered by some to be type 1 neovascularization. While
PCV is a sub-RPE vascular lesion, it does not have the clinical, structural, and
angiographic characteristics of type 1 lesions. This method of classification
would not rely on only one imaging technology as in the past with FA but also
on imaging techniques that emerged in recent years, refining our understanding
of the disease.

TYPE 1 NEOVASCULARIZATION
Gass defined type 1 neovascularization histologically as neovascularization
occurring beneath the RPE monolayer. It is by far the most common form of
neovascularization in AMD. In a study by Cohen et al. (11), occult CNV with or
without PED represented 56.6% of the 207 cases studied. With FA, these vessels
are usually described as occult or poorly defined. In a study by Lafaut et al. (12),
occult membranes were analyzed after surgical excision. All cases consisted of
thin fibrovascular membranes in the sub-RPE space. Only in two of the ten
specimens was an additional smaller fibrovascular component found in the
subretinal space.
FA terminology applied to type 1 lesions includes “late leakage of
undetermined source” or vascularized PED. ICG was found to be useful in
detecting choroidal neovascular membranes (CNVMs), especially if FA cannot
reveal a well-defined neovascular membrane (Figs. 6.1A and 6.2A). ICG is a
complementary imaging study in the diagnosis of occult lesions where masking
phenomena may prevent the visualization of the neovascular net. ICG is less
dependent on leakage as ICG dye bound to albumin is kept in the intravascular
space (13,14). This relatively new technique helped in deciding the eligibility of
treatment with photodynamic therapy (PDT) in some occult CNV cases (15).
Yannuzzi and Slakter (16,17) found that 39% and 44%, respectively, of patients
with occult CNV by FA showed well-demarcated hyperfluorescence by ICG
angiography. The ICG pattern of type 1 neovascularization can be classified into
three types depending on the size of the hyperfluorescent area: focal spots,
plaques, or a combination of both (18). In a postmortem histopathologic study of
an eye with ICG plaque, Chang et al. (19) confirmed the localization of plaques
documented by ICG to be between the RPE and an intact Bruch's membrane.

FIGURE 6.1 A. Type 1 CNVM. Left. FA showing leakage of undetermined origin. Right. ICG
angiography showing arteriolized pattern of choroidal neovascularization. B.Type 1 CNVM. Left. ICG
angiography showing arteriolized pattern of CNV. Right. Macular OCT showing a visible large pigment
epithelium detachment (PED).
FIGURE 6.2 A. Type 1 CNVM. Early- and late-phase FA: Leakage of undetermined origin. PED is
visible. B.Type 1 CNVM. Left. ICG: Bushy appearance of CNVM apparent with ICG. Right. OCT of the
same case. Pigment epithelium detachment with minimal subretinal fluid.

Spectral-domain optical coherence tomography (SD- OCT) allows

visualization of intraretinal structures as well as RPE morphology (Figs. 6.1B
and 6.2B). It identifies RPE detachment and CNV-related infiltration. According
to Malamos et al. (14), with the use of the RPE map, it is possible to identify the
location of the occult CNVM. If a membrane is hidden under the RPE, the latter
appears slightly prominent, flat at the top, and slowly declining at the margins.
These lesions are usually ill defined and convex with irregular surfaces. SD-
OCT can be more informative than is FA and allows better identification and
classification of membranes with occult components that may not be easily
detected and delineated. The 3D surface maps of the RPE further clarify the
exact level of the lesion and its microscopic morphology in combination with B-
scan morphology.
Type 1 membranes are the most common type associated with AMD, while
type 2 membranes are associated with focal lesions affecting the Bruch's
membrane and the RPE in diseases such as high myopia with lacquer cracks,
ocular histoplasmosis syndrome, serpiginous choroiditis, and choroidal ruptures
(20). In disorders like AMD, lipid-rich material, such as cuticular drusen,
contains numerous complement components that accumulate between the
Bruch's membrane and the RPE. Type 1 neovascularization grows in the
presence of such material and is not seen in disorders with focal disturbances of
the RPE–Bruch's membrane complex such as multifocal choroiditis and
panuveitis, myopia, and choroidal rupture (10). Type 1 pattern is rarely seen in
disorders like Best disease, vitelliform macular dystrophy, and pseudoxanthoma
elasticum where material accumulates above and below the RPE monolayer
(10).
Unlike classic lesions, occult-type lesions rarely cause visual symptoms
promptly, allowing some lesions to grow to a large size before being detected
clinically. They tend to have a variable and less aggressive natural course based
on the presenting acuities and long-term natural history. In the MARINA study
(21), patients with occult neovascular lesions presented better initial visual
acuities than in the ANCHOR study (22) patients who had classic lesions. This
was also observed in the VIP and TAP trials where many minimally classic
lesions and occult lesions with no classic component had relatively acceptable
visual acuity (23). According to Grossniklaus et al. (24), this benign course may
relate to the theory that in some eyes, type 1 neovascularization may grow as a
compensatory mechanism to provide nutritional support to the ischemic outer
retina by recapitulating the normal choriocapillaris. In his study, Engelbert et al.
(25) observed that only one eye out of eighteen eyes with type 1
neovascularization (6%) developed geographic atrophy overlying affected areas.
The exudation in type 1 neovascularization is predominantly in the form of
subretinal fluid. Subretinal fluid is less clinically aggressive than is cystoid
macular edema (CME). The presence of CME portends a poor prognosis, in that
it presumably indicates damage to the outer retinal barrier in the form of RPE
dysfunction and disruptions in the tight junctions that contribute to the external
limiting membrane (ELM) band on SD-OCT (3). However, in some cases, type 1
neovascularization may follow an aggressive course and cause a decrease in
visual acuity. These vessels may also erode through the RPE giving rise to type 2
neovascularization. In the TAP study, approximately 40% of eyes progressed
from minimally classic to predominantly classic lesions with 24% converting in
the first 3 months (26). This was also confirmed by Schneider et al. (27) when
23% of eyes with occult lesions having no classic components progressed to
predominantly classic membranes over a period of 6 to 12 months. Overall, the
conversion rate from occult with no classic to classic neovascularization at 1
year was 46% (39%–54%); no conversion in the reverse direction was ever
described (28).
The response to treatment with intravitreal anti–vascular endothelial growth
factor (VEGF) therapy may be less than the response observed in type 2 or type
3 neovascularization. Eyes with these type of neovessels may often continue to
manifest extrafoveal subretinal fluid. Type 1 neovascularization may be less
responsive to anti-VEGF treatment; nevertheless, the antipermeability effects of
these drugs are often sufficient to resolve most, if not all, of the fluid above the
RPE monolayer and may limit the continued growth of these vessels. Despite the
incomplete clearance of subretinal fluid, and sometimes the CME, many eyes
maintain stable visual acuity for up to 5 years on monthly anti-VEGF treatment
with no eyes experiencing sight-threatening submacular hemorrhages (25).
It was described by Spaide (29) that vessels may be detected by SD-OCT as
hyperreflective material lining the undersurface of the elevated RPE. The
contraction of this neovascular tissue, spontaneously or in response to
intravitreal anti-VEGF treatment, could produce dehiscence and tears in the RPE
layer. Such possibility can be predicted by the linear diameter of the PED as well
as the maximal PED height (30).

TYPE 2 NEOVASCULARIZATION
As mentioned previously, the type 2 pattern predominates in macular disorders
where there is a focal alteration of the RPE–Bruch's membrane complex such as
pathologic myopia with lacquer cracks (Fig. 6.3A and B), punctate inner
choroidopathy, multifocal choroiditis and panuveitis, histoplasmosis, and
choroidal rupture. It is seen also in entities where material is deposited above the
RPE such as vitelliform macular dystrophies, Best's disease, Stargardt's disease,
subretinal drusenoid deposits (reticular pseudodrusen), and pseudoxanthoma
elasticum (10).
FIGURE 6.3 A. Type 2 membrane in a case with high myopia. FA (early and late phases) showing leak in
a classic pattern. B. OCT of the same case. Disruption of the RPE is observed as well as a hyperreflective
area anterior to the RPE that corresponds to a type 2 CNVM.

Type 2 lesions usually start as type 1 neovascularization and then can

penetrate the RPE–Bruch's membrane complex to proliferate in the subretinal
space above the RPE monolayer to give the type 2 appearance (28).
Histopathology studies show that predominantly classic neovascularization
is mainly subretinal or combined subretinal with sub-RPE components. Classic
membranes were seen to contain not just capillaries but also larger caliber
vessels, whereas occult membranes contained predominantly, or only, capillaries
(12).
According to MPS criteria, CNV was considered well defined or classic
when a net of new vessels was identified at an early stage of FA with progressive
leakage in later stages (Fig. 6.4A and B). They fluoresce intensively against a
relatively dark background due to attenuation of the choroidal fluorescence by
the intervening RPE (Fig. 6.5A). About 17% (11) of all CNVM show
exclusively classic membranes, but it is possible that many of these harbor small
areas of type 1 vessels that are undetectable by FA alone (10). Occult lesions are
sometimes underestimated due to overlying classic membranes, but a PED seen
on OCT can explain the presence of an occult membrane. Classic membranes
can also be seen with ICG as an area of hyperfluorescence without marked
leakage activity. ICG images may underestimate the dimension of the
neovascular complex compared to morphologic imaging by SD-OCT (31).
FIGURE 6.4 A. FA showing CNVM (early and late phase) with leakage in a classic pattern. B. OCT of the
same case: Intact RPE with an overlying hyperreflective lesion representing a type 2 CNVM.
FIGURE 6.5 A. FA showing CNVM (early and late phases) with leakage in a classic pattern. B. OCT of
the same eye showing disruption of the RPE with an overlying hyperreflective lesion associated with
subretinal fluid.

On OCT, a high level of correlation was found between areas of classic CNV
seen on FA and the volume of subretinal tissue (32). It is possible to differentiate
a classic CNV using SD-OCT. The retinal thickness map specifically shows a
well-defined lesion with steep borders and a craterlike central depression (14).
Leakage and staining can be easily differentiated with this imaging technique
where hyperreflectivity represents visualization of fibrotic tissue and not edema.
With OCT, type 2 membranes can be localized above the RPE and beneath the
photoreceptor outer segments (10) (Fig. 6.5B).
The hyperreflective line, anterior to the RPE line, represents the inner
segment–outer segment junction of the photoreceptors (33). Disorganization of
this layer, overlying the inner segment–outer segment (IS/OS) junction, is often
accompanied by intraretinal cystic spaces. Intraretinal fluid rather than subretinal
fluid predominates in type 2 lesions and usually is associated with decreased
visual acuity (34,35). The differentiation between these two types of leakage can
be easily seen with OCT but is not possible with FA (10).
In patients with classic lesions, most vision loss occurs within the first 6
months with little vision loss after the first year. Predominantly classic lesions
included in the TAP trial were found to be smaller in size than the occult (type 1)
lesions included in the VIP trial. It is suggested that predominantly classic
lesions almost always cause visual symptoms promptly, perhaps allowing
identification of smaller lesion sizes, whereas occult lesions may or may not
cause prompt visual symptoms, which allows them to grow to larger sizes
(23,28).
Early in their evolution, type 2 vessels appear to be exquisitely sensitive to
intravitreal anti-VEGF agents. There may be a decrease in the volume of
subretinal fluid and fibrovascular tissue. In larger and more mature lesions, the
abnormal vessels remain as a hyperreflective band (subretinal fibrosis) causing
disorganization and thinning of the overlying receptors. This is seen as a
disruption of the IS/OS junction, loss of the ELM, and thinning of the outer
nuclear layer on SD-OCT. These eyes may continue to manifest intraretinal fluid
despite frequent treatment, presumably due to the loss of the outer blood–retinal
barrier in the form of RPE damage and disruption of the tight junctions that
contribute to ELM band on the OCT (10). Even in eyes with early lesions, the
disruption of the IS/OS layer may be indicative of the future visual result, and
visual recovery may be limited in these eyes because of the initial assault to the
photoreceptors (33).

TYPE 3: RETINAL ANGIOMATOUS

PROLIFERATION
It was a long-held belief that all neovascularizations in AMD originated from the
choroidal circulation. In 2001, Yannuzzi et al. (36) challenged this traditional
dogma based on the presumed origin and evolution of the neovascularization
process. They believed that IRN originating from the inner retinal circulation
produced a surrounding telangiectatic compensatory response to accommodate
an increase in vascular perfusion, and they termed this variant “retinal
angiomatous proliferation” (RAP). Thereafter, and due to a lingering uncertainty
as to the origin of these lesions, Freund et al. (37) have proposed naming this
vascular lesion “type 3 neovascularization” to distinguish and emphasize the
location of the neovascularization (intraretinal) independent of its origin. Type 3
neovascularization, or RAP, indicates proliferating vessels within and below the
retina itself. The origin of this neovascular subtype can be from either circulation
and may originate from both circulations simultaneously in the form of a retinal–
choroidal anastomosis (RCA). The presence of an RCA gives this entity many of
its characteristic clinical, SD-OCT, and angiographic features including
intraretinal hemorrhages and CME. Type 3 lesions have been observed
originating in areas with substantial photoreceptors, bringing the deep retinal
vasculature in close proximity to the underlying RPE, Bruch's membrane, and
choriocapillaries. Typically, the angiogenic response occurs overlying a focal
drusen/basal laminar deposit that may already be infiltrated by type 1 vessels.
Presumably, loss of or erosion through the intervening RPE and an appropriate
angiogenic milieu would allow the retinal and choroidal circulations to merge in
the form of RCA with a continued proliferative response. As these lesions
originate from both the retinal and choroidal circulations, they never originate
within the foveal avascular zone.
RAP is further subdivided into three stages, which describe the disease
progression. Stage I indicates the earliest clinical manifestations, in which RAP
begins in the deep retinal complex forming IRN. As the vessels evolve, they
extend beneath the neurosensory retina, becoming subretinal neovascularization
(SRN), or stage II. In stage III, an RCA is formed as SRN merges with choroidal
circulation, forming CNV (38–41) and the potential for a vascularized pigment
epithelium detachment (PED). FA is useful in revealing the presence of the
angiomatous intraretinal vascular complex and the extension of the associated
PED. However, other diagnostic techniques, such as indocyanine green (ICG)
angiography and OCT, have now proven to be as useful—or even more—to
demonstrate the presence of the RAP lesion.
Treating RAP has proven difficult due to the unique nature of the disease and
a lack of clinical trials focused on this form of neovascularization. To date, only
prospective and observational studies have been conducted, often with small
sample sizes, and no standard of treatment has been established. As with other
forms of neovascularization, the main treatment options for RAP include anti-
VEGF agents, PDT, focal laser, transpupillary thermotherapy, and surgical
ablation, or various combinations of these. To date, PDT with intravitreal
triamcinolone provided the most promising results initially, but by 12 months,
many of the effects had faded (42). Anti-VEGF agents may be beneficial in
decreasing vasogenic drive and retinal edema, when followed by PDT (43). ICG-
guided focal laser obliteration as salvage treatment may be an option, as
proposed by Bearelly et al. (44).

TYPE 4: POLYPOIDAL CHOROIDAL

VASCULARIZATION
Polypoidal choroidal vascularization (PCV) is recognized as a subset of type 1
CNV due to the location of PCV abnormalities, below the RPE. The various
clinical manifestations of PCV are shown in Figure 6.6. PCV is most frequently
characterized by a branching network of choroidal vessels and orange-red
polypoidal lesions (45). PCV vascular abnormalities lie between the RPE and
Bruch's membrane (46); therefore, PED is often seen in patients with PCV
(47,48). PCV has often been misdiagnosed as other forms of exudative AMD
due to similarities in clinical appearance on OCT and FA (49). Both PCV and