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Textbook of Vitreoretinal Diseases and Surgery 1st
Edition Edition S Natarajan Digital Instant Download
Author(s): S Natarajan , N Hussain
ISBN(s): 9788184486193, 8184486197
Edition: 1st Edition
File Details: PDF, 9.72 MB
Year: 2009
Language: english
Textbook of
Vitreoretinal Diseases
and Surgery
Textbook of
Vitreoretinal Diseases
and Surgery
Chief Editors
S Natarajan DO FMRF
Chief Managing Director
Aditya Jyot Eye Hospital Pvt Ltd
Mumbai, India
Nazimul Hussain MD DNB

Consultant Ophthalmologist
Vitreoretinal Specialist
Al Zahra Hospital
Sharjah, United Arab Emirates
Co-Editor
Supriya Dabir MD DNB
Vitreoretinal Consultant
Aditya Jyot Eye Hospital Pvt Ltd
Mumbai, India
Foreword
SS Badrinath and Tarun Sharma
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Textbook of Vitreoretinal Diseases and Surgery

© 2009, Jaypee Brothers Medical Publishers
All rights reserved. No part of this publication should be reproduced, stored in a retrieval system, or transmitted in any form
or by any means: electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the
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First Edition: 2009

ISBN 978-81-8448-619-3
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Printed at Ajanta Offset
Contributors
Stefan Mennel MD Chi-Chun Lai MD Ramesh Murthy MD FRCS

Department of Ophthalmology Associate Professor and Consultant, Pediatric
Philipps-University Marburg Director of Retina Ophthalmology and Strabismus
Robert-Koch-Sector 4, 35037 Department of Ophthalmology Oculoplasty and Ocular Oncology
Marburg, Germany Chang Gung Memorial Hospital LV Prasad Eye Institute
Chang Gung University LV Prasad Marg, Hyderabad
Jörg C Schmidt MD Taiwan Andhra Pradesh, India
Department of Ophthalmology
Philipps-University Marburg Hemanth Murthy MD Sima Das MS
Robert-Koch-Sector 4, 35037 Retina Institute of Karnataka Consultant, Pediatric
Marburg, Germany Bengaluru, Karnataka, India Ophthalmology and Strabismus
Oculoplasty and Ocular Oncology
Kuan-Jen Chen MD KS Sriprakash MS LV Prasad Eye Institute
Department of Ophthalmology Superintendent and Chief of LV Prasad Marg, Hyderabad
Chang Gung Memorial Hospital Vitreoretinal Surgery Andhra Pradesh, India
Chang Gung University Minto Eye Hospital
Taiwan Bengaluru Medical College and Santosh G Honavar MD FACS
Research Centre Director and Head
Nan-Kai Wang MD Bengaluru 560 002 Oculoplasty and Ocular Oncology
Lecturer and Karnataka, India LV Prasad Eye Institute
Attending Staff LV Prasad Marg, Hyderabad
Department of Ophthalmology Rajesh B Babu MS Andhra Pradesh, India
Chang Gung Memorial Hospital Consultant, Uveitis and Ocular
Chang Gung University Immunology Pramod S Bhende MS
Taiwan Narayana Nethralaya Consultant, Sankara Nethralaya
Bengaluru, Karnataka, India 18 College Road, Chennai
Wei-Chi Wu MD PhD Tamil Nadu, India
Associate Professor and Rajvardhan Azad MD FRCS FAMS
Attending Staff Chief, Vitreoretinal Services Chintamani Madhav Khare MS
Department of Ophthalmology Dr RP Centre for Consultant, Sankara Nethralaya
Chang Gung Memorial Hospital Ophthalmic Sciences 18 College Road, Chennai
Chang Gung University AIIMS, New Delhi, India Tamil Nadu, India
Taiwan
Avinash Pathengay FRCS Yashoda Ghanekar PhD
Consultant
Yih-Shiou Hwang MD Cell Biologists
Retina Vitreous Services
Lecturer and Attending Staff Pune, Maharashtra, India
LV Prasad Eye Institute
Department of Ophthalmology
GMR Varalakshmi Campus
Chang Gung Memorial Hospital Nazimul Hussain MS DNB
Visakhapatnam
Chang Gung University Consultant Ophthalmologist
Andhra Pradesh, India
Taiwan Vitreoretinal Specialist
Mayur Morekar MD Al Zahra Hospital, PO Box 3499
Ling Yeung MD Consultant Sharjah, UAE
Lecturer and Attending Staff Uvea and Immunology
Department of Ophthalmology LV Prasad Eye Institute S Natarajan DO
Chang Gung Memorial Hospital Kallam Anji Reddy Campus Chairman and Managing Director
Chang Gung University Banjara Hills, Hyderabad Aditya Jyot Eye Hospital Pvt Ltd
Taiwan Andhra Pradesh, India Mumbai, Maharashtra, India
Soumen Mondal MD Jatinder Singh Saumil Sheth MD FRCS

Aditya Jyot Eye Hospital Pvt Ltd Aravind Eye Hospital Consultant Vitreoretinal Specialist
Mumbai, Maharashtra, India Madurai, Tamil Nadu, India Aditya Jyot Eye Hospital
Mumbai, Maharashtra, India
Ramasamy Kim
Hemanth Trehan MD
Aravind Eye Hospital Bharat Ramchandani MD
Consultant Ophthalmologists
Madurai, Tamil Nadu, India Aravind Eye Hospital
Command Hospital
Bengaluru, Karnataka, India Madurai, Tamil Nadu, India
P Namperumalsamy
Saritha Katta PhD Aravind Eye Hospital Deepak Agarwal
Kallam Anji Molecular Genetics Madurai, Tamil Nadu, India Aravind Eye Hospital
Laboratory, LV Prasad Eye Institute Madurai, Tamil Nadu, India
Hyderabad, Andhra Pradesh, India Emmett T Cunningham Jr
Aravind Eye Hospital Anuradha Dhawan
Subhabrata Chakrabarti PhD Madurai, Tamil Nadu, India Aravind Eye Hospital
Scientist, Kallam Anji Molecular Madurai, Tamil Nadu, India
Genetics Laboratory, LV Prasad Eye Ashad Sivram DO
Institute, Hyderabad Aditya Jyot Eye Hospital Tunji Qluleye
Andhra Pradesh, India Mumbai, Maharashtra, India Aravind Eye Hospital
Madurai, Tamil Nadu, India
Inderjeet Kaur PhD
Scientist, Kallam Anji Molecular
Abhijit Datta MD Steffen Hörle
Aditya Jyot Eye Hospital
Genetics Laboratory, LV Prasad Eye Department of Ophthalmology
Institute, Hyderabad Philipps-University Marburg
Andhra Pradesh, India Robert-Koch-Sector 4, 35037
Angshuman Mukherjee DO DNB Marburg, Germany
Supriya Dabir MD Mumbai, Maharashtra, India
Vitreoretinal Consultant Priyanka Doctor MS DNB
Aditya Jyot Eye Hospital Aditya Jyot Eye Hospital Pvt Ltd
Anirudha Maiti MD Mumbai, Maharashtra, India
Anjli Hussain MD Mumbai, Maharashtra, India Ajay Dudani MD
Medical Retina and ROP Specialist Associate Professor
UAE Anupam Malpani MD KJ Somaiya Medical College and
Visiting Consultant Hospital, Mumbai
Anand Rajendran
Aditya Jyot Eye Hospital Maharashtra, India
Aravind Eye Hospital
Madurai, Tamil Nadu, India
Padmini P Badle
Dhananjay Shukla Dharmesh Kar MD KJ Somaiya Medical College and
Aravind Eye Hospital Aditya Jyot Eye Hospital Hospital, Mumbai
Madurai, Tamil Nadu, India Mumbai, Maharashtra, India Maharashtra, India
vi
Foreword
Science and technology related to medicine is a fast growing field.

Latest advances related to various diseases such as cell biology, molecular engineering, physical
sciences, application of mathematics and information technology are being added up to the knowledge
pool almost every year. So is true for vitreoretinal diseases, and the literature has started touching
the related aspects such as genetics, stem cell research and nanotechnology.
While the thickness of the neurosensory retina is just around 200 microns, the body of knowledge
on vitreoretinal diseases is gigantic. This book that summarizes the deliberations of the Textbook of
Vitreoretinal Diseases and Surgery takes us to a further level of understanding the current concepts in
the diagnosis and management of diseases affecting retina.
Starting with the first chapter on genetics of age-related macular degeneration with such intricately
woven information on the latest developments and trends in twin analysis, gene mapping and
screening, role of oxidative stress and inflammation, this book takes us through a journey via the
various vitreoretinal diseases, which have been a topic of debate and discussion in the recent past.
Delicately touching the medical and surgical aspects of adult and paediatric retinal diseases, lasers,
and diagnostic modalities, the information contained in the text is vast and enormous, with the
material organised as an integrated text.
Written by the experts in the field, each of the twenty-three chapters begins with an introductory
note, progressing gradually into the detailed but concise and up-to-date knowledge with well
illustrated figures, legends and references, to impart a visual impact on the reader. As a result, the
text presents factual and concise package and expert perspective with the latest in the field.
We wish to congratulate Chief Editors, Dr S Natarajan and Dr Nazimul Hussain and the
Co-Editor Dr Supriya Dabir, who undoubtedly have presented the information in the book with a
focused approach guided by their immense experience in academics.
The book will be a useful guide to the clinicians, scientists and students. It appears as a complete
quantum of basic, medical and surgical information, garnished with molecular genetics and stem cell
flavors.
SS Badrinath FRCSEd Tarun Sharma MD FRCSEd MBA

President Director
Medical Research Foundation Shri Bhagwan Mahavir Vitreoretinal Services
Chairman Emeritus Director, Fund Raising
Sankara Nethralaya Medical Director, Sankara Nethralaya
Preface
After the great triumph of the 8th International Advanced Vitreoretinal Surgery Conference held in
picturesque Kuala Lumpur early in 2008 with world renowned faculty, it became imperative for me
to share the thoughts, speeches and ideas which made the conference a resounding success.
As the Chairman of the Scientific Committee for this conference, I feel extremely privileged to
bring you this book which marks the first such successful compilation of the collection of scientific
sessions with bridging plenary lectures focusing on the updates in vitreoretinal diseases.
This anthology addresses the latest treatment modalities in both medical as well as surgical
retina. It also highlights the painstaking research in genetics, stem cell therapy, lasers etc. Apart
from this, focus has been on the utility of various advanced diagnostic modalities in day-to-day
practice.
This book is meant to cater to not only to nascent retinal surgeons but also practicing surgeons as
an update to their knowledge. Since the conference limits the audience to a few, this book is meant
to rejuvenate the information on the updates in the field of vitreoretinal surgery to all those unable
to attend the meet.
I would like to thank all the guests and invited speakers from across the world from the bottom
of my heart. Their time and contributions are greatly cherished and undoubtedly provide the icing
on the academic cake. A book of this stature would be incomplete without its scientific programme
and I am duty bound to felicitate all those who have provided scientific contributions. I would also
like to thank and appreciate Dr Nazimul Hussain and Dr Supriya Dabir who have worked tirelessly
with me and contributed immensely in completing the book.
Ultimately, they have justified the efforts of the Organising and Scientific Committees.
“A man’s learning is imperishable and a precious wealth

All other possessions are less golden”
—Thirukkural-Verse 400
Thank you for granting me the privilege of serving as the Chairman of the Committee.
S Natarajan
Contents
1. Genetics of Age-related Macular Degeneration ............................................................ 1

Saritha Katta, Subhabrata Chakrabarti, Inderjeet Kaur, Nazimul Hussain
2. Cell Therapy in Retinal Diseases .................................................................................. 15

Yashoda Ghanekar, Nazimul Hussain
3. Atypical Manifestations of Diabetic Retinopathy ...................................................... 31

Anand Rajendran, Dhananjay Shukla, Jatinder Singh, Ramasamy Kim
P Namperumalsamy, Emmett T Cunningham Jr
4. Anti-VEGF Therapy in Proliferative Diabetic Retinopathy ..................................... 39

Anand Rajendran, Bharat Ramchandani, Deepak Agarwal
Anuradha Dhawan, Tunji Oluleye
5. Vascular Endothelial Growth Factor: Inhibitors in Age-related

Macular Degeneration ..................................................................................................... 45
Supriya Dabir, Aniruddha Maiti, Abhijit Datta, S Natarajan
6. Role of Combination Therapy in Diabetic Macular Edema ..................................... 57

Saumil Sheth, Supriya Dabir, S Natarajan
7. Hemorrhage at the Macula: Classifications and Treatment Options ...................... 75

Stefan Mennel, Jörg C Schmidt
8. Retinal Stress by Vitreous Traction ............................................................................... 85

Jörg Christian Schmidt, Steffen Hörle, Stefan Mennel
9. Polypoidal Choroidal Vasculopathy ............................................................................. 93

Ling Yeung, Kuan-Jen Chen, Chi-Chun Lai
10. Vitreous Hemorrhage: Recent and Future Management ........................................ 107

Nan-Kai Wang, Wei-Chi Wu, Yih-Shiou Hwang, Ling Yeung, Chi-Chun Lai
11. Suprachoroidal Hemorrhage ........................................................................................ 119

Priyanka Doctor, Dharmesh Kar, Supriya Dabir, Anupam Malpani, S Natarajan
12. Imaging in Posterior Uveitis ......................................................................................... 131

Avinash Pathengay, Mayur Morekar
13. Optical Coherence Tomography in Age-related Macular Degeneration ............. 149

Hemanth Murthy
14. Newer Advances in Retinal Lasers .............................................................................. 165

Hemanth Murthy
15. Role of Biostains in Vitreous Surgery ........................................................................ 173

Hemanth Trehan, Avinash Pathengay
16. Wide-angle Viewing System in Vitreoretinal Surgery ............................................ 183

S Natarajan, Soumen Mandal, Nazimul Hussain, Angshuman Mukherjee
17. Microincision Vitrectomy .............................................................................................. 191

S Natarajan, Soumen Mandal, Nazimul Hussain, Ashad Sivaraman
18. Update on Retinopathy of Prematurity: Concept and Management .................... 201

Rajvardhan Azad, Anjli Hussain, Nazimul Hussain
19. Pediatric Retinal Detachment ....................................................................................... 225

Pramod S Bhende, Chintamani Madhav Khare
20. Pediatric Intraocular Tumors–I .................................................................................... 241

Ramesh Murthy, Sima Das, Santosh G Honavar
21. Pediatric Intraocular Tumors–II ................................................................................... 257

Ramesh Murthy, Sima Das, Santosh G Honavar
22. Ocular Manifestations of HIV/AIDS ........................................................................... 275

Rajesh B Babu, KS Sriprakash
23. Central Serous Chorioretinopathy .............................................................................. 293

Ajay Dudani, Padmini P Badle
Index ....................................................................................................................................................... 303
xii
Abstract
Age-related macular degeneration (AMD) is a late-onset complex disorder with multifactorial etiologies. Both genetic
and environmental factors play a role in the disease pathogenesis. AMD is the third leading cause of blindness in the
elderly. Familial aggregation, segregation studies and linkage analysis have provided both qualitative and quantitative
evidence on the genetic basis in AMD. Several candidate loci have been earlier mapped in AMD but variants in genes viz.
APOE, ABCA4, FBLN6 and EFEMP1 harboring these loci have accounted for only a small proportion of cases. Recent
screening of two major loci has led to the identification of the Complement Factor H (CFH) on 1q32 and LOC387715 and
HTRA1 on the 10q26 gene cluster. Single nucleotide polymorphisms (SNPs) in CFH (Y402H), LOC387715 (A69S) and a
promoter variant in HTRA1 have been associated with AMD in large case-control cohorts. These SNPs exhibited large
effect sizes and high disease odds for the risk genotypes across different populations. Interestingly, these associations
have been widely replicated across multiple ethnic groups worldwide indicating their potential role in the disease
pathogenesis. In this article, we would outline the genetics of AMD with special emphasis on CFH followed by other
genetic variants based on studies done by our group and colleagues worldwide. We would also provide a brief overview
on the possible molecular mechanisms leading to AMD.
Introduction
Visual impairment leading to blindness is a major impediment towards growth and development in
populations worldwide. The burden of blindness is a global challenge that needs to be tackled on a
war footing. Recent estimates of the World Health Organization (WHO) indicate that around 45
million people are blind and 135 million are visually impaired worldwide and 90% of these people
live in the developing countries. Among the blinding conditions, cataract accounts for 60% of global
blindness, followed by glaucoma (12.3%). Age-related macular degeneration (AMD) is the third
leading cause of blindness and accounts for 8.7% of the world population (WHO Fact sheet no. 282).
Although AMD is more common among the elderly in developed countries, it is also becoming a
cause of concern in the developing countries, with fast demographic changes in lifestyle and
senescence.
The Problem
AMD causes progressive impairment of central vision and is a leading cause of irreversible vision
loss worldwide. The overall prevalence of late stage AMD varies from 1.4 to 1.7% across different
epidemiological cohorts (Klein et al, 1992; Mitchell et al, 1995) and increases significantly with age.
The prevalence of AMD in India ranges from 1.84-2.7% (Nirmalan et al, 2004), similar to the global
prevalence. It is estimated that by the year 2020, around 8 million people will have vision loss due to
retinal complications including AMD (Bressler, 2002). As it leads to irreversible blindness, managing
AMD is a global public health challenge.
Risk Factors in AMD

Epidemiological surveys conducted on large case-controls cohorts have identified several demographic
and environmental risk factors in AMD. The Age-related Eye Disease Study (AREDS) conducted on
large Caucasian cohorts indicated gender, age and smoking as important risk factors in the
development of AMD (AREDS 2000, 2004). This was later replicated in many studies done on other
ethnic groups (Krishnaiah et al, 2005; INDEYE study 2007). These risk factors are briefly described
2 as follows:
Genetics of Age-related Macular Degeneration
GENDER
The incidences of early and late AMD are 2 and 7 times more in females above > 75 years of age,
compared to males below 75 years of age (Klaver et al, 1998). The high risk in females may be due to
the loss of a protective effect of estrogens in postmenopausal women.
RACE
The prevalence of AMD was shown to vary greatly among different races. All forms of AMD are
more prevalent in the white population than darkly pigmented races (Bressler et al, 2008; Friedman
et al, 1999). Although the exact mechanism is not known, it was suggested that may melanin protect
against the formation of lipofuscin, which is a marker of cellular senescence and promoter of oxidative
damage (Kawasaki et al, 2008). A recent study found no differences in macular or melanin pigment
densities between eyes with and without early AMD (Kayatz et al, 2001).
CIGARETTE SMOKING
Smoking has been postulated to cause AMD by depression of serum antioxidant levels and alteration
of choroidal blood flow and detoxification of the retinal pigment epithelium (RPE). It has been
hypothesized that decrease in luteal pigments in human retina due to cigarette smoking may cause
oxidative damage to the macula, thereby leading to an increased risk of developing AMD. Many
population-based studies have shown the association of smoking with increased risk of AMD
(Berendschot et al, 2002; AREDS 2000; Delcourt et al, 1998). It was also shown that prior and current
smokers are prone to develop AMD atleast 5 to 10 years before non-smokers. A risk ratio of 2 or
higher was observed for neovascular AMD (Klein et al). Stryker et al, reported that men who
smoked one pack of cigarettes per day had only 72% of the plasma α-carotene levels of nonsmokers,
even after adjusting for dietary differences between smokers and nonsmokers. The APEDS study
reported an odds ratio of 3.29 [95%CI, 1.42–7.57] for AMD in individuals with the history of cigar
smoking (Nirmalan et al, 2004).
ALCOHOL CONSUMPTION
Alcohol intake causes tissue damage by increasing the oxidative stress or affecting mechanisms that
protect against oxidative damage to retina. The inconsistent findings among various studies, however,
suggest that consumption of alcoholic beverages is not likely to be an important risk factor for the
incidence of AMD at this point of time (Eye Disease Case–Control Study Group; 1992).
DIET
Diet has been related to several chronic conditions including cancer, coronary heart disease, and
cataract. It may also have an important role in preventing and slowing the development of AMD
(Seddon et al, 1994).
LIGHT EXPOSURE
There have been conflicting reports on association of ultraviolet or visible light with AMD.
Blumenkranz and associates found a statistically significant difference in the incidence of AMD
between sun-exposed and sun-protected skin, whereas the Eye Disease Case-Control Study Group
did not find sunlight exposure a risk factor in AMD. Majority of studies however, found no
associations between estimated ambient UV-B exposure/sunlight exposure and age-related
maculopathy (Stryker et al, 1988). 3
HYPERTENSION
Systemic hypertension was found to be associated with neovascular AMD. Hyman et al, conducted
a case-control study of risk factors for neovascular and non-neovascular age-related macular
degeneration (AMD). It was observed that neovascular AMD was positively associated with diastolic
blood pressure greater than 95 mm Hg (odds ratio [OR] = 4.4), as opposed to non-neovascular AMD
(Khan et al, 2006). The association of hypertension with advanced AMD was also reported by AREDS
study [OR = 1.45] (AREDS, 2005).
GREATER BODY MASS INDEX

The association of greater body mass index with AMD has been reported by some studies (Klein et
al, 2003; van Leeuwen et al, 2003; Johnson et al, 2005). Johnson et al, reported that the risk of obesity
with AMD could be related to the physiologic changes such as increased oxidative stress, changes in
the lipoprotein profile, and increased inflammation. These changes would also result in increased
destruction and decreased delivery of lutein and zeaxanthin to the macula of the eye. Therefore,
association to AMD risk could be through indirect effects on changes in lutein and zeaxanthin status
and metabolism (Johnson et al, 2005).
CATARACT SURGERY
There are few studies, which reported cataract surgery as a risk factor for AMD. Wang et al, studied
the association of cataract surgery and 5-year incidence of late-stage age-related maculopathy in the
patients from the Beaver Dam and Blue Mountains eye studies. It was found that over a period of
five years either neovascular AMD or geographic atrophy developed in 6.0 to 7.5% of aphakic eyes
(10 of 168 right and 11 of 147 left eyes), compared with 0.7% of phakic eyes (40 of 5504 right and
37 of 5572 left eyes) (Abecasis et al, 2004). According to APEDS, prior cataract surgery was significantly
associated with increased incidence of AMD (adjusted OR 3.79; 95% CI, 2.1– 6.78) (Nirmalan et al,
2004).
Genetics of AMD
The genetic basis of AMD was relatively ignored for many years as other causes for the disease
were explored. Genetic epidemiological studies have revealed that genetic differences between
populations might play an important role in explaining the prevalence among diverse ethnic groups.
Higher concordance among the monozygotic twins, familial aggregation and segregation analyses
have suggested a strong genetic basis for the disease (Seddon et al, 1997, 2005; Klaver et al, 1998).
TWIN ANALYSIS
Twin studies allows the exact comparison of the relative contribution by genetic as well as non-
genetic factors on a trait. Such studies provide an exact quantification of the contribution of genetic
factors or heritability which is the proportion of variance of a trait that is attributable to genetic
factors. Such studies have provided more direct evidence of AMD heritability by comparing disease
concordance rates in monozygotic (MZ) vs. dizygotic (DZ) twins. In a large USA population based
survey of 840 elderly male twins, including 210 MZ twin pairs, 181 DZ twin pairs, and 58 singletons
268 twins were having signs of maculopathy, of which 106 had advanced disease. Analyses of this
4 twin data yielded heritability estimates of 0.67 for intermediate and advanced disease (grades 3, 4,
and 5), and 0.71 for advanced disease only, including geographic atrophy and exudative AMD
(grades 4 and 5). Hammond and colleagues evaluated concordance rates for early ARM in a
cohort of 506 female volunteer twin pairs (226 MZ twin pairs and 280 DZ twin pairs). ARM was
defined as the presence of soft drusen 0.63 mm in diameter in the macular area or the presence of
pigmentary changes along with any type of drusen. The concordance for ARM in MZ twins was 0.37
compared with 0.19 in DZ twins, and the heritability of ARM was estimated to be 45% (95% CI 0.35
to 0.53).
FAMILY BASED GENE MAPPING STUDIES

These studies aimed at identifying regions on the chromosome shared by the closely related affected
members in families using microsatellite markers or single nucleotide polymorphisms (SNPs) spread
across the whole human genome. This procedure is very straight forward and has been successfully
used for Mendelian traits. However, AMD being a late onset complex disorder suffered major
hurdles from the unavailability of the closely affected relatives (parent and siblings) and no clear cut
inheritance pattern.
Several such genome-wide linkage studies have identified a number of putative loci for AMD but
only a few of these regions have been replicated independently. The first locus for AMD at 1q25-31
was mapped by Klein et al,(1998) in a large family, however, the mutation Q5345R in the Fibulin
gene harboring this locus could not sufficiently prove its contributions to AMD pathogenesis.
Subsequently the susceptible loci have been mapped to chromosomes 3p, 3q, 4p, 5p, 5q, 6q, 8q, 9p,
9q, 10q,12q, 15q,16p,16q,17q,18p, 20q and 22q, but no causative mutation has been reported in the
genes located in these regions (Weeks et al, 2000; Seddon et al, 2003; Weeks et al, 2004; Majewski et
al, 2003; Schmidt et al, 2004, Haddad et al, 2006). The chromosomal regions at 1q31–32 and 10q26
were identified in several independent studies and confirmed by meta-analysis of six datasets and
these regions harbor the largest effect susceptibility variants for AMD (Swaroop et al, 2007).
CANDIDATE GENE SCREENING

Candidate genes for AMD were identified based on both genome-wide scan results and knowledge
about gene function/expression data. Initially, genes involved in the pathogenesis of retinal
degenerations similar to AMD were screened. Several Candidate genes responsible for macular and
retinal dystrophies (ELOV4 and ABCA4: Stargardt disease; TIMP3: Sorsby fundus dystrophy; and
Peripherin: Retinal degeneration) that share common features with AMD were extensively screened
for their involvement in AMD (Ayyagiri et al, 2001; Allikemets et al, 2001, 1999; Akimoto et al, 2001;
De La Paz et al, 1997; Shastry et al, 1999). But the variation in these genes could account for only a
small subset of AMD cases.
Currently, AMD candidate genes have been examined mainly by the way of case-control association
studies using SNPs in the known loci which point to the identification of the possible disease-causing
genetic variants or to the variations that might be lying in the close vicinity of the actual disease
causing allele. Such studies evaluate whether specific alleles occur at a significantly higher frequency
among the affected individuals vs. controls and can led to the identification of specific DNA signature
or haplotype that can help in predicting the risk of AMD. The advent of high throughput SNP arrays
has facilitated greatly the gene discovery especially for multigenic or complex diseases. Recently,
some major candidate genes have been identified using these arrays in large case-control cohorts
that explain a substantial proportion of AMD. 5
Complement Factor H (CFH) Gene

The polymorphism Y402H in the Complement Factor H (CFH/ARMS1) gene has been shown to be
significantly associated with AMD susceptibility. CFH on 1q32 is an important regulator of complement
system of innate immunity. A T→C substitution at 1277 nucleotide in exon 9 of CFH resulting in a
change of tyrosine to histidine (Y402H) increases an individual’s risk of having AMD by several
folds. The odds ratio for AMD reported by these studies ranged between 2.4- 4.6 for the risk allele
“C” and between 3.3-11.5 for those with the risk genotype “CC”. The association of the Y402H SNP
in AMD has been shown across different studies worldwide. Magnusson et al, (2006) further
investigated the association of CFH and AMD based on genotype-phenotype correlations and observed
that the Y402H allele confers a significant risk to both late stages (neovascular AMD, Geographic
Atrophy) and early stages of AMD (soft drusens) in US and European AMD patients. It was also
observed that the Y402H variant contributes to increased risk of advanced AMD through its
involvement on the development of soft drusen which are precursors of advanced AMD phenotypes.
This SNP has been implicated in most AMD populations worldwide, except in the Japanese (Gotoh
et al, 2006, Okamoto et al, 2006).
Hageman et al, (2005) analyzed haplotypes using eight intragenic SNPs in CFH and also the
immuno-histochemical status of drusen and sections of cadaveric eye in AMD patients. Their analysis
revealed a risk haplotype, which had almost two-folds higher frequency among the cases (50%) than
controls (29%). Two protective haplotypes for AMD were also identified among the controls. CFH is
an important regulator of complement system of innate immunity against microbial infection. This
regulation of complement activity is achieved by the binding of CFH to C3b (generated by the
cleavage of a chains of C3), thereby stopping the production of C5b-9 (the component of membrane
attacking complex). The Y402H residue located within the binding sites for heparin and C-reactive
protein. Altered binding of CFH to these proteins results in changes in CFH’s ability to suppress the
complement-related damage to the host cells (Clark et al, 2005, Johnson et al, 2006).
Discovery of complement factor H gene as a major candidate for AMD led to the investigation of
other complement pathway genes for their involvement in AMD pathogenesis. CFH is a member of
the RCA (Regulators of complement activation) gene cluster located on chromosome 1q32 spanning
21.45 cM and including more than 60 genes of which 15 are complement related. All these complement
genes are arranged in tandem through duplications and gene conversions. CFH lies in the close
vicinity of CFHR1 and CFHR5 genes that encode for five CFH related plasma proteins. A CFH
haplotype harboring the deletion of the ‘CFH-related’ genes CFHR1 and CFHR3 was found to be
protective against AMD but in small proportion of AMD cases.
Factor B and Complement Component

Gold et al, (2006), reported a strong association of variation in the Factor B (BF) and complement
component 2 (C2) genes with AMD. BF and C2 genes are located in the major histocompatibility
complex class III region (6p21). The L9H and R32Q variants in BF and E318D and an intron 10
variant in C2 were found to confer significantly reduced risk of AMD. When these haplotypes were
analyzed together with CFH variants it was shown that variation in these two loci can predict
clinical outcome in 74% of the affected individuals and 56% of the controls.
BF and C2 are expressed in the neural retina, RPE and choroids. Additionally the BF protein was
observed in ocular drusen and Bruch’s membrane. Glutamine at position 32 of this protein has been
6 shown to have reduced hemolytic activity as compared to wild type Arg32 (Lokki and Koskimies
1991). BF is an important activator of the alternative complement pathway and thus may result in
AMD by abnormal BF activity.
Recently two studies reported significant associations between AMD and variants in the
complement component 3 (C3) gene on chromosome 19p13. C3 plays an important role in activation
of both the classical and the alternative complement pathways and the plasma complement C3a
levels are significantly elevated in AMD cases compared to controls. A fourth recent study also
found ARM associated variants in the C7 and MBL2 (Gene ID 4153) complement pathway genes by
complement pathway focused analysis of an earlier genome-wide association scan.
Genes in the 10q26 Cluster

The second AMD locus mapped on 10q26 harbored three important candidate genes PLEKHA1
(OMIM 607772), hypothetical LOC387715 and HTRA Serine Peptidase 1 (HTRA1; OMIM 602194)
(Jakobsdottir et al, 2005). Resequencing of this cluster revealed a significant association of the A69S
(rs10490924) SNP in LOC387715 gene in two large AMD cohorts of German origin (Rivera et al,
2005). These results were further replicated in Caucasian (Conley et al, 2006; Ross et al, 2007; Seddon
et al, 2007), Japanese (Tanimoto et al, 2006) and Russian (Fisher et al, 2006) AMD patients. It has also
been shown that the presence of the rs10490924 SNP, along with an associated history of smoking,
strongly modifies the risk of AMD (Schmidt et al, 2006). The combined effect of the rs10490924 SNP
and smoking significantly enhanced the risk of AMD in some populations (Seddon et al, 2007; Wang
et al, 2007; Schaumberg et al, 2007) but this finding could not be replicated in a large dataset comprising
the AREDS (Age-Related Eye Disease Study) and CHS (Cardiovascular Health Study) cohorts (Conley
et al, 2006) The combined additive effect of the rs1061170 (CFH) and rs10490924 SNPs exhibited a
high population attributable risk percentage (PAR %) in AMD (Seddon et al, 2007; Schmidt et al,
2006).
Very recently, another SNP (rs11200638) located 512bp upstream of the transcription site of HTRA1
gene in the same 10q26 cluster, was implicated in three independent reports on Caucasian (Yang et
al, 2006; Cameron et al, 2007), Chinese (DeWan et al, 2006) and Japanese (Yoshida et al, 2007) AMD
subjects. It was also demonstrated that this SNP in the promoter region was in LD with rs10490924
that was a further 6.6 kb upstream of HTRA1 (Dewan et al, 2006) Earlier studies done on Chinese
(Dewan et al, 2006) , Japanese (Kondo et al, 2007; Yoshida et al, 2007) and Caucasian populations in
the US22 (Yang et al, 2006; Cameron et al, 2007) have demonstrated the association of rs11200638
(HTRA1) in AMD. It was also shown that the rs11200638 confers similar risk in dry and wet AMD
cases in the Caucasian populations.
While, the function of both these variants are yet uncharacterized, two locus odds ratios have
indicated significant risk conferred by the HTRA1 variant in conjunction with the CFH variant Y402H
(Cameron et al, 2007). Recent studies, however, have provided convincing evidence of the significant
involvement of the LOC387715 SNP, but not HTRA1, in the development of AMD (Kanda et al, 2007).
Although the underlying functions of LOC387715 are yet to be unveiled, it was suggested that
rs11200638 is a strongly associated marker in the vicinity of rs10490924 because of the strong LD
between these SNPs (Kanda et al, 2007; Dewan et al, 2006).
Functionally, the presence of LOC387715/ARMS2 mRNA has been demonstrated in the retina
and other cell lines; it localizes to the mitochondrial outer membrane in transfected mammalian cells
(Kanda et al, 2007). Although the functional implications of the rs11200638 SNP in AMD pathology
has been suggested based on the detection of HTRA1 in drusen of both wet (Dewan et al, 2006) and 7
dry AMD (Cameron et al, 2007) eyes and in promoter-based assays, this finding was not replicated
in another study (Kanda et al, 2007). Thus, the precise role of the LOC387715 and HTRA1 SNPs in
AMD is as yet unknown, and their interactions with different factors in the complement pathway
remain speculative. However, such speculations cannot be addressed unless extensive data on gene–
gene interactions in the background of other nongenetic factors leading to the pathophysiology of
AMD are elucidated (Swaroop et al, 2007)
APOE
In the past few years, association of variants in genes involved in lipid metabolism such as
Apolipoprotein E (APOE) have increased our understanding of the underlying mechanism of AMD
development. Based on common pathogenic features including lipid deposition (drusen and plaque
formation), thickening of connective tissue (Bruch’s membrane and arterial inner lining) and elevated
levels of CRP in serum, a common mechanism for the development of AMD and atherosclerotic
cardiovascular diseases was proposed (Freidman 2000). However, it was noted that the effect of
APOE alleles on AMD risk was contrastingly different than that of atherosclerosis and cardiovascular
diseases (Baird et al, 2004; Zareparsi et al, 2004). Association of APOE in AMD has been reported by
several groups (Klaver et al, 1998; Schmidt et al, 2002; Baird et al, 2004; Zareparsi et al, 2004) showing
a reduced risk of AMD with APOE-e2 allele and higher risk with allele APOE-e4. However, APOE
polymorphisms have exhibited varied geographical and ethnic variations across AMD patients
worldwide (Kaur et al, 2006).
Toll like Receptor 4 (TLR4)

A recent study by Zareparsi et al, (2005) implicated the TLR4 gene (9q32-33) in AMD pathogenesis.
Toll like receptors are involved in innate immunity and pathogen recognition (Cook et al, 2004),
linked to regulation of cholesterol efflux and participates in phagocytosis of photoreceptor outer
segments by the RPE (Kiechl et al, 2002; Castrillo et al, 2003; Kindzelskii et al, 2004; Blander et al,
2004). The D299G polymorphism in TLR4 was associated with a 2.65 folds increased risk of AMD,
thereby suggesting that altered TLR4 signaling by this variant may influence phagocytic function of
RPE which in turn may contribute to RPE damage. It was also shown that TLR4-D299G had an
additive effect on AMD risk (OR = 4.13, p = 0.002) with allelic variants of APOE and ATP binding
cassette transporter-1 (ABCA1), which are involved in cholesterol efflux. However, the effect of
TLR4, APOE and ABCA1 variants on AMD susceptibility was in contrast to that seen in atherosclerosis.
But TLR4 polymorphisms have not been explored extensively across different world AMD populations
compared to other candidate genes.
Mechanisms of AMD Development

Several pathogenic mechanisms have been proposed to understand the complex etiologies of AMD
development including RPE cell death, oxidative damage of cellular components, mitochondrial
dysfunction and accumulation of toxic components such as lipofuscin and advanced end
glycation products (Haddad et al, 2006). It was thus proposed that variations in genes involved in
inflammation, oxidative stress and cholesterol metabolism play significant role in the pathogenesis
8 of AMD.
ROLE OF COMPLEMENT ACTIVATION AND INFLAMMATION IN THE PATHOGENESIS OF AMD

Inflammation has been implicated in several diseases of aging like Alzheimer’s disease, stroke and
cardiovascular disease. Interestingly all these diseases share lots of similarities with AMD with
respect to pathological features and common biomarkers for the disease such as increased levels of
CRP, CFH and Homocysteine in plasma of patients, presence of serum amyloid P component in
drusens (the levels of which are elevated in plasma of Alzheimer patients) and association with CFH
(Y402H) and APO E variation, etc.
Protein profiling studies of the drusen also revealed the presence of immunoglobulins, components
of the complement pathway (C3, C5b-9 complex) (Johnson et al, 2000), molecules involved in acute
phase response to inflammation (e.g. Amyloid P component and 1-antitrypsin), proteins that modulate
immune response (vitronectin, clusterin, apolipoprotein E, membrane cofactor protein, complement
receptor 1), major histocompatibility complex class II antigens and HLA-DR and cluster differentiation
antigens (Mullins et al, 2000). Accumulation of all these proteins and cellular debris suggest that cells
are subjected to a chronic sublethal complement attack mediated by choroidal dendritic cells and
macrophages resulting in the further degradation of Bruch’s membrane (Johnson et al, 2001, Hageman
et al, 2001). These studies gave a strong evidence for the role of inflammation and dysfunction of
complement pathways in the pathogenesis of AMD.
ACCUMULATION OF LIPOFUSCIN IN THE RPE

Lipofuscin are autoflourescent lysosomal storage bodies that accumulate in the postmitotic
metabolically active cells throughout the body due to normal aging process. These bodies originate
primarily from the degradation of different intracellular organelle and the incomplete degradation
of extracellular material taken into the cells by phagocytosis. RPE lipofuscin are formed due to
ingestion of photoreceptor outer segments as evident from the presence of retinoids, high
concentration of DHL-Lipids, etc. Accumulation of lipofuscin in RPE has been associated with the
development of AMD though evidence for the same has not been provided.
ROLE OF OXIDATIVE STRESS MEDIATORS AND MODIFICATIONS IN AMD PATHOGENESIS

The evidence for oxidative stress playing a major role in AMD pathogenesis includes increased risk
of AMD in smokers, presence of oxidative modified proteins in the drusen/Bruch’s membrane/
RPE / choriodal tissues and slower progression of AMD in patients fed on regular supplementation
of antioxidant vitamins and Zinc. Several studies have pointed out that oxidative protein modifications
serve as a primary catalyst for macular degeneration, the reactive oxygen species/oxidative products
being provided by photo-oxidative rich milieu in the retina particularly the lipids and retinoid rich
photoreceptor segments. Proteome study of drusens also demonstrated the elevated levels of
oxidatively modified proteins like μ-carboxyethyl pyrrole (CEP) adducts in the drusen, Bruch’s
membrane and plasma of AMD patients. It was found that the mean level of CEP adducts was 1.5
fold higher (p = 0.004) and that of antibody titers in plasma was 2.3-fold higher (p = 0.02) in patients
with AMD compared with normal age-matched controls (Gu et al, 2003). Of individuals (n = 13)
exhibiting both antigen and autoantibody levels above the mean for non-AMD controls, 92% had
AMD. Based on the results, CEP immunoreactivity and autoantibody titer may have diagnostic
utility in predicting AMD susceptibility.
These CEP adducts are generated from oxidation of polyunsaturated fatty acids (PUFAs)
particularly docasohexanoic acid (DHA) present in photoreceptor outer segments and have been 9
shown to induce choroidal neovascualrization (CNV) in retinal tissues. In-vitro treatment of human
RPE cells with CEP dipeptide or CEP-HSA did not induce increased VEGF secretion and suggesting
that anti-CEP therapeutic modalities might be of value in limiting CNV in AMD (Ebrahem et al,
2006). CNV in retinal tissues can also be stimulated by the complement components (C3a and C5a)
present in the drusen. It was shown that genetic ablation of receptors for C3a or C5a reduces VEGF
expression, leukocyte recruitment and CNV formation after laser injury. These experiments suggested
that antibody-mediated neutralization of C3a or C5a or pharmacological blockade of their receptor
could be therapeutic modalities for AMD (Nozaki et al, 2005).
Conclusions
Genetic association studies have led to the implication of several candidate genes in AMD in the last
few years. These association studies have been meaningful as they have been widely replicated
across multiple populations with varied ethnic backgrounds in clinically well characterized cohorts
(Todd 2006). Moreover, the effect sizes of these variants, particularly the CFH (Y402H), LOC387715
(A69S) and HTRA1 have been substantially large that permitted a proper association amidst varied
sample sizes in different studies. In future, more such studies are required across wider geographical
regions to identify candidates that contribute to the AMD pathogenesis. Genetic typing of AMD
patients would also permit clinicians to develop correlations with genotypes for estimating disease
risk and progression. Identification of susceptible gene variant(s) would allow early intervention in
subjects ‘at-risk’ of developing AMD for a better prognosis. While the underlying biological functions
of these candidate genes and their interactions are yet to be characterized, large multicentre studies
with these variants should be undertaken with respect to the treatment modalities in order to
understand the therapeutic mechanisms in subjects carrying the risk genotype(s).
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50. Nozaki M, Raisler BJ, Sakurai E, Sarma JV, Barnum SR, et al. Drusen complement components C3a and C5a
promote choroidal neovascularization. Proc Natl Acad Sci, USA 2006;103: 2328-33.
51. Okamoto H, Umeda S, Obazawa M, Minami M, Noda T, et al. Complement factor H polymorphisms in Japanese
population with age-related macular degeneration. Mol Vis 2006;12:156-8.
52. Rivera A, Fisher SA, Fritsche LG, Keilhauer CN, Lichtner P, et al. Hypothetical LOC387715 is a second major
susceptibility gene for age related macular degeneration contributing independently from complement factor H to
disease risk. Hum Mol Genet 2005;14: 3227-36.
53. Ross RJ, Bojanowski CM, Wang JJ, Chew EW, Rochtchina E, et al. The LOC387715 and age related macular
degeneration: Replication in three case-control samples. Invest Ophthalmol Vis Sci 2007;48:1128-32.
54. Schmidt S, Klaver C, Saunders A, Postel E, De La Paz M, et al. A pooled case-control study of the apolipoprotein E
(APOE) gene in age-related maculopathy. Ophthalmic Genet 2002;23:209-23.
55. Schmidt S, Saunders AM, De La Paz MA, Postel EA, Heinis RM, et al. Association of the Apolipoprotein E gene with
the age-related macular degeneration: Possible effect modification by family history, age, and gender. Mol Vis
2000;6: 287-93.
56. Schmidt S, Scott WK, Postel EA, Agarwal A, Hauser ER, et al. Ordered subset linkage analysis supports a susceptibility
locus for age-related macular degeneration on chromosome 16p12. BMC Genet 2004;5:18.
57. Schmidt S, Hauser MA, Scott WK, Postel EA, Aggarwal A, et al. Cigarette smoking strongly modifies the association
of LOC387715 and age-related macular degeneration. Am J Hum Genet 2006;78: 852-64.
58. Seddon JM, Ajani UA, Mitchell BD. Familial aggregation of age-related maculopathy. Am J Ophthalmol 1997;123:
199-206.
59. Seddon JM, Ajani UA, Sperduto RD, Hiller R, Blair N, et al. Dietary carotenoids, vitamins A, C, and E, and advanced
age-related macular degeneration. Eye Disease Case-Control Study Group. JAMA 1994;272: 1413-20. Erratum in:
JAMA 1995; 273: 622.
60. Seddon JM, Cote J, Page WF, Aggen SH, Neale MC. The US twin study of age related macular degeneration:
Relative roles of genetic and environmental influences. Arch Ophthalmol 2005;123: 321-7.
61. Seddon JM, Santangelo SL, Book K, Chong S, Cote J. A genome-wide scan for age-related macular degeneration
12 provides evidence for linkage to several chromosomal regions. Am J Hum Genet 2003;73: 780-90.
62. Seddon JA, Francis PJ, George S, Schultz DW, Rosner B, et al. Association of CFH Y402H and LOC387715 A69S with
progression of age-related macular degeneration. JAMA 2007; 297:1793-1800.
63. Seitsonen S, Lemmela S, Holopainen J, Tommila P, Ranta P, et al. Analysis of variants in the complement factor H,
the elongation of very long chain fatty acids-like 4 and the hemicentin 1 genes of age-related macular degeneration
in the Finnish population. Mol Vis 2006;12: 796-801.
64. Sepp T, Khan JC, Thurlby DA, Shahid H, Clayton DG, et al. Complement Factor H variant Y402H is a major risk
determinant for geographic atrophy and choroidal neovascularization in smokers and nonsmokers. Invest
Ophthalmol Vis Sci 2006;47: 536-40.
65. Shastry BS, Trese MT. Evaluation of the peripherin/RDS gene as a candidate gene in families with age-related
macular degeneration. Ophthalmologica 1999;213:165–70.
66. Simonelli F, Frisso G, Testa F, di Fiore R, Vitale DF, et al. Polymorphsim p.402Y>H in the complement factor
H protein is a risk factor for age-related macular degeneration in an Italian population. Br J Ophthalmol 2006;90:1142-
5.
67. Souied EH, Leveziel N, Richard F, Dragon-Durey MA, Coscas G, et al. Y402H Complement factor H polymorphism
associated with exudative age-related macular degeneration in the French population. Mol Vis 2005;11:1135-40.
68. Stone EM, Lotery AJ, Munier FL, Heon E, Piguet B, et al. A single EFEMP1 mutation associated with both Malattia
Leventinese and Doyne honeycomb retinal dystrophy. Nat Genet 1999;22:199-202.
69. Tanimoto S, Tamura H, Ue T, Yamane K, Maruyama H, et al. A polymorphism of LOC387715 gene is associated
with age-related macular degeneration in the Japanese population. Neurosci Lett 2006;414: 71-4.
70. The Eye Disease Case – Control Study Group. Risk factors for neovascular age-related macular degeneration. Arch
Ophthalmol 1992;110:1701-8.
71. Todd JA. Statistical false positive or true disease pathway? Nat Genet 2006;38: 731-3.
72. Tomany SC, Cruickshanks KJ, Klein R, Klein BE, Knudtson MD. Sunlight and the 10-year incidence of age-related
maculopathy: the Beaver Dam Eye Study. Arch Ophthalmol 2004;122: 750-7. Erratum in: Arch Ophthalmol. 2005;
123: 362.
73. Tomany SC, Wang JJ, Van Leeuwen R, Klein R, Mitchell P, et al. Risk factors for incident age-related macular
degeneration: Pooled findings from three continents. Ophthalmology 2004;111:1280-7.
74. Weeks DE, Conley YP, Mah TS, Paul TO, Morse L, et al. A full genome scan for age-related maculopathy. Hum Mol
Genet 2000;9:1329-49.
75. Weeks DE, Conley YP, Tsai HJ, Mah TS, Schmidt S, et al. Age-related maculopathy: a genome-wide scan with
continued evidence of susceptibility loci within the 1q31, 10q26, and 17q25 regions. Am J Hum Genet 2004;75:
174-89.
76. World Health Organization. Fact sheet no. 282. Magnitude and causes of visual impairment. http://www.who.int/
mediacentre/factsheets/fs282/, 2004.
77. Yang Z, Camp NJ, Sun H, Tong Z, Gibbs D, et al. A variant of the HTRA1 gene increases susceptibility to age-
related macular degeneration. Science 2006;314: 992-3.
78. Yoshida T, DeWan A, Zhang H, Sakamoto R, Okamoto H, et al. HTRA1 promoter polymorphism predisposes
Japanese to age-related macular degeneration. Mol Vis 2007;13: 545-8.
79. Zareparsi S, Branham KE, Li M, Shah S, Klein RJ, et al. Strong association of the Y402H variant in complement factor
H at 1q32 with susceptibility to age-related macular degeneration. Am J Hum Genet 2005;77:149-53.
80. Zareparsi S, Buraczynska M, Branham KE, Shah S, Eng D, et al. Toll-like receptor 4 variant D299G is associated with
susceptibility to age-related macular degeneration. Hum Mol Genet 2005;14:1449-55.
81. Zareparsi S, Reddick AC, Branham KE, Moore KB, Jessup L, et al. Association of apolipoprotein E alleles with
susceptibility to age-related macular degeneration in a large cohort from a single center. Invest Ophthalmol Vis Sci
2004;45: 1306-10.
13
Introduction
Cell therapy is an exciting area of research that is aimed at regeneration of tissues or organs in
patients with irreparable damage to these tissues or organs. Cell therapy is no longer restricted to
transfer of cells from a healthy subject to a patient, as in bone marrow transplant or corneal transplant.
We can now envisage cell therapy that involves generation of patient-specific cells for transplantation,
manipulate them in vitro into the desired cell type and transplant these cells into a patient for functional
restoration of the damaged tissue. Although many cell therapies are currently being tested in animal
models and their clinical application might take much longer, results from these studies are encouraging
and suggest that regeneration of custom-made organs might indeed be possible in real life.
Cell therapy in the form of simple blood transfusion and bone marrow transplant has been in use
for a long time. This was limited to collecting healthy cells from matched donors and introducing
these cells into the patient. The cells were not propagated or manipulated in any way in vitro prior to
transplantation. The first culture and transplantation of dermal epithelial cells was performed to
replace damaged skin in burns patients1, 2 and taking cues from these experiments, similar approach
was used to regenerate corneal epithelium in patients with limbal stem cell deficiency.3 Simultaneously
embryonic stem cells were first established in 19814,5 and further understanding of mechanisms
underlying self-renewal and differentiation of stem cells into various types of cells led to designing
of new therapeutic strategies.
Degenerative diseases of retina like retinitis pigmentosa (RP), age-related macular degeneration
(AMD) and diabetic retinopathy lead to irreparable damage to the retina and are a major cause of
blindness in developed countries (Figure 2-1). Several genetic disorders such as retinitis pigmentosa
or Leber congenital amaurosis also lead to retinal degeneration and blindness. A therapeutic approach
that can regenerate retinal cells or the entire retina would help improve visual acuity and quality of
life of these patients. Use of cell therapy for retina however is not easy as the retina consists of
several cell types and retinal disorders can occur due to damage to any of these cell types. The
neural retina consists of rod and cone photoreceptors, bipolar cells, ganglion cells, horizontal cells,
Müller cells and amacrine cells that are arranged in a definite three-dimensional structure. The
neural retina rests on the retinal pigment epithelium (RPE), which is in close contact with
photoreceptors and is vital for maintenance of photoreceptor homeostasis. Degeneration of any
type of cells in the neural retina or RPE can lead to retinal degeneration and loss of vision. Retinal
disorders could also occur due to uncontrolled growth of endothelial cells leading to
neovascularization in the retina that can eventually lead to retinal detachment in diseases like diabetic
retinopathy and AMD. The current attempts at retinal cell therapy are therefore aimed at regeneration
of cells in neural retina, regeneration of RPE and re-establishment of correct retinal vasculature. For
treatment of inherited retinal disorders, the cell therapy also might involve manipulations to correct
the genetic defect by gene therapy.
Cell therapy is an interdisciplinary field that requires inputs from the field of stem cell biology,
regenerative biology, material science, developmental biology, genetic engineering and clinicians.
The success and feasibility of any cell therapy depends on a number of factors, such as availability of
suitable source of stem cells, expansion and manipulation of cells to generate the desired type of cell,
introduction of cells in vivo, survival and integration of cells to reconstruct degenerated tissue and
restoration of its physiological function (Figure 2-2).
An appropriate source of stem cells that can generate and sustain cells/tissue of interest is an
16 important starting point for cell therapy. Stem cells are the cells that have the ability to renew
Cell Therapy in Retinal Diseases
FIGURE 2-1: Fundus photograph showing wet (Top left) and geographic atrophy (Top right) age-related macular
degeneration, and proliferative diabetic retinopathy (bottom)
themselves and also differentiate into any other cell of the body. They could be embryonic stem cells,
which are derived from the inner cell mass of blastula or adult stem cells, which are present in the adult
tissues such as bone marrow, liver, muscles, skin and cornea. Embryonic stem cells are totipotent, i.e.
they have the ability to differentiate into any other cell type. Adult stem cells on the other hand have
limited capacity to divide and can differentiate only into specific types of cells. They can be either
multipotent stem cells that can differentiate into many types of cells, such as the bone marrow stem
cells, or unipotent stem cells that can differentiate into only one type of cell, e.g. limbal stem cells. For
retina, several sources of stem cells such as embryonic, fetal and adult stem cells from eye and bone
marrow have been investigated as discussed below. An autologous source of cells would be preferred
in order to avoid complications of rejection associated with graft rejection, but it may not be possible
to use an autologous source if retinal degeneration is due to an inherited disorder.
The next important issue is the ability to culture these stem cells in vitro to expand them. The
number of cells available for transplantation, especially if they are from adult tissues, is often limited
and the cells need to be cultured in vitro to obtain sufficient number of cells. Expansion of cells while
maintaining their characteristics such as their stemness or their differentiation status is a challenge
and requires standardization of culture conditions. This involves identification of correct culture 17
FIGURE 2-2: Different steps in cell therapy
medium, growth factors, feeder cells and it may also involve standardization of correct matrix that
will help cellular growth and differentiation or harvesting of cell at the time of transplantation.
After expansion, stem cells might be directly introduced in the animal to allow it to differentiate
in the niche provided by the recipient or the cells might be coaxed to undergo differentiation in vitro
to generate the cell or cells of interest. Directed differentiation of a stem cell into a cell of interest is
extremely challenging and requires understanding of mechanisms underlying the development of
that particular tissue during embryogenesis. The differentiation of a stem cell into the desired cell
type is regulated by both intrinsic information present within the cell and the cues provided by the
niche it is residing in. Therefore for directed differentiation, stem cells need to be cultured under
specific conditions that would allow their differentiation into cells of interest. Extensive
experimentation to understand the developmental biology and cell differentiation of the tissue of
interest needs to be carried out using various animal models to understand the conditions required
for such in vitro manipulations. This is particularly complicated for tissues like retina, which is made
up of several cell types. For treatment of genetic disorders, the defective copy of the gene needs to
be replaced with the wild type or the correct copy of the gene at this stage.
The correct route of introduction of these differentiated cells in vivo also needs to be investigated.
In retinal cell therapy, the mode of introduction, whether subretinal or intravitreal, is important.
Once introduced in the body, the transplanted cells are monitored for their survival. The cells are
also monitored to check if they maintain their state of differentiation. If undifferentiated cells have
been introduced, whether they can differentiate in vivo into the desired cell type needs to be
monitored. The cells should also integrate well in the tissue. In the retina, it is important that
18 transfected cells establish synaptic connections for restoration of retinal function. Similarly in the
case of RPE cells, the cells should integrate and establish contact with photoreceptors in order to
maintain their homeostasis. All these steps are important in ensuring that the transplanted cells
function well in vivo and are a true substitute for the degenerated or damaged tissue.
Last but not the least transplanted cells should not cause any undesirable changes in the recipient.
Cell therapy involves removal of stem cells from their normal niche and introduction into a new
environment. Changes in the stem cell niche could lead to unsafe situations like uncontrolled cell
division leading to tumor formation. The application of cell therapy to humans should be performed
only after ensuring that the cell therapy is free of such safety issues and it restores normal
functioning.
Sources of Stem Cells for Retinal Therapy

Both ocular and extraocular sources of stem cells have been explored for their potential to restore
retinal function. The ocular tissues investigated so far are retinal cells, RPE cells, ciliary cells, iris
pigment epithelial (IPE) cells and limbal cells. The advantage of using ocular cells is they arise from
the same progenitor cells, with the exception of limbal cell, and share common pathways of
differentiation during early development. These cells therefore would require less manipulation in
vitro. The extraocular cells investigated so far are embryonic stem (ES) cells, bone marrow stem
cells, cells from central nervous system (CNS) and cord blood cells. ES cells, bone marrow cells and
cord blood cells have better capacity to self-renew, can differentiate into several cell types, are
easier to culture and therefore might be preferred over ocular stem cells.
STEM CELLS FROM OCULAR SOURCE

Retinal Stem Cells
Retinal progenitor cells give rise to all cells of the retina. They generate ganglion cells, horizontal
cells, cone photoreceptors and amacrine cells during early embryogenesis and rod photoreceptors,
bipolar cells and Muller glia during late embryogenesis and the early postnatal days. While many
studies have shown that a particular stem cell can be differentiated into a retinal progenitor cell or
retinal neurons, only transplantations of fetal or neonatal retinal cells in degenerating retina have
shown integration and at least partial restoration visual function, suggesting that committed retinal
progenitors from fetal or neonatal cells are most suitable for therapeutic purpose. This can perhaps
be attributed to the fact that immature neurons from fetal tissues have better ability to reconnect
with neurons and cells from fetal tissue are already committed to a particular fate and require less
manipulation in vitro to direct their differentiation.
Fetal retinal sheets consisting of retina and RPE have been transplanted in subretinal space of
Royal College of Surgeons (RCS) rats, which is a model for retinal degeneration.6 These fetal sheets
integrated well and restored normal morphology of rat retina. Functional studies were not performed
in this report. Xenotransplantation of neonatal mouse retinal cells into opossum also resulted in
integration of retinal cells into all layers of the retina and the transplanted cells expressed neuronal
and retinal markers.7 This study indicated that the host age is important for integration, as cells
injected in 5-10 days postnatal retina integrated well while those injected in older animals survived
in vitreous but did not integrate. In another study, sheets of rat embryonic retinal cells were used
for transplantation in rat model of retinal degeneration and it led to some restoration of visual
function, as detected by electrophysiological experiments.8 The restoration was restricted only to 19
the superior colliculus region. A more systematic study in mouse to check cells at what stage are able
to integrate in adult retina showed that postnatal P1-P7 retinal cells which express retina-specific
transcription factor Nrl, but not embryonic cells, could integrate well in the retina.9 The integrated
cells formed functional synaptic junctions and also improved visual function as seen by pupillometry
and extracellular field potential recordings from ganglion cell layer. This study identified the specific
ontogenic stage of transplanted cells that is important for integration of retinal cells and can restore
retinal function.
Human studies have also been carried out which show quite encouraging results. Transplantation
of fetal retina in patients with retinitis pigmentosa showed that fetal retina integrates well into the
degenerating retina and can improve visual acuity.10, 11 A recent report of clinical trial from the same
group shows that transplantation of fetal retinal sheets led to improvement in visual acuity of 7 out
of 10 patients suffering from RP and AMD.12 No adverse events were reported in these studies and
the grafts survived well. This is encouraging although its application to large population could have
ethical and practical problems. However with the information available now it might be possible to
differentiate stem cells in vitro to a similar ontogenic stage before transplanting these cells into the
retina.
Studies show that retinal stem cells (RSCs) present in neonatal retinas can certainly be isolated,
propagated and differentiated in vitro to generate retinal cells. This is important, as it would be
helpful if cells from one fetal retina could be used for several transplantations. Neonatal/fetal tissues
would likely have less number of stem cells/progenitor cells and ability to increase their number
before transplantation would increase success rate of this treatment. Using epidermal growth factor
(EGF), retinal progenitors from neonatal rats could be differentiated into both early and late neuronal
cells.13 Similar studies were also performed in pigs, where progenitor cells from neural retina of pig
could be cultured in vitro and when injected subretinally in laser-injured retina, could integrate and
express photoreceptor cells markers.14 Mouse retinal progenitors from postnatal day 1, when
transplanted into hosts lacking rhodopsin, were able to develop into mature neurons, express
recoverin, rhodopsin or cone opsin and integrate into the retina.15 Interestingly these cells could also
rescue cells in the outer nuclear layer, with improvement in light-mediated behavior as compared to
uninjected animals. On the other hand, subretinal injection of radial glial cells from retina of new-
born mice into rd1 or VPP mice led differentiation of injected cells into either ganglion cells or glial
cells.16 Another study has shown that incubation of mouse radial glia cells with FGF-2 and B27 also
can lead to differentiation of these cells into photoreceptors with high yield.17 More extensive studies
need to be carried out to investigate whether these cells are functionally as competent as normal
retinal cells.
RPE Cells
RPE cells also have been investigated for their potential to generate retinal cells, as both neural
retina and RPE develop from neuroepithelium and follow similar developmental signal during initial
development. Indeed rat RPE cells from early embryonic stages have the potential to develop into
retinal neurons.18 In the presence of bFGF, RPE cells from stages E12-E14 could differentiate into
cells expressing markers of rod cells, amacrine cells and RGC cells. Although RPE cells appear to
have the potential for differentiation into retinal cells, they are difficult to harvest, this might hinder
their use for cell therapy.
On the other hand, homologous transplantation of RPE cells has shown promising results. Replacing
20 degenerating RPE cells by transplanting healthy, autologous RPE cells from peripheral retina has
been used a therapeutic approach for treating AMD. Dissociated RPE cells19 as well as RPE explants
from the mid-periphery have been transplanted in patients with neovascular AMD20, 21 and this led
to some improvement in visual acuity. The procedure however can lead to complications such as
retinal detachment and improvement in visual acuity can be transient.22
Ciliary Stem Cells

The ciliary margin zone in lower animals such as Xenopus, the adult ciliary margin zone harbors
retinal progenitors, which are capable of regenerating all types of retinal cells through out their
life.23 A similar structure that ensures continuous regeneration of retinal cells is unfortunately not
present in mammals but this observation prompted researchers to investigate the potential of ciliary
body as a source of retinal progenitors. Studies indeed showed that ciliary epithelium of mouse,
specifically the pigmented ciliary margin, harbors stem cells that could differentiate into
photoreceptors, bipolar neurons and Muller glia.24 Such cells have also been reported in ciliary margin
of rat25 and pig.26 These cells are also present in human ciliary body and could differentiate into early
and late retinal neurons.27 Stem cells have been isolated from cadaveric human pars plana and pars
plicata, from eyes from subjects as old as 70 years indicating that progenitor cells are present in the
human retina through out life. In vitro these cells could differentiate into all cell types of neural
retina as well as RPE, although the frequency of each cell type varied.28 Moreover these cells could
integrate into postnatal mouse retina and embryonic chick retina where they differentiated into
photoreceptors. In mouse the percentage of photoreceptor precursors from pars plana increased
significantly during retinal injury induced by N-methyl-N-nitrosourea, suggesting that in vivo the
ciliary stem cells could indeed play a role in retinal repair.29 A similar phenomenon was observed in
response to ganglion cell death after optic nerve axotomy.30 Ciliary epithelial cells can also be directed
towards photoreceptor differentiation by transfection of transcription factor Crx.31 Further studies
need to be carried to investigate whether these cells are functionally competent to replace retinal
cells.
Iris Stem Cells

Adult iris tissue is easy to harvest by routine iridectomy and can express neuronal antigens when
cultured in vitro, which makes it a viable option for generating photoreceptors. When cultured iris
pigmented epithelial (IPE) cells were transfected with transcription factor Crx, expression of
photoreceptor-specific genes was observed in IPE cells.32 Postnatal IPE cells, when co-cultured with
embryonic retinal cells, were able to express photoreceptor and Muller glia-specific proteins,
suggesting a potential of these cells for retinal cell therapy.33 IPE cells engineered to express pigment
epithelium-derived factor (PEDF) were also able to protect degenerating photoreceptor in RCS rats,
suggesting they can also exert a trophic effect for rescue of degenerating retina.34
The potential of IPE as a substitute for RPE has also been investigated with promising results.
Autologous transplantation of IPE in subretinal space in rabbit showed that IPE cells survive well in
the subretinal space and also show fragments of rod outer segments in their phagosomes, suggesting
that they could functionally replace RPE in degenerative diseases like AMD.35 Transplantation of
autologous IPE cells harvested by iridectomy have also been transplanted subretinally in patients
with RPE degeneration or after traumatic loss of RPE cells and this led to either improved or stable
visual acuity.36 In addition IPE cells transduced with viral vector expressing BDNF can rescue
photoreceptors against phototoxicity both in vitro37 and in vivo in rats.38 21
Limbal Stem Cells

Limbal tissue surrounding the cornea harbors stem cells that normally regenerate the corneal
epithelium. Limbal cells are easy to harvest, harbor stem cells and can be cultured in vitro, which
makes them a good source of cells for cell therapy. Interestingly rat limbal cells have been directed
to differentiate into a neural progenitors when incubated with noggin, an inhibitor of BMP-mediated
signaling.39 Further studies showed that co-culture of limbal cells with postnatal day 1 retinal cells
led to expression of photoreceptor-specific proteins such as opsin, rhodopsin kinase, arrestin and
IRBP.40 These neural progenitors, when injected intravitreally in mice with retinal damage, were
able to incorporate into the retina. Similar properties have also been observed in human limbal
cells.41 Whether these incorporated cells are functional in vivo needs to be further investigated.
STEM CELLS FROM EXTRAOCULAR SOURCE

Embryonic Stem Cells
Embryonic stem (ES) cells have several advantages over adult stem cells. Embryonic cells are totipotent
and can be differentiate into any other type of cell. They can also be cultured and expanded more
easily than adult stem cells. It is however also associated with ethical issues. Due to debates on
ethical issues on use of embryonic stem cells, research on ES cells is restricted or banned in some
countries. Also, being allogenous, once transplanted into a patient, embryonic stem cells could express
MHC proteins and cause rejection of graft. Despite these problems, laboratory studies on mouse
and human embryonic stem cells have proved valuable for providing information about differentiation
of retinal cells, which can be extrapolated to differentiation of adult stem cells into retinal progenitor
cells. These studies have helped in understanding the nature of molecular signals required for retinal
development and show that the niche provided by the developing embryo is important for
differentiation of retinal cells.
A structure resembling the eye has been generated in vitro from human embryonic stem cells by
co-incubation with PA6 stromal cells and FGF-2, dexamethasone and cholera toxin. 42 This
structure had lens-like refractile structure surrounded by cells expressing markers of retinal cells,
which in turn was surrounded by RPE-like cells. Thus the structure showed organized morphology
resembling the structure of eye. Cells expressing markers of a particular type were clustered together
although these clusters were not exactly arranged the way different cells are arranged in the eye.
Further studies by the same group showed that further treatment of these cells with Wnt2b led to
proliferation of retinal progenitor cells and when transplanted into the enucleated optic cup of chick
embryos, they were able to form a single mature layer of RPE-like cells.43 The cells also differentiated
into cells expressing markers of ganglion cells in some cases. Cells expressing retinal markers have
also been generated by co-incubation of mouse ES cells with developing retina.44 Recent studies
have used more defined factors and shown that mouse ES cells can be differentiated into retinal
precursors in a step-wise manner.45 In this study mouse ES were incubated with cells with Wnt
antagonist Dkk1 and nodal antagonist LeftyA to generate rostral brain progenitors, followed by
treatment with activin and serum that led to differentiation of rostral cells into retinal precursors.
These precursors generated cells with photoreceptor phenotype when co-cultured with embryonic
retinal cells. These experiments suggest that ES cells are a feasible alternative for retinal cell
transplantation.
Human ES cells have also been differentiated into retinal progenitors in a similar step-wise manner,
22 using a different strategy.46 In this study, human ES cells on feeder cells were first treated with
noggin, followed by incubation with fibroblast growth factor (FGF) and epidermal growth factor
(EGF) in serum-free medium. This treatment promoted differentiation of ES cells into neural
progenitors and these neural progenitors also expressed several markers of retinal cells. In another
study human ES cells were treated with noggin, Dkk1 and insulin-like growth factor (IGF) to generate
embryoid bodies, followed by incubation with bFGF for three weeks to generate retinal progenitors.47
This treatment was more efficient and generated retinal progenitors with high efficiency, up to 80%.
The cells were also able to integrate into degenerating mouse retina. In addition both human and
mouse ES cells have been shown to differentiate into RPE and other types of neurons when cultured
on denuded, gelatin-coated human amniotic membrane.48 This method suggests an alternative to
use of xenogenic feeder cells as has been reported previously.42, 44
The studies mentioned above use fetal calf serum or fetal tissues as feeder layers to direct
differentiation of ES cells into photoreceptors. The differentiation medium used here is not defined
and would involve use of xenogenic agents and hence may not be suitable for transplantation in
human patients. Recent study by Osakada and coworkers reported the use of a completely defined
medium to generate photoreceptors and RPE cells from mouse, monkey and human ES cells.49 The
authors used a combination of purified signaling molecules in a step-wise manner and combined this
with selection of cells based on progenitor-specific markers at each step to finally generate
photoreceptors from ES cells. They first generated neural retinal precursors from mouse ES cells
using serum-free suspension culture in the presence of Wnt and nodal antagonists as described
earlier.50 From these cells, retinal progenitors were selected based on expression of retinal progenitor
marker Rx. These cells were further cultured on laminin-fibronectin coated dishes along with DAPT,
an inhibitor of notch signaling. Around 22% of the treated cells expressed Crx, photoreceptor-
specific marker and were post-mitotic. 10% of these cellls expressed cone-specific markers. A further
incubation of these cells with combination of aFGF, bFGF, taurine, sonic hedgehog and retinoic acid,
led to generation of 17% cells that expressed rod-specific markers. A similar approach was also used
for generation of human and monkey photoreceptors. RPE cells were also generated from monkey
ES cells using defined medium, only this time selection was done for expression of Mitf, marker for
RPE progenitor, instead of Rx.49 It remains to be seen whether these cells can establish connections
with neurons and whether they are functionally active.
ES cells can also provide a niche for degenerating retinal cells that can prevent further damage
under certain conditions. Mouse ES cells differentiated to neural lineages have been shown to delay
retinal degeneration in mouse models. The ES cells differentiated into neuronal cells after removal
LIF and addition of retinoic acid, these neuralised cells were introduced in mnd mice, which suffer
from lysosomal storage disorder that causes retinal degeneration.51 These cells could integrate into
the retina, reduce lysosomal storage bodies and delay photoreceptor degeneration. This suggests
that neuralised ES cells could prevent or delay disease progression if not completely rescue the
disease.
While use of ES cells for therapeutic purpose in humans is questionable at this stage, development
of two important techniques that can reprogram differentiated adult cells into stem cells have caused
excitement among scientists and such cells could be further differentiated into retinal progenitors
using the information that is already available from research on embryonic stem cells. The first
technique is that of somatic nuclear transfer, which involves transfer of nucleus from an adult cell
into an enucleated oocyte (Figure 2-3A). The combination of factors present in the oocyte can
reprogram the adult nucleus and the resulting cell acquires characteristics of a totipotent stem cell
which, when transferred into a surrogate mother, can develop into a complete animal. Dolly the
23
A B
FIGURES 2-3A and B: Reprogramming of adult nucleus. A: Reprogramming by somatic cell nuclear transfer.
B: Reprogramming by introduction of four transcription factors to generate induced pluripotent cells
sheep was the first animal developed by this technique.52 The technique although promising, again
requires use of oocytes, which may not be ethically acceptable and the success rate of this technique
is low.
The second and more recent breakthrough in stem cell biology is the generation of induced
pluripotent cells (Figure 2-3B). For the first time scientists were able to completely dedifferentiate a
differentiated dermal fibroblast to generate a pluripotent stem cell by transfection of four transcription
factors; Oct3/4, Klf4, c-myc and Sox2.53 When introduced into a blastula, these cells could give rise to
all tissues of the fetus, including germ cells, indicating that reprogrammed adult cells behaved like
embryonic stem cells.54 This technique will allow generation of stem cells from patient’s own cells,
which can be further manipulated to generate cells of our interest. It also does not require use of
oocytes or embryonic tissue and the use of induced pluripotent cells should therefore be free of ethical
issues. Use of these cells will also avoid graft rejection. Such cells can be induced into photoreceptors
as described49 and then introduced into patients with retinal degeneration to prevent further damage.
At the same time such therapeutic approaches should be used with caution as ES cells have
unlimited potential to self-renewal and introducing these cells in a different environment could
trigger their division through some unidentified factor. Indeed animals generated using induced
pluripotent stem cells had a higher rate of tumor formation.54 Also neural precursors generated
from mouse ES cells led to teratoma formation in 50% mice when injected in mouse retina.55 These
health and safety concerns need to be addressed before application of stem cell therapy.
Bone Marrow Stem Cells

Due to their plasticity, feasibility of using autologous cells and their ability for self-renewal, potential
24 of bone marrow cells for regenerative medicine has been explored extensively for several disorders.
In vivo bone marrow hematopoietic stem cells can differentiate into cells of lymphoid and myeloid
origin while bone marrow mesenchymal cells form endothelial cells, chondrocytes, skeletal muscles
and osteocytes. In addition in vitro they can also be differentiated into cardiocytes, renal cells,
pancreatic cells and hepatocytes. Bone marrow cells can also differentiate into neurons in vitro56 and
as retina is also of neural lineage, the potential of bone marrow cells has been explored for retinal
cell therapy. In addition potential of bone marrow cells for revascularization of retina and for trophic
support for survival retinal cells has also been explored.
Bone marrow cells can differentiate into retinal cells both in vitro and in vivo. When chimeric mice
with hematopoietic cells expressing GFP, were subjected to laser-induced Bruch’s membrane rupture,
GFP-positive astrocytes, vascular endothelial cells and RPE cells were found in the retina.57 Systemically
injected bone marrow stromal cells also migrated to the subretinal space in rats with NaIO¯induced
3
RPE degeneration and expressed RPE65.58 These studies indicate a role for bone marrow cells in
retinal wound repair in vivo and their ability to home the site of retinal injury. CD90+ marrow
stromal cells have also been used to generate retinal progenitor cells using a combination of activin
A, taurine and EGF in vitro.59 After incubation with this cocktail, these cells expressed markers of
photoreceptors such as rhodopsin, opsin and recoverin. When injected into the subretinal space of
RCS rats, these cells integrated into the retina and formed structures similar to photoreceptor layer.
However restoration of cone and rod function by these cells need to be further examined. Bone
marrow stromal cells also acquired RPE-like characteristics when transduced with adenovirus and
co-cultured with RPE cells.60 When injected subretinally into RCS rats, these cells integrated into
host RPE and prevented further photoreceptor degeneration of RCS rats for up to two months after
injection. This effect was enhanced when the stromal cells were transduced with PEDF prior to
injection, suggesting a role for PEDF in this trophic support by stromal cells.60
Interestingly bone marrow cells can also rescue retinal degeneration by contributing to angiogenesis
in the retina. Lineage negative (Lin¯) hematopoietic cells contain a population of endothelial cells.61
Intravitreally injected Lin¯ cells incorporated into the retinal vasculature of neonatal mice and also
in the vasculature of injury-induced angiogenesis in adults.62 Importantly these cells could also rescue
retinal vascular degeneration in rd1 and rd10 mice, which are models of retinitis pigmentosa, and
which in turn could rescue photoreceptors.63 The vasculature preferentially rescued cones from
degeneration and this was accompanied by increased expression of anti-apoptotic genes. A detectable,
although abnormal, electroretinogram recording was also observed in injected mice. Retinal
vasculature thus appears to be an important regulator of retinal integrity and restoration of retinal
vasculature could be a feasible approach for treating retinal degeneration.
CNS Stem Cells

In the adult central nervous system, stem cells and neural progenitors exist in the areas like ventricular
zone, subventricular zone and hippocampus. As retina originates from CNS and retinal precursors
develop from neural progenitors, it is logical to use neuronal stem cells (NSCs) for retinal repair.
NSCs from hippocampus were able to incorporate into mechanically injured rat retina, although
these cells differentiated into neuronal cells but not into photoreceptors. 64 However in
xenotransplantation of neonatal murine brain progenitor into opossum the murine cells were able to
incorporate in opossum retina and expressed neuronal and retinal markers.7 Adult hippocampal
neural progenitors cells have also been transplanted in opossum at different stages, from postnatal
to adult, and these studies show that survival and integration of NPCs was influenced by age of the
recipient and maximum integration was observed in the developing retina.65 These studies indicate 25
that the retina itself can provide the correct cues for differentiation of neuronal cells into retinal cells
till a certain stage of development. The studies carried out using NSCs so far do not provide any
evidence for functional rescue of retinal cells during retinal degeneration.
Cord Blood Stem Cells

Cord blood stem cells are another attractive source of stem cells that can be explored for regeneration
of retinal cells. Cord blood cells are easily available and can be cultured in vitro for long periods.
Like bone marrow cells, they are multipotent and can differentiate into a variety of cell types.66 Most
importantly they can differentiate into neurons.66, 67 Recently lineage-negative cord blood cells were
also reported to undergo differentiation in vivo to generate cells expressing retinal markers, when
they were injected into subretinal space in mice.68 Further studies need to be carried out to investigate
whether these cells can integrate and generate functional equivalents of retinal cells.
Future Perspectives
Cell therapy holds great promise as potential therapeutic approach for treatment of retinal disorders.
Studies carried out so far provide evidence that stem cells from a number of sources have the
potential to differentiate into cells with retinal phenotype. These cells when transplanted in animal
models can also survive and integrate into the retina in some cases but the evidence for restoration
of retinal function is available only for a few of these studies. This suggests that more stringent tests
investigating function of retinal cells should be performed in addition to investigating the expression
of various retinal markers. Exactly which characteristics of transplanted cells and recipient’s retina
are important for functional integration is not clear yet. But fetal and neonatal retinas probably hold
the answers to these questions. Fetal/neonatal retinas provide both progenitor cells that can restore
some visual function when transplanted into a degenerating retina and a niche that can differentiate
stem cells into retinal cells. The challenge ahead of us is to generate retinal cells, which are at the
appropriate stage of differentiation so that they are capable of restoring retinal function. At the
same time caution should be exercised to ensure the safety of these cell therapies and to avoid
undesirable outcomes of cell therapy. With the advances in stem cell biology, it is also now possible
to generate patient-specific pluripotent stem cells that are capable of generating all types of cells, if
the correct milieu is provided. It remains to be seen whether these cells can be coaxed in vitro to
differentiate into retinal cells that can integrate and restore retinal function.
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29
Introduction
As the diabetic population burgeons globally, so will the unusual presentations of the disease become
increasingly manifest. The retinopathy of diabetes will be no exception to this rule. This essay attempts
to chronicle the various atypical manifestations of diabetic retinopathy (DR) hitherto reported. The
protean manifestations can be due to the inherent variability of DR, systemic and ocular diseases
that may modify its course or as a result of exceptional novel features.
The Inherent Variability of Diabetic Retinopathy

FLORID DIABETIC RETINOPATHY
A rare complication, occurring in less than 1% of cases of proliferative diabetic retinopathy (PDR),
florid DR is a rapidly progressing, bilateral, severely ischemic retinopathy with poor vision that
usually occurs in young, type 1 poorly controlled diabetic patients. 1-3 Extensive panretinal
photocoagulation with early vitrectomy when mandated will help improve their visual outcome.2,3
FEATURELESS RETINA
Featureless retina is a unique, rare type of PDR wherein retinal neovascularization may present
without the characteristic preproliferative background retinal lesions such as microaneurysms,
hemorrhages, cotton-wool spots and intraretinal microvascular abnormalities. The absence of these
features may be explained by the presence of extensive areas of capillary non-perfusion seen in these
cases which leads to the disappearance of these lesions in these severely ischemic zones.4 Although
it may appear atrophic at first glance, fluorescein angiography may reveal undetected areas of
neovascularization with extensive capillary non-perfusion (Figures 3-1A and B).
FIGURES 3-1A AND B: (A) Fundus photograph of an eye that apparently looks like a case of mild nonproliferative
diabetic retinopathy. (B) Fluorescein angiogram of the eye reveals extensive capillary nonperfusion and leakage from
neovascular tissue
PERIPHERAL ABNORMALITIES IN DIABETIC RETINOPATHY

Occasionally, cases of diabetic retinopathy (2-3%) show predominant involvement of the periphery
32 with relative sparing of the posterior pole.5,6 Such patients are at risk of developing peripheral
Other documents randomly have
different content
— Voinhan kantaa vettä ja halkoja, ellen pysty lattioita pesemään.
Kun ihmeellinen seurue astui sisään ovesta, riensi Plotina, joka

ikkunasta oli nähnyt tulijat, vastaan.
— Mitä te aiotte? kysyi hän aivan ymmällään.
— Me tulemme pitämään suursiivousta, ettei Sohvi-täti toru sinua,

sanoi Kaarina päättävästi.
— Mutta isä ei pidä siistimisestä. Sinä tiedät, Kaarina, kuinka

hyväluontoinen isä on, mutta vain pölyriepua nähdessäänkin hän
ihan vimmastuu, ja tuulettaminen on hänelle kauhistus. Hän väittää,
että naiset ovat keksineet suursiivoukset ja muut sellaiset hommat
vartavasten kiusatakseen miehiä.
Manne ratkesi ulvovaan nauruun, mutta Kaarina heitti ikkunan

tarmokkaasti auki sanoen: — Kyllä hän nyt tällä kertaa saa sen
koetuksen joka tapauksessa kestää. Onko hän kotona nyt?
— Ei, hän lähti kävelyttämään Klioa.
— Ja kauanko hän tavallisesti viipyy sillä matkallaan?
— Tunnin tai pari.
— Hyvä. Me ryhdymme heti toimeen. Manne, tee tuli takkaan ja

kanna vettä kattilaan. Sinä, Sikke, pölytät huonekalut. Plotina siistii
pöydän ja minä kirjahyllyn. Rivakasti toimeen. Pidä sinä, Plotina,
silmällä, milloin isäsi palaa kävelymatkalta ja anna varoitusmerkki
meille.
Nopeasti kuin henki olisi ollut kysymyksessä ryhtyi kukin
määrättyyn tehtäväänsä. Sikeitä ja Kaarinalta sujui työ helposti.
Sikke oli luonnostaan käytännöllinen, ja Kaarina oli usein auttanut
äitiään isän työhuoneen järjestämisessä, mutta Plotina rukka, joka
oli varsin vasenkätinen kaikessa, mikä koski jokapäiväisiä askareita,
ja sen lisäksi pelkäsi isänsä yllättävän heidät, teki kaikki nurinpäin ja
sai tuskin mitään aikaan. Manne hääri nenä noessa keittiössä ja
helisteli hellanrenkaita, niin että olisi luullut melun kuuluvan toiselle
puolen kylää.
— Vesi on lämmintä, ilmoitti hän, joutukaa pesemään lattiaa. Olen

kuulevinani Klion määkinän.
Tytöille tuli hätä käteen. Plotina kaatoi likinäköisyydessään

mustepullon papereille ja oli vaipua maahan kauhistuksesta.
— Älä nyt hätäile, sanoi Sikke leveällä, tyynellä tavallaan ja pisti

paperit piiloon. Isäsi on niin hajamielinen, että luulee itse
kaataneensa mustepullon.
— Voi voi, enhän minä saata isää pettää, vaikeroi Plotina, en ole
vielä koskaan pettänyt häntä.
Kaarina latoi kuumeentapaisesti kirjat paikoilleen, ja Sikke pesi

polvillaan lattiaa otsa hiessä.
— Puhtaaksi minä tämän nyt luutuan, vaikka sata maisteria olisi

tulossa, hän vakuutti rauhallisesti.
Manne pujahti ulos ja houkutteli maisterin kasvitarhaan

näyttämään hänelle komeita kaalinkupujaan. Ukko teki tuon
tuostakin lähtöä sisään, mutta aina poika keksi jotakin uutta
katselemisen aihetta kasvitarhassa. Viimein, kun kaikki porkkanat,
kurpitsalavat ja punajuuret oli moneen kertaan katsottu, ei Manne
enää keksinyt mitään syytä pidättääkseen historioitsijaa. Silloin hän
epätoivoissaan salaa päästi Klion irti ja kului hetken aikaa, ennenkuin
maisteri ja hän saivat sen kiinni.
— Pelastakoot tytöt nyt nahkansa. Enempää en minä voi, mutisi

Manne ja juoksi ketterästi edellä avaamaan ovea ukolle.
Tytöt olivat kuulleet heidän askelensa ja päätä pahkaa

pelastautuneet Plotinan huoneeseen. Maisteri Saramaa liikkui
epäluuloisen näköisenä huoneessaan haistellen ilmaa. Se oli
raittiimpaa kuin tavallisesti, hän oikein kaipasi rakasta tupakansavua,
ja muutoinkin — huone näytti niin merkillisen oudolta. Mutta Manne
seurasi hänen kintereillään ja puhua pakisi lakkaamatta, niin että
ukko ei ennättänyt tehdä huomioitaan rauhassa. Ja hetken aikaa
muristuaan hän unohti epäluulonsa syventyen kirjoihinsa.
Sillä aikaa tytöt häärivät muissa huoneissa. Kanttorin Alma oli

ikkunasta huomannut heidän oudot puuhansa eikä voinut hillitä
uteliaisuuttaan, vaan pujahti naapurin portista sisälle. Sikeltä, joka
oli tyhjentämässä likavesiämpäriä, hän sai kuulla tyttöjen puuhista ja
tarjoutui heti apulaiseksi.
— Sinä olet näppärä käsitöissä, sanoi Kaarina, parsipas nuo reiät

sohvan päällisessä ja paikkaa kiikkutuolin matto. Tuosta tuolista
pistää jouhet esille, korjaa sekin.
Alma juoksi kotiin noutamaan lankaa ja neulaa, sillä Plotina oli niin
hämmennyksissään kaikesta siitä väkivaltaisuudesta, mitä hänen
kodissaan harjoitettiin, ettei löytänyt omia ompeluvehkeitäänkään.
Palatessaan Alma toi mukanaan pari pöytäliinaa ja puhtaan
päällyspeitteen.
Ahkerissa käsissä sai pieni puutteellinen koti pian aivan toisen

ulkonäön. Ikkunaverhot parsittiin ja pölytettiin, pöydillä oli Alman
tuomat sievät liinat, Plotinan vuoteelle, jonka hän aikoi luovuttaa
tädilleen, levitettiin puhdas päällyspeite. Vaatekomerosta Kaarina
löysi pienen sievän maton.
— Onko tämäkin tädin kangaskaupasta, kysyi hän nostaen mattoa

Plotinan nähtäväksi.
— On.
— Se kelpaa mainiosti vuodematoksi. Minä levitän sen tähän sinun

vuoteesi eteen.
Manne lähetettiin noutamaan voileipiä, tytöillä oli niin kiire,

etteivät ennättäneet käydä syömässäkään. Iloinen puheensorina
kuului maisterin huoneeseen, ja työssään häiriintyneenä tämä
pistäysi katsomassa, mitä oli tekeillä. Ihmeekseen hän huomasi
oudon näyn. Huone, jossa vain aniharvoin oli muita kuin sen arka
emäntä, oli nyt täynnä hilpeitä vieraita. Sikke seisoi pöydällä vasara
kädessä ja nauloja suussa ja viritti verhoja ikkunan eteen. Manne
istui hajasäärin lattialla kiilloittaen kynttiläjalkoja, Alma oli polvillaan
sohvan edessä parsinneula kädessä, ja Kaarina hieroi mustepilkkuja
pöydästä, tuolilla oli lautasellinen voileipiä ja pari kylmää perunaa.
Maisteri miltei vaaleni kauhusta nähdessään tätä kotirauhan

häiritsemistä.
— Mitä tämä oikein merkitsee? sopersi hän luoden nuhtelevan
katseen hämmentyneeseen tyttäreensä.
— Me tulimme vähän auttamaan Plotinaa, kun kuulimme, että

saisitte vieraita, sanoi Kaarina iloisesti.
— Onko se nyt välttämätöntä tämmöinen siistiminen, minusta

täällä oli siistiä kyllä ennenkin?
— Auta armias! pääsi Siken vilpittömiltä huulilta. Täällähän oli kuin

———
— Tuollaiset vanhat rouvat ovat tavallisesti vähän turhantarkkoja,

kiirehti Kaarina keskeyttämään häntä, ja kun Plotinalla on niin paljon
tärkeämpää työtä, tarjouduimme hänelle apulaisiksi. Häiritsemmekö
setää?
— Ette erikoisesti, mutisi maisteri ja vetäytyi takaisin

huoneeseensa harmitellen itsekseen hameväen ihmeellistä halua
aikaansaada turhanpäiväisiä selkkauksia.
— Kas niin, sanoi Kaarina luoden viimeisen tarkastavan silmäyksen

ympärilleen, nyt täällä luullakseni alkaa olla järjestyksessä. Vai mitä
arvelette tytöt?
— Oi, täällähän on ihmeen hienoa, huudahti Plotina kiitollisena.
— Jotakuinkin siistiä, kuului Alman arvostelu.
— Mutta eikö teidän mielestänne ole epärehellistä, että meidän

kotimme näyttää aivan erilaiselta, kuin mitä se arkioloissa on? kysyi
Plotina arasti.
— Ei laisinkaan, vakuutti Sikke, ainahan siistitään vieraita varten.
— Ja sitäpaitsi, sanoi Kaarina, voit päättää, että tästälähin pidät

kotisi siistinä.
— Luuletko minun siihen kykenevän? kysyi Plotina avuttomana.
— Miksikä et, uhraa joka päivä parikin tuntia kodin järjestyksessä

pitämiseen, sen kyllä voit tehdä, vaikka oletkin lukutoukka.
— Minä koetan, mutta minulta käy kaikki niin hitaasti ja

takaperoisesti, huokasi Plotina.
Kun tytöt olivat lähdössä, veti Kaarina Plotinan erikseen ja kysyi:
— Suo anteeksi, että sekaannun asioihinne, mutta haluaisin niin

mielelläni olla sinulle avuksi, ja sen vuoksi kysyn: mitä aiot antaa
tädillesi ruuaksi?
— Onhan meillä perunoita, vihanneksia ja vuohenmaitoa.
— Hyvä, mutta osaatko sinä keittää?
— Olenhan minä keittänyt meidän kaikki ateriamme.
— Mitä arvelet siitä, jos minä huomenna tulisin auttamaan sinua

päivällisen valmistamisessa? Rouva Tulla päästää kyllä minut tunniksi
tai pariksi, hän on nykyjään hyvin kiltti, ja he lähtevät huomenna
koko perhe nimismiehelle Klaaran päiville.
— Voi, Kaarina, kuinka sinä olet hyvä. Kyllä minä olisin iloinen, jos
tulisit, pelkään, ettei Sohvi-täti ole niin vaatimaton ruuan suhteen
kuin isä. Minulta usein ruoka palaa pohjaan tai unohtuu suola.
Tohtorinna Holstin mielestä oli välttämätöntä, että tytöt rinnan
lukujen kanssa oppivat käytännöllisiä toimia. Kaarina oli siis tottunut
liikkumaan keittiössä, vaikkei hän mikään ensiluokan kokki ollutkaan.
— Minä arvelen, sanoi hän seuraavana päivänä istuessaan

Mökissä, keittiön puulaatikolla, että keitämme vihanneslientä,
sipulilaatikkoa ja korppuvanukasta, minä osaan näet parhaiten
valmistaa näitä ruokalajeja, sillä ne olivat isän mieliruokaa.
— Niin, ja ajattelepas, Alma toi tänään tänne hauen, jonka Freedu

oli saanut verkolla. Eikö se ollut kiltisti tehty?
— Olipa kyllä. Laitamme siis haukea munakastikkeen kera.
Tytöt riensivät puutarhaan poimimaan liemeen tarvittavat ainekset.

Plotina oli hermostunut, hän pelkäsi täsmällistä tätiään ja oli
tavallista kömpelömpi. Kaarina ihmetteli mielessään, olisiko rouva
Simola laisinkaan saanut päivällistä sinä päivänä, ellei hän olisi tullut
auttamaan.
— Kuulepas nyt, Plotina, sanoi hän, kun kello läheni neljää.

Pelkään pahoin, että tulette myöhään, ellette jo mene laivalle. Ja se
olisi ikävä. Ihmiset joutuvat aina pahalle tuulelle, ellei kukaan ole
heitä vastassa, kun he saapuvat vieraalle paikkakunnalle.
— Plotina, Plotina, kuului sivuhuoneesta hätäinen ääni, ja maisteri

pisti ovesta pörröisen päänsä. Minä en löydä kaulusta enkä
kalvosimia. Olen etsinyt niitä kaikkialta, yksin paperikoristakin.
Plotina riensi hätään. Kaarina kuuli piironginlaatikkoja vedettävän

ja suljettavan ja vähän väliä hermostunutta torumista: — Se on sitä
naisväen komentoa. Järjestetään, järjestetään, ja seuraus on, ettei
mitään löydy. Nähtävästi kadonneet esineet kumminkin lopuksi
löydettiin, sillä jonkin ajan kuluttua maisteri ilmestyi esiin yllään
musta kulunut sortuutti, hyvin vanhanaikainen kaulus kaulassaan ja
tukka harjattuna.
— Kyllä he nyt myöhästyvät, tuumi Kaarina pistäessään

korppulaatikon uuniin. Luulenpa, että jään tänne katsomaan, kuinka
Plotina raukka suoriutuu leikistä. Voinhan olla muka Plotinan
apulainen, niin en häiritse ketään läsnäolollani. — Hän sieppasi
naulasta Plotinan huivin, jota tämä käytti vuohta lypsäessään.
Sidottuaan sen päähänsä ja ison esiliinan vyölleen hän aivan hyvin
saattoi käydä pienestä sievästä palvelustytöstä. Iloisesti hyräillen hän
nosti patoja ja kattiloita. Sen ohessa hän kattoi pöydän kolmelle
hengelle ja poimi puutarhasta kukkia saadakseen päivällispöydän
niin hauskan näköiseksi kuin niillä pienillä apukeinoilla saattoi, mitä
hänellä oli käytettävinään.
Hän oli juuri kyyryllään uunin edessä katsoakseen, oliko vanukas

jo kypsä, kun keittiön ovi kuului aukeavan. Katsahtaessaan taakseen
hän huomasi lihavahkon, englantilaismalliseen kävelypukuun puetun
keski-ikäisen naisen, joka matkalaukku vasemmassa ja sateenvarjo
oikeassa kädessä seisoi ovella.
Kaarina kohosi seisaalleen ja niiasi syvään.
— Eikö herrasväki ole kotona? kysyi rouva äreästi.
— He lähtivät laivasillalle maisterin kälyä vastaan, sanoi Kaarina ja

niiasi uudestaan. Myöhästyivät tietysti ja menivät toista tietä, ajatteli
hän itsekseen.
Rouva ojensi käskevällä liikkeellä matkalaukkunsa Kaarinalle.

— Vie tämä makuuhuoneeseeni. Missä teidän eteisenne on?
— Rouva on hyvä ja seuraa minua, sanoi Kaarina nöyrästi. Häntä

alkoi huvittaa palvelustytön osa.
Hän auttoi päällystakin rouvan yltä ja vei hänet Plotinan

huoneeseen.
Tarkastelevin silmin mittasi rouva Simola huonetta.
— Hm, sanoi hän puoliääneen, tytöllä näkyy olevan verraten siistiä

ja puhdasta — tuopas minulle lasi vettä, huusi hän keittiöön.
Kun Kaarina oli ojentanut hänelle vesilasin, kysyi rouva Simola:
— Oletko ollut talossa kauankin?
— En, en kauan, vastasi Kaarina. Samassa juolahti hänen

mieleensä, että hän ehkä saattoi tehdä ystävilleen palveluksen. Hän
lisäsi siis empien ja nöyrästi: — Mutta minä pidän paljon neidistä ja
maisterista, on vain niin ikävä, että heillä on niin paljon vaikeuksia.
Rouva Simola loi tyttöön paheksuvan katseen.
— Minun mielestäni on nenäkästä ja sopimatonta, että sinä

sekaannut isäntäväkesi asioihin.
— Niinpä kyllä, myönteli Kaarina nöyrästi, mutta minä pidän heistä

paljon, ja mieltäni pahoittaa, kun näen, miten heidän täytyy taistella
puutetta vastaan, vaikka heillä on rikkaita sukulaisia.
— Jos sinä olet niin perehtynyt isäntäväkesi asioihin, kuin tahdot

näyttää, tiedät ehkä myös, että heidän niin kutsutut vaikeutensa
ovat suureksi osaksi omaa syytä. Eräs läheinen sukulainen tarjoutui
pitämään huolta Plotinasta, jos hän olisi suostunut muutamiin varsin
järkeviin ehtoihin.
— Nämä ehdot saattoivat kyllä olla järkeviä, mutta oikeamielisiä

ne eivät olleet. Tuon läheisen sukulaisen olisi pitänyt käsittää, että
tyttäret eivät mielellään eroa isästään, ja muutoinkin olivat ehdot
tuiki sopimattomat Plotinan luonteelle. Jos minä olisin ollut tuo
läheinen sukulainen, olisin auttanut maisteria ja hänen tytärtään
tavalla, joka heillekin olisi ollut mieluinen. Kaarina oli puhunut
innokkaasti, mutta vaikeni äkkiä hämillään. Rouva Simola katseli
häneen terävästi.
— Oletko sinä talon palvelustyttö.
— E-e-n, apulainen vain.
— Joka tapauksessa en ole halukas keskustelemaan kanssasi niin

arkaluontoisesta seikasta kuin isäntäväkesi toimeentulosta.
Kaarina niiasi.
— Kuten suvaitsette, rouva. Hän poistui keittiöön juuri parahiksi

estääkseen kastikkeen pohjaanpalamista.
Muutaman minuutin kuluttua saapuivat maisteri ja Plotina. He

olivat sekä hämillään että pahoillaan ja vastaanottivat onnettoman
näköisinä ankaran sukulaisensa esitelmän aiheesta: mitä
täsmällisyyden puute ja saamattomuus saavat aikaan.
— Se on näetkös, Kristoffer hyvä, ollut sinun elämäsi suuri virhe,

ettet koskaan ole ollut täsmällinen. Ja mihin se on johtanut? Täällä
sinä nyt istut viheliäisessä maalaiskylässä yksinäisenä ja
unohdettuna ja pengot vanhoja kellastuneita papereita sen sijaan,
että olisit opettajana jossakin arvossapidetyssä oppilaitoksessa tai
ehkäpä yliopiston professorina. Toiset sinua typerämmät ovat
päässeet kiipeämään paljon korkeammalle.
— Sinä olet, Jumala paratkoon, aivan oikeassa, Sohvi-käly, huokasi

maisteri ja näytti onnettomalta kuin koulupoika rehtorinsa edessä.
— Rakas täti, oikeastaan se oli minun syyni, että myöhästyimme,

uskalsi Plotina kohottaa ääntään isän puolustukseksi.
Rouva Simola kääntyi äkkiä tyttöön.
— Sen pahempi, sinun pitäisi olla isääsi ymmärtäväisempi. Mutta

sinä tietysti istuit myöskin nenä kirjassa. Ei kai tässä talossa muuta
tehdäkään kuin luetaan. Ja tuo kauhean nenäkäs palvelustyttö tai
apulainen lukee kai myöskin romaania keittiössä, koska ei
päivällinenkään ole valmis. Olen tietysti nälissäni matkustettuani viisi
pitkää tuntia epämukavassa ja likaisessa laivassa.
Plotina riensi pelästyneenä keittiöön.
— Kutsu vain pöytään, ruoka on valmis, sanoi Kaarina.
— Etkö sinä tule syömään?
— En, minä olen tällä kertaa vain sinun keittiöapulaisesi.
Plotina koetti estellä, mutta Kaarina pisti ruokatarjottimen hänen

käteensä. — Kas niin, ei mitään vastaväitteitä, mene sisään nyt vain.
Plotina totteli hämmentyneenä. Sohvi-tädin tuima katsanto lauhtui,

kun höyryävä vihannesliemi ilmestyi pöytään. Mielihyvällä hän totesi
sen olevan varsin maukasta. Syötyään vielä aimo annoksen kalaa ja
jälkiruokaa hän tyytyväisenä siirtyi juomaan kahvia Plotinan
huoneeseen, jonne Kaarina oli kattanut sievän kahvipöydän.
— Myönnän, sanoi Sohvi-täti viedessään höyryävää kahvikuppia

huulilleen, ettei tämä teidän kotoinen järjestyksenne sentään ole niin
perin nurinkurinen, kuin miksi sitä olin kuvitellut. Ehkäpä sinusta
vielä tulee ihminen, Plotina. Kunpa sinulla vain olisi toinen nimi! En
ymmärrä, kuinka äitisi saattoi suostua tuohon hirvittävään
pakanalliseen nimeen. Plotina, miksi ei yhtä hyvin platina tai tina.
— Anteeksi, käly rakas, huomautti maisteri säveästi, Plotina oli

älykäs roomalainen keisarinna, ja hänen mukaansa onkin
tyttäremme saanut nimensä.
— Loruja, minä en näe mitään järkeä siinä, että lapsille pannaan

pakanallisten keisarinnojen nimiä. Ja onko sinusta sitten hänessä
mitään ruhtinaallista? Minun mielestäni hän on pieni mitätön
tyttölapsi. No niin, onnettomuus on nyt kerran tapahtunut, ja lapsi
raukka on itse siihen viaton.
Kaarina naureskeli itsekseen keittiössä, missä hän pesi astioita,

Sohvi-tädin puheille. Olipa vahinko, ettei hän enää uskaltanut mennä
hänen kanssaan juttelemaan. Plotinan täti näytti varsin rehdiltä
ihmiseltä. Hän asetti astiat kaappiin ja viittasi Plotinan keittiöön.
— Minun täytyy mennä, Tullan väki on jo ehkä palannut kotiin.

Pidä hyvä huoli illallisesta ja muista kaikin mokomin tehdä vuode
mukavaksi. Toivoisinpa, ettet näyttäisi noin apealta.
— Sohvi-täti on niin peloittavan tarkkanäköinen, kuiskasi tyttö,

huomasin hänen ainakin viisi minuuttia tarkastavan erästä
mustepilkkua pöydässäni.
— Ehkä hän on parempi kuin miltä näyttää, lohdutti Kaarina
ystäväänsä.
Kaarina olisi mielellään seuraavana päivänä pistäytynyt Mökillä

kuulustelemassa, miten asiat siellä luistivat. Mutta hän ei päässyt
tunneiltaan, oli luettava entistä ahkerammin, ehtojen suoritusaika
läheni. Maunoa ja Erkkiä hän ei tavannut kuin kerran, silloinkin vain
pikimmältään laivalaiturilla, kun he olivat poislähdössä. Veljekset
pyysivät häntä hartaasti kotiinsa heidän kanssaan.
— Jätä koko Tullan joukko ja tule pois meille, houkutteli Erkki. Äiti
ottaa sinut avosylin vastaan, meillähän ei ole tyttöjä koko talossa,
kaksi poikaa vain. Pidämme sinua kuin kukkaa kämmenellä.
— Etkö todella voisi lähteä mukaamme, ehdotti Maunokin. Me

voisimme siinä tapauksessa jäädä tänne pariksi päiväksi, kunnes olet
lähtövalmis.
Kaarina pudisti surumielisesti hymyillen päätään. — Kiitos, ystävät,

mutta se on todella mahdotonta; en voi jättää oppilaitani pulaan. Se
olisi väärin, eikö totta, Mauno?
— Olet ehkä oikeassa, mutta tule sitten, kun olet heistä päässyt.
Laiva vihelsi, ja pojat heittivät hyvästit. Kaarina katsoi ikävöiden

poistuvan Ainamon jälkeen. Koskahan koitti sekin aika, jolloin hän
pääsisi lähtemään? Oli enää pari viikkoa lukukauden alkuun.
Illalla hän sai kaksi kirjettä postista. Toinen oli kirjoitettu suurin,
miehekkäin kirjaimin ja kuului seuraavasti:
"Sohvi Simola pyytää ilmoittaa Plotinan keittiöapulaiselle,

että tuo läheinen sukulainen on huomannut viisaimmaksi
antaa jokaisen seurata omaa taipumustaan, eikä tahdo
pakottaa ketään vieraalle alalle. Ja rauhoittaakseen
hyväsydämisen apulaisen huolia hän ilmoittaa, että Plotinan ja
hänen isänsä vaikeuksia tullaan huojentamaan. Samalla
ehdottaa Sohvi Simola, että nuori keittiöapulainen, kun hän
vasta tahtoo auttaa ystäviänsä, näyttelisi omaa osaansa.
Suorin tie paras."
Kaarina punehtui sekä ilosta että mielipahasta. Rouva Simola oli

siis keksinyt hänen pienen sotajuonensa, Plotina oli tietenkin
ilmaissut hänet. Ja hän oli oikeassa — suorin tie on paras. Oliko hän
mahtanut hyvin paheksua Kaarinaa? Ehkäpä pitää häntä petollisena?
Mitähän äiti ja isä mahtavat sanoa, kun saavat tästä kuulla? Isä on
niin tinkimätön, mitä oikeaan ja väärään tuli. — Huh, oikein poskia
poltti!
Mutta se oli kuitenkin ihanaa, että Mökin asukkaat olivat päässeet

ainakin toistaiseksi rahahuolistaan. Sohvi-täti oli varmaankin kelpo
ihminen.
Hän avasi toisen kirjeen, se oli äidiltä.
Rakas Kaisu-tyttöni, sanottiin siinä. Vihdoinkin olemme

päässeet niin pitkälle, että uskallamme lähettää sinulle
ilosanoman, jota jo pari viikkoa olemme salanneet, koska
olemme pelänneet sinua turhilla toiveilla pettävämme. Tänä
aamuna ilmoitti laitoksen ylilääkäri, että isä nyt on terve.
Viikon kuluttua saatamme lähteä kotiin päin matkustamaan
rakkaiden lastemme luo. Isä liikkuu jo ilman sauvaa, joskin
vielä jossakin määrin kankeasti. Käsiään hän liikuttaa aivan
vapaasti. Mieli on reipas ja valoisa. Tohtori arvelee, että hän
huoleti voi ryhtyä työhönsä jälleen. Sinä arvaat, kuinka
onnellinen isä on. Sillä juuri toimettomuus häntä on
kiusannut; ja pelko siitä, että me joutuisimme kärsimään
hänen pakollisesta työttömyydestään, on vaikuttanut hänen
tautiinsakin.
Saavumme Suomeen noin 24. päivän paikkeilla tätä kuuta.

Silloin toivomme tapaavamme sinut ja lapset Lanterissa.
Saamme viettää vielä kokonaisen ihanan viikon yhdessä,
ennenkuin sinä ja Kati lähdette kouluun. Olemme nimittäin
päättäneet isän kanssa lähettää Katinkin tänä syksynä
maailmalle.
Tervehdimme rakasta tyttöämme häntä hellästi syleillen.
Äiti ja Isä.
Kaarina vaipui tuolille istumaan ja kätki kasvot käsiinsä.
— Jumala, kuinka hyvä sinä olet, kuiskasi hän. Minä saan siis
nähdä isän ja äidin, Lanterin ja kaikki viikon kuluttua.
Hän kavahti ylös ja syöksyi suin päin alas portaita. Siken, joka
arkihuoneessa parhaillaan järjesti vastapestyjä vaatteita kaappiin,
hän oli syleilyllään rutistaa.
— Sinäpäs — muuta ei Sikke saanut sanotuksi.
— Äiti ja isä palaavat kotiin viikon kuluttua, riemuitsi Kaarina. Ja

minä pääsen Lanteriin.
— Kylläpä sinusta on hauska lähteä meiltä pois, sanoi Manne

hiukan katkerasti. Eipä sinulla tosiaan ole täällä kissanpäiviä
ollutkaan.
— Olisit jäänyt siihen asti, kun koulut alkavat, niin olisimme

matkustaneet yhdessä, virkkoi Iisa.
— Tehän lähdette kuitenkin jo viikon lopulla ehtoja suorittamaan.

Mitä minä täällä sitten enää tekisin!
Iloissaan ei Kaarina malttanut pysyä sisässä. Hän houkutteli tytöt

mukaansa kävelylle. Maantiellä he tapasivat Plotinan.
— Kaarina, minulla on niin paljon sinulle kertomista. Etkö tule

meille?
— Kävellään mieluummin. Saatetaan tytöt kotiin, niin tulen sitten

saattamaan sinua. Minullakin on uutisia.
Kaarina näytti Sohvi-tädin kirjettä.
— Sinä tietysti ilmaisit minut.
— Niin, enhän voinut muutakaan, kun hän kysyi nimeäsi.

Nähtävästi hän epäili, ettet ollut mikään tavallinen apuihminen.
Kaarina, täti ei ollut lainkaan niin peloittava kuin luulin. Me istuimme
illalla kahden minun huoneessani, hän kertoi minulle paljon äidistäni.
Luulen nähneeni kyyneliä hänen ankaroissa silmissään. Sitten hän
rupesi kyselemään olojamme ja teki sen niin lyhyesti ja täsmällisesti,
että minun ei auttanut kierrellä, totuus tuli ilmi kaikessa
alastomuudessaan. "Herranen aika, lapsi", huudahti hän silloin,
"miksi et ole minulle mitään kaikesta tästä kirjoittanut? Olen itsekäs
vanha nainen, mutta niin kovasydäminen en sentään ole, että
antaisin ainoan sisareni lapsen kärsiä puutetta. Mene nyt
nukkumaan, minä tahdon lähemmin ajatella asiaa." Seuraavana
aamuna hän piti pitkän nuhdesaarnan meille
epäkäytännöllisyydestämme, mutta lopuksi hän ilmoitti päättäneensä
varata minulle kuukausirahan sillä ehdolla, että käyttäisin sen
taloudellisiin menoihin enkä joutaviin kirjoihin ynnä muihin
hassutuksiin, kuten hän lausui. — Koko päivän hän sitten oli oikein
leppeä ja ystävällinen, isä raukallekin. Luulenpa melkein hänen
hiukan pitäneen minusta, lopetti Plotina kertomuksensa. Eihän se ole
ihme, lisäsi hän vaatimattomasti. Olihan äitini hänen sisarensa. Ja
ajatteles, lähtiessään hän pisti kouraani sadanmarkan setelin. Eikö se
ole suurenmoista, minulla ei koskaan ole ollut hallussani kymmentä
markkaa suurempaa rahaa.
Plotinan kasvot hehkuivat mielihyvästä. — Mutta minun täytyi

kertoa hänelle siitä suursiivouksestanne. Hän olisi muutoin luullut
minua paremmaksi kuin mitä olen. Hän nauroi sydämellisesti
puuhillenne.
Plotinan päätettyä kertomuksensa toi Kaarina esille iloiset

uutisensa.
— Lähdetkö niin pian? sanoi Plotina apeasti. Ja sitten minä en

ehkä koskaan enää saa sinua nähdä.
— Tottahan, lohdutti häntä Kaarina. Sinun täytyy ensi kesänä tulla

meille.
— Tiedäthän, etten voi jättää isää.
— Hän tulkoon mukaan.
— Hän ei kahteenkymmeneen vuoteen ole matkustanut täältä

minnekään. Mutta tietysti iloitsen siitä, että sinä pääset omiesi luo ja
että isäsi on terve. Sinä olet ollut isälle ja minulle kuin päivänsäde
varjossa kasvaville kukille.
He astuivat elokuun hämärässä vastaleikattujen ruispeltojen

sivuitse, kotiin palaavan karjan kellot kilahtelivat leppoisasti,
lähestyvän syksyn tuntua oli ilmassa. Heille itselleen käsittämätön
alakuloisuus valtasi tuokioksi tyttöjen mielen. He erosivat
kyynelsilmin ymmärtämättä miksi.
*****
Viimeinen ilta oli käsissä. Tullan nuoret olivat palanneet kotiin

suoritettuaan ehtonsa. Kunnallisneuvos oli tyytyväinen ja rouva
hyvillään. Tytöt olivat kutsuneet ystävänsä viettämään Kaarinan
lähtöiltaa. Pentti oli tuonut hänelle kukkakimpun ja Hilja pussillisen
piparkakkuja matkaeväiksi. Rouva Tulla oli juhlallisesti kiinnittänyt
hänen rintaansa hopeasoljen, jossa oli enemmän metallia kuin aistia.
— Kunnallisneuvokselta ja minulta pieni muisto, sanoi hän

arvokkaasti. Kaarina kiitti liikutettuna. Rouva Tulla oli tuottanut
hänelle monta karvasta hetkeä, mutta nythän oli kaikki ohi. Äiti
kullan sylissä unohtuisi kaikki paha ja ikävä. Hän ei tuntenut mitään
kaunaa kunnallisneuvoksetarta kohtaan.
Plotina oli kutsuttu toisten mukana. Hänellä oli uusi kaunis puku.
Kaarina arvasi sen olevan Sohvi-tädin lahjoittaman.
— Jäännöspalojen aika on onneksi ohi, kuiskasi hän

veitikkamaisesti
Plotinalle.
— Isän viimeinen kirjoitus on hyväksytty arvossapidettyyn
aikakauskirjaan, kuiskasi Plotina vastaukseksi. Hän on ylen
onnellinen.
Vieraiden lähdettyä istuivat nuoret vielä hetkisen yhdessä.
— On niin ikävä, kun sinä lähdet, Kaarina, sanoi Sikke. Eikö totta,
Iisa, me olemme paljon pitäneet Kaarinasta.
Iisa nyökäytti päätään.
— Soisin, että olisimme olleet ystävällisempiä häntä kohtaan.
— Mikäs esti, sanoi Manne. Hän ojensi ujostellen tuohesta

kutomansa puukontupen Kaarinalle. — Tuossa on, jos huolit. Ja
häveten liikutustaan hän viheltäen poistui huoneesta.
Aikaisin seuraavana aamuna Kaarina seisoi Ainamon kannella ja

katseli jälkeen jäävän kirkonkylän punaisia ja kellertäviä rakennuksia.
Vajaat kolme kuukautta oli kulunut siitä, kun hän oli astunut jalkansa
kylän laivalaiturille arkana, pelko ja jännitys mielessä. Miten paljon
hän olikaan kokenut sinä aikana hyvää ja pahaa, iloa ja surua. Hän
muisteli Siken jäähyväissanoja:
— Kaarina, sinä olet ollut hyvä meille, ja Iisa oli lisännyt:
— Parempi kuin mitä olisimme ansainneet.
Kaarina hymähti. Jos hän oli ollut hyvä, oli se Päivättären

silmälasien ansio. Miten isä olikaan sanonut? Näe hyvää ja kaunista
siinäkin, missä ihmissilmä huomaa vain vikoja ja moitittavaa.
Ehkä se olikin elämän salaisuus.

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Textbook of Vitreoretinal Diseases and Surgery

First Edition: 2009

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Soumen Mondal MD Jatinder Singh Saumil Sheth MD FRCS

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“A man’s learning is imperishable and a precious wealth

1. Genetics of Age-related Macular Degeneration ............................................................ 1

2. Cell Therapy in Retinal Diseases .................................................................................. 15

3. Atypical Manifestations of Diabetic Retinopathy ...................................................... 31

4. Anti-VEGF Therapy in Proliferative Diabetic Retinopathy ..................................... 39

5. Vascular Endothelial Growth Factor: Inhibitors in Age-related

6. Role of Combination Therapy in Diabetic Macular Edema ..................................... 57

7. Hemorrhage at the Macula: Classifications and Treatment Options ...................... 75

8. Retinal Stress by Vitreous Traction ............................................................................... 85

9. Polypoidal Choroidal Vasculopathy ............................................................................. 93

10. Vitreous Hemorrhage: Recent and Future Management ........................................ 107

11. Suprachoroidal Hemorrhage ........................................................................................ 119

12. Imaging in Posterior Uveitis ......................................................................................... 131

13. Optical Coherence Tomography in Age-related Macular Degeneration ............. 149

14. Newer Advances in Retinal Lasers .............................................................................. 165

15. Role of Biostains in Vitreous Surgery ........................................................................ 173

16. Wide-angle Viewing System in Vitreoretinal Surgery ............................................ 183

17. Microincision Vitrectomy .............................................................................................. 191

18. Update on Retinopathy of Prematurity: Concept and Management .................... 201

19. Pediatric Retinal Detachment ....................................................................................... 225

20. Pediatric Intraocular Tumors–I .................................................................................... 241

21. Pediatric Intraocular Tumors–II ................................................................................... 257

22. Ocular Manifestations of HIV/AIDS ........................................................................... 275

23. Central Serous Chorioretinopathy .............................................................................. 293

Index ....................................................................................................................................................... 303

Risk Factors in AMD

GREATER BODY MASS INDEX

FAMILY BASED GENE MAPPING STUDIES

CANDIDATE GENE SCREENING

Complement Factor H (CFH) Gene

Factor B and Complement Component

Genes in the 10q26 Cluster

Toll like Receptor 4 (TLR4)

Mechanisms of AMD Development

ROLE OF COMPLEMENT ACTIVATION AND INFLAMMATION IN THE PATHOGENESIS OF AMD

ACCUMULATION OF LIPOFUSCIN IN THE RPE

ROLE OF OXIDATIVE STRESS MEDIATORS AND MODIFICATIONS IN AMD PATHOGENESIS

FIGURE 2-2: Different steps in cell therapy

Sources of Stem Cells for Retinal Therapy

STEM CELLS FROM OCULAR SOURCE