Retinal Degeneration: Causes, Diagnosis, and Treatment [1 ed.] 9781608764426, 9781607410072

Citation preview

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Eye and Vision Research Developments Series

RETINAL DEGENERATION: CAUSES, DIAGNOSIS AND TREATMENT

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

Eye and Vision Research Developments Series Eye Cancer Research Progress Edwin B. Bospene (Editor) 2008. ISBN: 978-1-60456-045-9 Non-Age Related Macular Degeneration Enzo B. Mercier 2008. ISBN: 978-1-60456-305-4 Optic Nerve Disease Research Perspectives Benjamin D. Lewis and Charlie James Davies (Editors) 2008. ISBN: 978-1-60456-490-7 New Topics in Eye Research Lauri Korhonen and Elias Laine (Editors) 2009. ISBN: 978-1-60456-510-2 Eye Infections, Blindness and Myopia Jeffrey Higgins and Dominique Truax (Editors) 2009. ISBN: 978-1-60692-630-7

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Retinal Degeneration: Causes, Diagnosis and Treatment Robert B. Catlin (Editor) 2009. 978-1-60741-007-2

Eye and Vision Research Developments Series

RETINAL DEGENERATION: CAUSES, DIAGNOSIS AND TREATMENT

ROBERT B. CATLIN

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

EDITOR

Nova Biomedical Books New York

Copyright © 2009 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Library of Congress Cataloging-in-Publication Data Retinal degeneration : causes, diagnosis, and treatment / editor, Robert B. Catlin. p. ; cm. Includes bibliographical references and index. ISBN 978-1-60876-442-6 (E-Book) 1. Retinal degeneration. I. Catlin, Robert B. [DNLM: 1. Retinal Degeneration. WW 270 R43805 2009] RE661.D3R475 2009 617.7'35--dc22 2009001303

Published by Nova Science Publishers, Inc.    New York

Contents Preface

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Chapter 1

vii Physiopathology of Retinal Degeneration in Rd1 Mouse Model of Retinitis Pigmentosa: TGF-Β1, Proteinases and Oxidative Stress Mechanisms Satpal Ahuja, Poonam Ahuja-Jensen, A. Romeo Caffe, Magnus Abrahamson, Per Ekstroma and Theo van Veen

1

Chapter 2

Progressive Retinal Dystrophies Ilene Tsui, J. Mie Kasanuki and Stephen H. Tsang

43

Chapter 3

Usher Syndrome Carmen Nájera, Elena Aller, Teresa Jaijo and José M. Millán

61

Chapter 4

Molecular Genetics of Norrie Disease, Familial Exudative Vitreoretinopathy and Retinopathy of Prematurity Barkur S. Shastry

89

Chapter 5

Genetic Risk Factors in Age-Related Macular Degeneration Barkur S. Shastry

Chapter 6

Genetic Variations of ARMS2/HTRA1 Locus in 10q26.13 and Age-Related Macular Degeneration Dequan Chen

119

Pharmacological Monotherapy for Neovascular Age-related Macular Degeneration Bradley T. Smith, Daniel P. Joseph and M. Gilbert Grand

131

The Benefits and Risks of Cataract Surgery in Patients with Age-Related Macular Degeneration Dalia Zaliuniene and Vytautas Jasinskas

141

Chapter 7

Chapter 8

107

Contents

vi Chapter 9

Early Detection and Diagnosis of Age-Related Macular Degeneration: From Slit Lamp Examination to Advanced Imaging Techniques and Psychophysical Tests Michael Waisbourd, Yair Manor, and Anat Loewenstein

Chapter 10

Retinal Degenerations Associated with Systemic Drug Toxicity Pedro Romero-Aroca, Baget-Bernaldiz and Alicia Traveset-Maeso

Chapter 11

Non-Rod Non-Cone Photoreception in Humans: Roles in Vision and Disease Farhan Husain Zaidi

213

In Vivo Visualization of Photoreceptor Layer and Lipofuscin Accumulation in Stargardt’s Disease / Fundus Flavimaculatus by Optical Coherence Tomography Giuseppe Querques, Domenico Martinelli, Lea Querques, Gisèle Soubrane and Eric H Souied

237

Chapter 12

Chapter 13

Chapter 14

Chapter 15

Visual Training in Retinitis Pigmentosa Patients: Neural Plasticity and Function Recovery Enzo M. Vingolo, Serena Salvatore, Pier Luigi Greng and Paolo Limoli

Index

191

249

Serum-Free Retinal Explant Culture System and Comparative Rescue Effects of LEDGF, GST, CNTF, BDNF, NGF, Bfgf and Antioxidants in the Rd1 Mouse Model of Retinitis Pigmentosa Satpal Ahuja, MVSc, Poonam Ahuja-Jensen, A. Romeo Caffé, Maria Thereza Perez, Per Ekström and Theo van Veen

263

Future Outlook in the Treatment of Age-Related Macular Degeneration Olli Arjamaa

301

Short Communications

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

155

311

Posterior Capsular Opacification in Retinitis Pigmentosa Patients Enzo Maria Vingolo, Serena Salvatore, Sonia Cavarretta, PierLuigi Grenga and Roberto Grenga

313

Pedical Omental Transplant V.K. Agarwal and P.S. Hardia

323

Antioxidants in Age Related Macular Degeneration Pedro Durães Serracarbassa

335 341

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Preface Retinal tissue may degenerate for a number of reasons. Among them are: artery or vein occlusion, diabetic retinopathy, R.L.F./R.O.P. or disease (usually hereditary). Retinitis pigmentosa, retinoschisis, lattic degeneration, and macular degeneration are characterized by progressive types of retinal degeneration. This new book presents the latest research in the field. Chapter 1 - The rd1 (retinal degeneration) mouse retina shows degeneration homologous to a form of retinitis pigmentosa with a rapid loss of rod photoreceptors and deficiency of retinal blood vessels. Due to Pde6brd1 gene mutation, β subunit of phosphodiesterase (PDE) of rd1 retina has an inactive PDE which elevates cGMP and Ca2+ ions level. In vitro retinal explants provide a system close to the in vivo situation, so both approaches were used to compare the status of oxidative stress, transforming growth factor-β1 (TGF-β1), sialylation, galactosylation of proteoglycans, and different proteinasesendogenous inhibitors systems participating in extracellular matrix (ECM) remodeling/degeneration and programmed cell death (PCD)/apoptosis in wt and rd1 mouse retinas. Proteins and desialylated sulfated glucosaminoglycan parts of proteoglycans in ECM of rd1 retina were, respectively, decreased and increased due to enhanced activities of proteinases. Desialylation increases the susceptibility of cells to phoagocytosis/apoptosis, decreased neurogenesis and faulty guidance cues for synaptogenesis. In vivo activities of total proteinases, matrix metalloproteinase-9 (MMP-9) and cathepsin B were increased in rd1 retina on postnatal day 14 (PN14), -21 and -28, due to relatively lower levels of tissue inhibitor of MMPs (TIMP-1) and cystatin C, respectively. This corresponded with increased in vitro secretion of these proteinases by rd1 retina. Cells including end-feet of Mueller cells in degenerating rd1 retina showed intense immunolabeling for MMP-9, MMP-2/TIMP-1, TIMP-2 and cathepsin B/cystatin C, and proteinases pool was increased by Mueller cells. Intense immunolabeling of ganglion cell (RGC) layer for cathepsin B and of inner-plexiform layer of both PN2/PN7 rd1 and wt retinas indicated importance of cathepsin B in synaptogenesis and PCD of RGC. Increased levels of TGF-β1 in vitro transiently increased the secretion of MMPs and cathepsins activities by wt explants which activate TGF-β1 and remodel the ECM for angiogenesis and ontogenetic PCD. Whereas, lower level of TGF-β1 and persistently higher

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

viii

Robert B. Catlin

activities of MMPs and cathepsins in rd1 retinas and conditioned medium, suggested that proteinases degraded TGF-β1 and ECM and caused retinal degeneration. Lower activities of glutathione-S-transferase and glutathione-peroxidase in rd1 retina contribute to oxidative stress which damages membranes and increased the expression, release/secretion of proteinases relative to their endogenous inhibitors. Participation of oxidative stress in rd1 retinal degeneration was further confirmed from the partial protection of rd1 photoreceptors by in vitro and/or in vivo supplementation with glutathione-Stransferase or a combination of antioxidants namely lutein, zeaxanthin, α-lipoic acid and reduced-L-glutathione. Treatment with combination(s) of broad spectrum proteinase inhibitor(s) and antioxidants needs investigation. Chapter 2 - Retinal degenerative diseases are the leading cause of irreversible blindness in western countries today. Our knowledge of the underlying pathophysiology and hence, targets for therapeutic intervention, are evolving, but still limited. While many retinal degenerations are believed to have a multifactorial etiology, we now know that there is a genetic component that is at least partially responsible for the clinical manifestations seen in many of these diseases, such as retinitis pigmentosa and age-related macular degeneration (AMD). Chapter 3 - Usher syndrome (USH) is an autosomal recessive disease characterized by hearing loss and retinitis pigmentosa (RP). It is both clinically and genetically heterogeneous and its prevalence makes it the most common association of deafness and blindness of genetic origin. From a clinical point of view, USH is categorized into three types. Usher type I (USH1) is the most severe form and is characterized by severe to profound congenital deafness, vestibular areflexia, and prepuberal onset of progressive RP leading to blindness. Type II (USH2) displays moderate to severe hearing loss, absence of vestibular dysfunction, and later onset of retinal degeneration. The less frequent type III (USH3) shows progressive post-lingual hearing loss, variable onset of RP, and variable vestibular response. To date, five USH1 genes have been identified. In the majority of populations the most commonly mutated gene is MYO7A (USH1B), which represent 50–60% of the total USH1 patients, followed by CDH23 (USH1D), PCDH15 (USH1F), USH1C, and USH1G. Defects in MYO7A also cause autosomal dominant non-syndromic sensorineural deafness (DFNA11), autosomal recessive deafness (DFNB2), as well as atypical forms of Usher syndrome, which are clinically similar to Usher syndrome type III. Among the three genes implicated in USH2, mutations in the USH2A gene account for 70–80% of the USH2 cases, and this gene is also implicated in isolated RP without associated deafness. The other two implicated genes are VLGR1 (USH2C) and WHRN (DFNB31). USH3 is rare except in some founder populations, USH3A being the gene responsible for this type. Our group has been working in the clinics, on the epidemiology and genetics of this syndrome, carrying out the complete clinical study of each patient and the search for mutations in the different implicated genes, to identify a genotype-phenotype correlation, and to contribute to the understanding of the genetic basis of the disease, in order to establish a rational therapy in a next future. Chapter 4 - Blindness or visual impairment is a devastating health problem and has always been a major public health concern. Many ocular disorders involve neovascularization of the retina and it is the most common cause of blindness. Disorders such as familial

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Preface

ix

exudative vitreoretinopathy (FEVR), Norrie disease (NDP), retinopathy of prematurity (ROP), persistent fetal vasculature syndrome (PFVS) and Coats’ disease can cause blindness in early childhood and all of them involve abnormal vascularization of the peripheral retina and retinal detachment. Although the exact reason for the abnormal vascularization of the peripheral retina is not completely understood, during the last decade rapid progress has been made in identifying the genes and their variants responsible for these ocular disorders. As a result, interestingly all of the above disorders are found to be associated with mutations in a group of genes responsible for the highly regulated Wnt signaling pathway. This pathway is well known in making decisive role in embryonic patterning, cell fate determination and regulating ocular growth and development by activating the transcription of specific target genes. However, the relationship between phenotype and genotype in hereditary retinal diseases is still a mystery. It is not clear why certain genetic defects cause a more severe clinical symptom in some patients while in others the same genetic alteration causes a less severe phenotype. Although gene replacement therapy, prenatal diagnosis and carrier detection have not been tried extensively for ocular disorders such attempts are now feasible for many inherited eye disorders because of the availability of animal models. Additionally, further investigations on the basic Wnt signaling mechanism may lead to a better understanding of the retinal degeneration and novel therapeutic approach to prevent or treat these debilitating blinding diseases. Thus, for patients and clinicians the future holds more optimism than ever before. Chapter 5 - Age-related macular degeneration (AMD) is a disorder of the retina, retinal pigment epithelium (RPE) and choriocapillaris. It is a heterogeneous group of genetically complex progressive degenerative disorder and causes changes in the macular region, that is responsible for seeing fine details clearly. The condition is painless and is the leading cause of irreversible blindness in the elderly population. The most common characteristics of AMD are the development of drusen and atrophy of RPE (patchy loss of RPE). Heritability of AMD ranges from 46-71% and it has a penetrance of 0.05% before the age of 50 years and 11.8% after 80 years of age. The pathogenesis of the disorder and the biochemical pathways involved are not understood. Epidemiological, familial aggregation and twin studies suggest that genetic and environmental factors play an important role in determining the onset of disease. Among environmental factors, smoking and age have been consistently found to be associated with AMD. Over the past several years, linkage and association studies have identified several chromosomal regions that are likely to predispose individuals to AMD. Among these, the most consistently identified genetic loci lie on chromosomes 1q31 and 10q26 regions. Molecular genetic analyses of chromosome 1q31 region have identified a common variant (Y402H) in the complement factor H (CFH) gene as a major genetic risk factor for AMD in Caucasian population. Subsequent association studies on chromosome 10q26 region have also revealed a common polymorphism (A69S) in the coding region of the LOC387715 gene and a promoter polymorphism (-512 bp) in the adjacent (about 7 kb downstream of LOC387715 gene) HTRA1 gene. The LOC387715/HTRA1 polymorphisms are considered as second major risk factors contributing to AMD pathogenesis. These results have been replicated by several independent studies in different populations. Additionally, various forms of AMD are found to be associated with variations in factor B (BF) and complement component 2 (C2) that are located on chromosome 6p. A comprehensive study

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

x

Robert B. Catlin

of variants at 3 loci suggests an independent contribution of three loci to disease risk and no evidence of epistasis between CFH and LOC387715 genes has been reported. For other candidate genes, variations did not account for a significant fraction of patients. Although these studies have not provided any benefit for the treatment of the disorder, further research on additional genetic and environmental factors may contribute to the better understanding of the onset and progression of AMD. This may eventually result in better treatment and diagnosis. Chapter 6 - Age-related macular degeneration (AMD), a central retinal complex trait disease, is involved with genetic and environmental risk factors. Chromosomal 10q26.13 region was linked to the risk of AMD by early family-based genome-wide scan studies, and the AMD risk signal was first associated with three genes in the region, including PLEKHA1, ARMS2 (hypothetical) and HTRA1. Later SNP association studies have locked the most significant AMD-susceptibility signal in this region onto the locus of ARMS2 and HTRA1, and two single nucleotide polymorphisms (SNP) in this ARMS2/HTRA1 locus – rs10490924 in the hypothetical ARMS2 exon 1 and rs11200638 in the downstream HTRA1 promoter region have been consistently associated with AMD in different study cohorts across the world, including Caucasian, Chinese, Indian and Japanese populations. This suggests that either ARMS2 or HTRA1 or both play a critical role in AMD development. However, genetics has not been successful in differentiating the roles of the two genes in AMD susceptibility due to high or almost 100% linkage disequilibrium (LD) in this ~ 7 kb region. The controversy over whether either one or both genes are a must in AMD development remains un-resolved so far. To fully determine the roles of the two genes in AMD risk, we believe that the following two lines of questions must be answered: (1) Is ARMS2 existent as a real gene in nature? ESTs in the GenBank+EMBL+DDBJ database, 2 NIH-MGC clones and RTPCR amplified cDNA bands suggest that it can transcribe into an mRNA(s). Then what’s the mRNA sequence(s)? Is it exactly like what NCBI database predicted? Is it encoding a native protein(s)? Is the native protein exactly like what the database predicted? If yes, what’s the normal function of the native protein? How genetic variations in ARMS2/HTRA1 locus affect its function during AMD development? (2) Whether and how HTRA1 is involved in AMD risk? No nonsynonymous coding SNPs but promoter region SNPs and other types of SNPs of HTRA1 have been found to be associated with AMD, suggesting that HTRA1 expression level change may be a mechanism for the involvement of HTRA1 in AMD risk if it has a role in nature. It remains controversial as to whether the HTRA-rs11200638 risk allele A increases HTRA1 expression due to the presence of reports with supporting and denying data. Nevertheless, how do all the genetic variations in ARMS2/HTRA1 locus possibly affect HTRA1 expression level (increase, decrease or no effect)? AMD is a central retinal disease that mainly causes the irreversible central vision blindness in older individuals (more than 55 years old)[1;2]. In the United States, AMD leads to significant visual impairment for approximately 7.5 million elderly Americans[3;4]. AMD is known to be a complex trait involved with genetic and environmental risk factors. These include: (a) three chromosomal loci – CFH in 1q32[5-24], ARMS2/HTRA1 in 10q26.13 [8;18;25-49] and C2/BF in 6p21.3 [3;50]; (b) biomarkers of systemic inflammation such as C-reactive protein [51-56]; and (c) smoking [31;57-64]. Over half of the risk for AMD appears to be explained by genetic factors with environmental and lifestyle exposures.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Preface

xi

Chapter 7 - A shift has occurred in the treatment options of choroidal neovascularization (CNV) due to age-related macular degeneration (AMD). Prior to ocular photodynamic therapy (PDT) the only available treatment was laser photocoagulation. The advantages of PDT were limited to slowing the rate of visual acuity loss. However, compared with laser photocoagulation this was accomplished with less collateral damage to the overlying retina. Pegatanib sodium (Macugen), the first purely pharmacological treatment of AMD showed similar efficacy to PDT and ushered in a new method of treatment for CNV. Pharmacologic therapies directed at vascular endothelial growth factors (VEGF), the major stimulus for CNV growth, have now become the standard of care. Ranibizumab (Lucentis) is a recently FDAapproved monoclonal antibody to VEGF that can stabilize and even improve visual acuity in a significant number of patients. Favorable results have also been reported for a related monoclonal VEGF antibody, bevacizumab (Avastin). The following provides an overview of current therapies used to treat CNV due to AMD. Chapter 8 - Both cataract and age-related macular degeneration (ARMD) are not unusual findings in the aging eye. They are two the most common causes of irreversible visual loss in developed world. The number of cataract surgeries is steadily increasing in most of these countries. However, the benefits (and risks) of cataract surgery in patients with ARMD are uncertain. Some investigators found that cataract surgery benefits patients with ARMD, ensuing in improved visual function and quality of life in most patients , whereas others reported that in patients with ARMD, improvement in visual outcome after cataract surgery can be limited. Furthermore, recent studies found even progression from early to late stages of ARMD in eyes after cataract surgery. There are several possible reasons that might explain, either individually or in concert, the association between cataract surgery and late ARMD: 1) cataract and ARMD simply share one or more common risk factors, including age, diet, light exposure, inflammation, and/or genetic factors, 2) cataract surgery can increase photo-oxidative damage to the retina, 3) the surgery may also increase intraocular inflammation, 4) hypodiagnosis of ARMD in persons with lens opacity. However, it is not possible to draw an extensive conclusion concerning the effect of a cataract surgery on the development of early ARMD or on the progression of pre-existing ARMD, because in most of the previous studies, different study designs, classifications of ARMD, and the length of follow-ups were used. Currently, there is no conclusive evidence to support a relation between progression of ARMD and cataract surgery. However, some investigators have concluded that there is certain evidence from observational studies to support an association between cataract surgery and subsequent onset of late ARMD, respectively progression of early to late ARMD Additional clinical trials with sufficient statistical power, a well-defined length of the study period, an adequate control variables, such as age and severity of cataract or ARMD to prove or disprove the reasons of possible influencing factors, such as types of lens, intraocular inflammation, and genetic factors are needed to be assessed. A differential risk of ARMD and differential response to cataract surgery due to genetic diversity of the patient populations are still not reported.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

xii

Robert B. Catlin

Conclusion. We must be selective and treat only the patients who are to get most benefit from cataract surgery or who may have the lowest risk level for late ARMD. The development of guidelines for the surgical management of this group of patients is needed. Chapter 9 - Early detection of age-related macular degeneration (AMD), the leading cause of blindness and visual impairment in the developed world, is now of utmost importance in the era of preventive micronutrients and anti angiogenetic treatment strategies. According to epidemiological studies, about 30% of adults above age 75 show signs of AMD (Klein et al., 1992). Moreover, prevalence is expected to double in the coming decades, coincident with the increase in the elderly population (Friedman et al., 2004). Chapter 10 - Introduction. A variety of systemic drugs causes retinal toxicity, the visual function effect in the major cases is minimal or reversible, nevertheless, permanent or progressive visual loss may occur. In the present study we revised the literature of the systemic toxicity drugs, and rapport the results of our experience as Retina and Oncologic reference Center. Focusing special attention to the chloroquine, tamoxifen, and aminoglycosids. Methods. A search of the bibliographic databases (MEDLINE) was conducted; selected relevant studies were scrutinized and included in the review. We revised also the patients submitted to chloroquine , hydroxychloroquine, tamoxifen and other oncologic agents in our Hospital and also revised all toxic retinal degenerations of several drugs in the last 20 years. Results. We observed six different forms of retinal drug toxicity: disruption of the retina and retinal pigment epithelium associated to chloroquine derivatives and oncologic drugs; crystalline retinopathy associated most frequently to tamoxifen use; cystoid macular edema with the use of nicotine acid, prostaglandin topical drugs and aminoglycosids; vascular damage associated to cisplatinum , talc and oral contraceptives; retinal folds with use of antibiotics , hydrocholothiazide and metronidazole; finally we may appoint two drugs that causes visual disturbances produced by a probably retinal toxicity but without characteristic fundus abnormalities as the digoxin and the methanol. Conclusions. Although there are thousands of systemic medications, only a small number produce retinal changes, but the extensively use of some agents as derivative chloroquine and oncology agents, and the increasing use of intravitreal injections, the patients with retinal toxicity degenerations may increase in the next future. Furthermore the mechanism by which toxicity develops is not always understood, because retinal toxicity may occur when the agent is used at therapeutic levels. In conclusion Ophthalmologists’ need to maintain a high attention to the deleterious changes observed in a patients in treatment with systemic drug use. Chapter 11 - The fascinating discovery of a new fundamental class of photoreceptor, which is neither rod nor cone, in the inner retina of some mammals has recently been complemented by parallel discoveries in humans. Studies using human subjects with rod and cone dystrophies have unveiled the existence of a similar system of inner retinal photoreception in rodless coneless humans to that in some mammals [Zaidi FH et al. Shortwavelength light sensitivity of circadian, pupillary, and visual awareness in humans lacking an outer retina. Current Biology 2007 December 18; 17(24): 2122-8]. These and related studies and their significance are described in the first part of this chapter. The roles the

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Preface

xiii

receptor is likely to have in retinal degenerations and functionally related conditions are discussed in the second part of the chapter. The novel receptor is a subgroup of retinal ganglion cells called photoreceptive retinal ganglion cells (pRGC) or giant gangion cells, which reside in the inner retina and which in distinction to classic (rod and cone) outer photoreceptors use melanopsin as its photopigment. The pRGC receptor is the driver of the body’s neuro-endocrine circadian rhythms via secretion of pineal melatonin which it directly regulates, it makes a substantial component to pupillary reactions especially in humans, and it also contributes to behavioural alertness. Of cardinal importance is that recently the pRGC has been shown in humans to mediate conscious sight, a role for it that had not been described in animals. The response it elicits can be markedly different to that found in blindsight. Studies with rodless coneless humans, in whom vision is still found due to the persistence of the pRGC response, create a major revision to existing models for understanding visual perception. This is into a classic pathway which is found in the outer retina and originates in rods and cones, and an alternate pathway arising from the inner retina and which is driven by pRGCs. The latter can even function in the absence of rods and cones, but are also regulated to some degree by inputs from the outer retina. This new fundamental delineation of retinal function has important implications for how retinal degenerations may be understood, defined and classified. The clinical relevance of this novel retinal photoreceptor system is discussed in relation to several areas. The novel photoreceptor’s role in conscious sight redefines how blindness is evaluated. Implications for ophthalmic and orbital surgery are discussed. Candidate diseases of the inner retina and optic nerve which may directly reflect dysfunction in pRGCs are considered. The question is considered as to whether dysfunction in the pRGC manifests as inner retinal dystrophies affecting retinal ganglion cell function in an analogous way to outer photoreceptor dystrophies of rods and cones. The implications of the pRGC’s discovery in humans and its roles in vision are discussed around key topics of interest to several groups. For example how vision from pRGCs, as opposed to rods and cones, seems to account for otherwise inexplicable clinical findings in a variety of conditions. Chapter 12 - Purpose: Retinal flecks are commonly observed in both Stargardt’s disease (STGD) and fundus flavimaculatus (FFM). The aim of our study was to determine the precise localisation of these flecks within the retinal layers using Stratus optical coherence tomography (OCT3, Humphrey-Zeiss, San Leandro, California). Moreover we assessed photoreceptor (PR) morphology in patients with STGD and FFM using high definition OCT (HD-OCT, OCT 4000 Cirrus, Humphrey-Zeiss, San Leandro, California). Finally, we tried to investigate the relationship between PR layer morphology and localization of retinal flecks, as evaluated respectively by HD-OCT and OCT, and best corrected visual acuity (BCVA). Methods: This was a prospective observational case series. A complete ophthalmologic examination, including best corrected visual acuity (BCVA) and OCT (OCT3 and/or HDOCT) was performed in 40 consecutive patients with STGD/FFM. Results: A total of 76 eyes were included in the study. Using OCT (OCT3 and/or HDOCT), we observed hyperreflective deposits which we classified in two types: type 1 lesions located in the inner part of the retinal pigment epithelium layer and type 2 lesions located at the level of the outer nuclear layer. Moreover, HD-OCT was capable of visualizing regions of

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

xiv

Robert B. Catlin

transverse PR loss in the foveal region. BCVA impairment showed a statistically significant correlation to the presence of complete loss of PR layer in the foveal region (p 0.05) to detect light at any of several other longer or shorter wavelengths (420, 460, 500, 515, 540, 560, and 580 nm). On questioning she described what she saw as a conscious appreciation of “brightness”, which was reported only for the 481 nm stimulus. This is shown in figure 4. These detection probabilities remained unchanged when corrected for multiple testing (Bonferroni).

Figure 4. Conscious Sight in the Absence of Rods and Cones. Data, figures and legend adapted from Current Biology (Zaidi FH et al, Current Biology 2007 Dec 18; 17(24): 2122-8) [1].

The above figure shows the most fascinating data which are the results of the psychophysical testing of the apparently ‘blind’ female subject that demonstrated her

222

Farhan Husain Zaidi

conscious perception of light at 481 nm ( p < 0.001) but failure (p > 0.05) to detect light at any of several longer or shorter wavelengths of light (420, 460, 500, 515, 540, 560, and 580 nm). These results mirror the spectrally tuned response of the pupil (figure 2), and suggest that the subject's detection and conscious awareness of light also arise from pRGCs and not rods and cones. Each histogram represents the percentage of correct responses out of 20 trials for both left and right eyes (360 trials in total).

Differences from Blindsight Blindsight is a form of sight arising from extra-geniculostriate pathways. It has most often been described in subjects with damage to the primary visual cortex (V1) who are often regarded as having no conscious perception of the stimulus presented [22]. A major feature of most cases of blindsight is that it is a subconscious visual awareness in subjects who genuinely believe that they cannot see. Some classifications of blindsight permit conscious appreciation of features to a visual stimulus like motion, but not to brightness. Conscious appreciation of brightness is the main feature of conscious sight which distinguishes it from the phenomenon of blindsight. Although superficially the responses in the female rodless coneless subject resemble cortical blindsight in that the female subject was able to detect a stimulus with a rate of success above chance using forced choice methodology, the data in fact represent a markedly different phenomenon. This is as the female subject saw the ‘brightness’ of the light stimulus, and when questioned she was fully conscious of seeing this brightness in the 481 nm light source. Hence vision mediated by the pRGC is different to blindsight, for unlike blindsight it incorporates a conscious percept of sight, indeed a conscious percept of how bright the light source is. There is of course the possibility that in addition to conscious sight, some aspect to blindsight might also be mediated by photoreception from the pRGC, and in this context the findings in the context of the male subject described later are potentially relevant.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Could Artefact be Responsible? Many of the roles for the human pRGC that have been conclusively found using these rodless coneless humans, including the novel discovery therein of the human form of the receptor, have to some extent been predictable from other studies, especially those of other mammals in whom the pRGC had been discovered some years before. But what is exceptionally novel is the discovery using these subjects of a form of ‘rodless coneless’ sight, which, further, seems to arise from photoreception by the RGCs, a function for the pRGC that had hitherto not been discovered in any organism. One question that must be considered before drawing the conclusion that conscious sight can arise from pRGCs (and in the absence of rods or cones) is whether these responses to light could have arisen from a small number of surviving rods and/or cones rather than from the pRGCs? Although visually evoked potentials (VEP), electroretinogram (ERG), and ocular coherence tomography (OCT) analysis cannot absolutely preclude the persistence of a tiny residual population of rods

Non-Rod Non-Cone Photoreception in Humans: Roles in Vision and Disease

223

and/or cones, there was no functional evidence of any significant rod or cone activity, hence any effect from these putative outer retinal elements is negligible. This is as both the λmax of ~480 nm and the correspondence of the pupil action spectrum to a single opsin- and vitamin A-based photopigment (figure 2) template very strongly implicate phototransduction by the pRGC subsystem alone. Responses from rods and cones would not have peak spectral sensitivities in the region of 480nm and their action spectra do not fit the data, while these same data fit very well the pRGCs responses from other mammals (figure 2). Furthermore, the persistence of circadian photoentrainment also suggests the responses as arising from pRGCs (figure 1). Hence to summarise, the signals from these ‘rodless coneless’ retinas are certainly those expected from pRGCs, and these signals are also coming from the right place – the intact inner retina (in the presence of an absent outer retina). One final potential issue needs to be addressed. The stimuli required to elicit sight in this subject were extremely intense, necessitating specially designed high output fibreoptic sources linked to narrow band width neutral density and interference filters. So is it possible that the responses were artefactual, arising in some way from the heat generated from the intense light source shone on and into the eye being confused by the subject with sight? This could not have been the case however as the subject ‘saw light’ only with the blue 481 nm stimulus being turned on, and their pupil reacted most to 481 nm light, and not at all to long wavelength light. A 481 nm stimulus is short wavelength or blue light, or that part of the visual spectrum which imparts its energy to human tissue as rotational forces to electrons, and which contrasts in this respect with long wavelength (red) light which is that part of the visual spectrum which imparts its energy as heat. Further, there was no conscious visual response above or below 480 nm.

Neuroanatomical Organisation of this New Type of Sight

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Despite the major implications of these studies to how the most basic components of the visual system at the level of the retina and optic nerve are understood, a question remains, however as to which neuronal pathways mediate these effects of light – which parts of the brain are involved? Neuroanatomical investigations in rodents show that melanopsincontaining ganglion cells project to a range of retinorecipient nuclei, including major projections to: i. hypothalamic suprachiasmatic nuclei (SCN) - the endogenous circadian pacemaker. ii. the intergeniculate leaflet of the thalamus, an area that is closely linked to normal circadian function and conveys photic and nonphotic signals to the SCN. iii. the ventrolateral preoptic area - the on/off control switch for sleep and wake states. iv. the olivary-pretectal nucleus implicated in the pupillary constriction response. v. the superior colliculus - mediating visual and auditory sensorimotor reponses [23,24]. vi. the dorsal lateral geniculate nucleus (dLGN). In non-human primates this subgroup of ganglion cells has a peak spectral sensitivity (λmax) of 482 nm [3,24]. It may thereby provide the neuroanatomical substrate in support of the essentially identical

224

Farhan Husain Zaidi short-wavelength visual awareness response observed in the rodless coneless female human subject [1]. Moreover, recent imaging studies in humans are beginning to identify brain regions associated with light-induced improvements in performance and cognition and show preferential short-wavelength activation of the thalamus and the anterior insula, structures strongly implicated in arousal and memory function [25-27].

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Behavioural Reactions to Light and Photoreceptive Retinal Ganglion Cells The male rodless coneless subject was studied over three years by workers in the United States testing the spectral sensitivity of the circadian, neuroendocrine, and neurobehavioral axes [1,2,28-30]. It was found that the residual pRGCs had a direct effects on melatonin suppression and waking-electroencephalogram(EEG) power density as an objective correlate of alertness. As with the model female subject the experimental methodology necessitated prolonged experiments. For the male subject some experiments necessitated testing over more than three years. The results are summarised in figure 5. It was first confirmed that the male subject retained a normal melatonin-suppression response to bright-white light exposure on two separate occasions three years apart [1,6]. Workers then conducted a 14 day study in hospital comparing the effects of 6.5 hr exposure to 460 nm and 555 nm monochromatic light on circadian phase resetting, melatonin suppression, and enhancement of arousal [28,29]. In order to compare the relative contribution of the novel photosensitive retinal ganglion cells (pRGCs) and classical (rod/cone) photoreceptors, workers used two light sources that would differentially stimulate these systems: a monochromatic “blue” light source with a peak emission (λmax) at 460 nm and hence close to the λmax of human pRGCs (~480 nm), and a monochromatic light source with a λmax at 555 nm corresponding to the peak of human photopic vision [31,32]. Since the subject exhibited a 24-hr sleep-wake pattern and an entrained aMT6s rhythm, it was predicted that the pRGC/melanopsin-driven system would be intact and that the shortwavelength stimulus would elicit full circadian, neuroendocrine, and neurobehavioral responses, whereas the lack of classical photoreception would preclude any response to midwavelength 555 nm light. The direct effects of exposure to green (555 nm) and blue (460 nm) monochromatic light on melatonin suppression (A) and waking-EEG power density (B) as an objective correlate of alertness are shown in figure 5. Exposure to the 555 nm light caused no suppression of melatonin as compared to the corresponding clock time the previous day, whereas exposure to the 460 nm light suppressed melatonin (total suppression by AUC = 57%) and maintained the suppression effect throughout the entire 6.5-hr exposure, shown in (A) in figure 5. The 460 nm light source also caused an elevation of alpha activity (8–10 Hz) in the waking EEG, indicative of a more alert state, shown in (B) in figure 5. Only alpha frequencies exhibited a wavelength-dependent difference during the second half of the light exposure, shown in (C) in figure 5. The data are consistent with the short-wavelength sensitivity for the acute effects of light in sighted subjects under similar conditions [28-30].

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Non-Rod Non-Cone Photoreception in Humans: Roles in Vision and Disease

225

Figure 5. Short-Wavelength Light Sensitivity for Melatonin Suppression and Enhancement of EEG Alpha Power in a Blind Man. Data, figures and legend adapted from Current Biology (Zaidi FH et al, Current Biology 2007 Dec 18; 17(24): 2122-8) [1].

A New Model for Vision: Inner Retinal and Outer Retinal Photoreceptors The data from the rodless coneless human experiments is transformative [1,2]. Figure 6 summarises the new model for vision which comes from these experiments [1]. It shows that the retina has in fact two parallel pathways for vision: an outer ‘classic’ system with photoreception originating in rods and cones, and an ‘alternate’ inner retinal photoreceptor

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

226

Farhan Husain Zaidi

system centred upon pRGCs. This is different to the old ‘duplex’ model of the retina which recognised only rods and cones, both outer retinal elements, as photoreceptors for visual perception. The alternate pRGC system is responsive to large changes in environmental brightness, giving rise to both conscious vision and behavioural changes to blue light, in addition to contributing significantly to pupil reactions especially in humans (most likely the sustained as well as the initial phasic pupil reaction) and to be the prime input for circadian photoentrainment [1,2,33,34]. There may be also be some interplay at the level of the retina in the visual information being processed between the outer and inner photoreceptor systems - particularly in view of known inputs from S-cones onto pRGCs in some other mammals. It is however important to bear in mind that rodless coneless humans show that the pRGC system is quite capable of functioning independently to rods and cones and even in their absence can mediate conscious vision of blue light [1]. The pRGC represents the first stage of phototransduction of light by retinal elements. Previously it was often thought by some workers that the design of the retina was perturbing in that the photoreceptors were at the back, furthest from contact with light rays coming from the environment - however the discovery of the pRGC shows that the inner retina is indeed where the first photoreceptor layer is found and is where phototransduction occurs first. However at present it is not conclusively known whether the information from the environment processed by pRGCs reaches the brain for visually-dependent tasks before that from rods and cones. This is as pRGCs show a delayed onset of firing in terms of action potentials, and then display rapid firing of several action potentials rather than the long hyperpolarisation phase of rods and cones. At the very least, from the experiments with rodless coneless humans, it is known that the contribution of pRGCs to vision is that of a gross detector of sudden and large changes of brightness in the environment [1]. However the pRGC probably has more subtle visual roles as well. It is worth noting that the rodless coneless subjects studied, both in terms of their conscious sight and their behavioural alertness, had very long-standing degenerations of the retina. Although the ganglion cell layer was anatomically intact in these exceptional subjects, who obviously retained sufficient function to elicit visual and other responses, this is not usually the case in subjects with these diseases. Some degree of functional loss in the pRGCs was probably present even in these model subjects, accounting in part at least for the very high intensity of light required to elicit the visual and pupillary responses from pRGCs. If this is the case, as is likely from the natural history of rod and cone dystrophies, then it is one line of evidence suggesting that pRGCs may contribute to vision at lower levels of environmental light as well. In fact although a rod-cone break in terms of dark and light adaptation is welldocumented, it does not preclude the pRGC having a role in mesopic vision, allowing the transition from seeing in brightness to seeing in the dark. This is theoretically plausible as the pRGC is known from the rodless coneless subjects to be active in very bright photopic conditions, while the sensitivity to blue light that was also found suggests that the receptor might also be active in scotopic conditions were such an experiment designed. This would be of use in nature for some mammals in the wild, for example in mammals living deep underwater. The remarkable phenomenon of colour constancy, wherein a colour, say yellow, appears to be the same hue of yellow despite dramatic changes in the overall brightness of light in the

Non-Rod Non-Cone Photoreception in Humans: Roles in Vision and Disease

227

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

environment, is very likely to be related to the pRGC. This also would be an example of the more subtle role the receptor may have in conscious vision beyond that which has been established of a rudimentary brightness detector for the visual world [1]. The relationship of the pRGC to circadian photoentrainment and sleeping and waking periods in response to night-day cycles is obviously linked to cycles of seasonal activity in animals, an extreme example of which is winter hibernation in temperate regions of the Earth. This also suggests that the visual perception now known to be associated with the pRGC might be active, in addition to bright photopic conditions, under mesopic and scotopic conditions of environmental light intensity as well.

Figure 6. A New Model for Vision. Classic (rod and cone) and alternate (pRGC) pathways for phototransduction of light in the mammalian retina which give rise to conscious visual percepts.

Practical Implications for Vision Research and Ophthalmology How Sight and Blindness are Assessed – Retinal Blindness and Orbital Surgery The pRGC was until recently erroneously thought of purely as a non-image-forming receptor. The recent discovery of the pRGC in humans, together with its role in conscious

228

Farhan Husain Zaidi

vision, necessitate that the clinical diagnosis of “complete” blindness should assess the state of both the outer and inner retinal photoreceptor systems, at least in select cases [1,2]. Common measures of vision used in clinical practice may be insufficient to detect an intact inner photoreceptor system and many patients throughout the world will have been erroneously classified as having no perception of light, which is analogous to saying they are completely blind. Attempting to elicit responses to narrow bandwidth blue light in the region of 480 nm is required to thoroughly exclude any contribution to sight from pRGCs. However in other cases this distinction will be artificial, as the number of diseases that rigidly spare inner retinal function while wiping out outer retinal function is limited, mainly because most severe and chronic retinal diseases cause atrophy over time of the surrounding areas owing to trans-synaptic degeneration of neighbouring retinal elements. In evaluation of complete retinal blindness, assessment of both the inner and outer pRGC systems is particularly critical prior to bilateral and unilateral enucleation or evisceration of the globe. Other situations met in orbital surgery are the management of cryptophthalmos and the management of anophthalmos. Residual phototransducing retinal elements are known to be found in many of these patients and their intactness determines clinical management and surgical rehabilitation of the orbit. It is also a direct area for clinical application of the findings of basic scientific work.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Preservation of Circadian Photoentrainment and Melatonin Administration If supposedly ‘blind’ individuals are found to be light sensitive in terms of pRGC function, then this information helps to at least ensure that they expose their eyes to sufficient daytime light to maintain normal circadian entrainment and sleep/wake rhythmicity. Evaluation of complete blindness prior to bilateral enucleation, and in cryptophthalmos and anophthalmos, are also important because if light-responsive eyes are removed or individuals do not expose their eyes to a robust light-dark cycle, subjects may develop a debilitating circadian-rhythm sleep disorder, which may be particularly profound in its effects on performance and development if the subjects are children, who are also more difficult to diagnose in this context [10,11]. Patients with diseases of the inner retina that result in retinal ganglion cell death (e.g. glaucoma) are at particular risk and should be counselled about the effects of pRGC loss in the context of circadian rhythm. The association between blindness and circadian rhythm disturbance is starting to be appreciated by clinicians. Where complete blindness results, appropriately timed melatonin treatment may be warranted in order to establish entrained circadian rhythmicity [35,36].

Counselling and Legal Implications The implications of testing for activity in the pRGC system in a number of clinical scenarios, including those just mentioned, will undoubtedly come to acquire medicolegal significance. Patients with serious sight loss and ‘apparent’ blindness using routine clinical tests may realistically expect to be counselled and fully informed about what actual vision

Non-Rod Non-Cone Photoreception in Humans: Roles in Vision and Disease

229

they have and what potential vision they have from the alternate pRGC system under environmental blue light conditions. Furthermore, the potential for future techniques under development, such as gene therapy and in the longer term stem cell treatment targeted at the inner retina and ganglion cells, needs also to be considered in the broader context of visual perception from inner retinal photoreception. This is especially important in view of some visual benefit with such treatments aimed at the outer retinal elements [37-40].

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Considerations in the Diagnosis of Retinal Diseases and the Evaluation of Responses to Treatment It is noted by a number of scientists and clinicians using optical coherence tomography (OCT) in studies of various treatment modalities for macular oedema that changes of retinal thickness and visual acuity do not necessarily correlate despite relative preservation of the foveal avascular zone [41,42]. While measurements of retinal thickening from optical coherence tomography generally parallel visual acuity results, several such cases do not despite the relatively intact foveal avascular zones. In many of these cases the findings of optical coherence tomography imaging are strikingly similar to those described in the study of rodlesss coneless subjects previously detailed, in that the outer retinal elements appear chronically damaged (in this case from oedema rather than primary degeneration) while the inner retina, containing the pRGCs, is well-preserved. Such subjects show much better visual acuity than would be expected however for this degree of chronic dysfunction and respond well to treatment. Both the ‘better than expected’ vision in such cases and the ‘better than expected’ response to treatment can plausibly be explained by intact pRGCs affecting prognosis. pRGCs would seem also to have other roles in macular oedema. Macular oedema is a common outcome of several retinal pathologies and also exhibits diurnal variation, for example macular oedema from central retinal vein occlusion is greater in the morning [43]. While nevertheless seemingly complex, many of the systemic factors responsible for this diurnal variation are in turn dependent, ultimately, on circadian photoentrainment originating within the inner retinal pRGCs. Another effect of this is in measurement of retinal thickness using OCT. Measures of normality within OCT clinical software programmes incorporate calculations for diurnal variation. However these may be less representative if diurnal variation is disrupted, for example as may occur if retinal disease is extensive enough for the inner retina to be damaged (which is far from uncommon in such patients), and for circadian photoentrainment to hence be disrupted [44].

Diseases of Ganglion Cells: The Retina and Beyond Several retinal diseases affect the inner retina where the pRGC is located. A wide variety of pathologies from retinal artery and vein occlusions through to myelinated nerve fibres are relevant to consider. The discovery of the pRGC in humans and its visual and other roles are hence directly relevant to understanding the aetiology of these conditions. These conditions may also form important models to study the role of pRGCs further. Comparatively little is

230

Farhan Husain Zaidi

known about the precise topography of pRGCs in the inner retina, and these diseases can help provide more understanding. Glaucoma is a disease that is a primary disorder of ganglion cells. A large number of varieties exist with different aetiologies. Subtypes of glaucoma may be caused by dysfunction in pRGCs and study of this relationship is an obvious important avenue of future research given the worldwide prevalence of glaucoma, its effect on vision, and its treatability. It is also possible that at least some effects of glaucoma are associated with damage to pRGCs. There is much to suggest this. For example, a poorly understood phenomenon is that visual loss in glaucoma is often associated with loss of sensitivity to blue light, to which pRGCs are sensitive and which may therefore be a consequence of pRGC dysfunction. Furthermore, the loss of the pRGC, whose role includes circadian photoentrainment, would seem to have a role in explaining the changes in diurnal variation associated with glaucoma. The latter is of relevance therapeutically since many drug treatments for glaucoma aim to reduce fluctuations in intra-ocular pressure [45-49]. Indeed alterations in melatonin receptor expression as well as changes in endogenous melatonin production are already known to be associated with glaucoma [50]. Neuroprotective strategies, important in the pharmacological treatment of glaucoma, including normal tension glaucoma, assume refreshed importance in view of the discovery of the pRGC in humans. It is of course important to recollect that the pRGC is unique for a mammalian photoreceptor in extending from the retina through the orbit to the brain. Hence any of the large variety of orbital conditions compressing or infiltrating the optic nerve, as well as neuro-ophthalmic lesions (indeed optic neuropathies in general), are other obvious candidates for productive study.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Optical Implications of Inner Retinal Photoreception - Cataract and Refractive Surgery, Intraocular and Prescription Lenses, and Age-Related Macular Degeneration In the past few decades there has been a vogue for intraocular and prescription lenses that block short wavelength light. The putative benefit these lenses offer has been to reduce the risk of age-related macular degeneration (the blue light hazard theory), though this risk remains unproven. Various different lenses are available, miscellaneously blocking ultraviolet light, violet light blockers reducing exposure to both violet (400-440 nm) light and ultraviolet (200-400 nm) radiation, and blue blockers attenuating blue light (440-500 nm). A more convincing case for the user of these lenses, and one which has been proven, is on the basis of improved circadian photoentrainment from greater activation of pRGCs if ultraviolet light is blocked and blue light photoreception increased [51]. Since circadian photoentrainment decreases with age owing to increasing opacity of the crystalline lens, and possibly from reduction in pupil size, artificial lenses that allow more visible short wavelength light into the eye (blue or violet) can boost photoentrainment in the retina. Out of these lenses the violet blocking lenses offer similar ultraviolet and blue photoprotection but better scotopic and melanopsin photoreception than blue blocking lenses [51]. Meanwhile sunglasses provide about 50% more ultraviolet and blue photoprotection than either violet or

Non-Rod Non-Cone Photoreception in Humans: Roles in Vision and Disease

231

blue blocking intraocular lenses [51]. However ultraviolet blocking intraocular lenses provide the older pseudophakic patient with the best possible rhodopsin and melanopsin sensitivity by maximising photoreception from both pRGCs and S-cones (including visual inputs from both cells), and also maximising any possible photoprotection for age-related macular degeneration on the basis of the blue light hazard theory. The effects of both visual and pupillary responses from pRGCs on retinal light and dark adaptation are also directly relevant to one further research area, which is the response of the pupil following refractive surgery [1,14,33]. Pupil size, dependent on the state of retinal adaptation, is an important factor in determining the area of optical ablation, and has yet to be optimised.

Conclusion The discovery of the ganglion cell photoreceptor in human retinas and the multiple roles that have been discussed, most novelly in visual perception, have led to a fundamental transformation in understanding how the eye and brain function [1,2]. The human chapter of the pRGC story only began to be told to a worldwide audience in 2007 [1,2]. As these new core concepts filter through to various fields and disciplines, a widening set of research areas with scope for genuine impact in basic and applied vision science seem likely to build upon these fascinating experimental discoveries [1,2]. Indeed in view of these advances it is now biologically plausible that groups of diseases will be regarded as dystrophies of the inner retinal photoreceptors, that is the photoreceptive ganglion cells, in an analogous manner to the genetic and phenotypic recognition of outer photoreceptor (rod and cone) dystrophies. This will be in addition to the numerous areas where, self-implicitly, the discovery of human photoreceptive retinal ganglion cells, especially their newly discovered role in visual perception, will be of significant importance to clinical practice and clinical research.

References

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

[1]

[2] [3]

[4]

Zaidi FH, Hull JT, Peirson SN, Wulff K, Aeschbach D, Gooley JJ, Brainard GC, Gregory-Evans K, Rizzo JF 3rd, Czeisler CA, Foster RG, Moseley MJ, Lockley SW. Short-wavelength light sensitivity of circadian, pupillary, and visual awareness in humans lacking an outer retina. Current Biology, 2007 Dec 18; 17: 2122-2128. Van Gelder RN. Non-visual photoreception: sensing light without sight. Current Biology, 2008 Jan 8; 18: R38-R39. Hattar S, Liao HW, Takao M, Berson DM, Yau KW. Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity. Science. 2002 Feb 8; 295: 1065-1070. Foster RG, Provencio I, Hudson D, Fiske S, De Grip W, Menaker M. Circadian photoreception in the retinally degenerate mouse (rd/rd). Journal of Comparative Physiology. A, Neuroethology, Sensory, Neural, and Behavioral Physiology, 1991 Jul; 169: 39-50.

232 [5]

[6]

[7]

[8]

[9] [10]

[11] [12]

[13] [14]

[15]

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

[16]

[17]

[18] [19]

Farhan Husain Zaidi Klein T, Martens H, Dijk DJ, Kronauer RE, Seely EW, Czeisler CA. Circadian sleep regulation in the absence of light perception: chronic non-24-hour circadian rhythm sleep disorder in a blind man with a regular 24-hour sleep-wake schedule. Sleep, 1993 Jun; 16: 333-343. Czeisler CA, Shanahan TL, Klerman EB, Martens H, Brotman DJ, Emens JS, Klein T, Rizzo JF 3rd. Suppression of melatonin secretion in some blind patients by exposure to bright light. The New England Journal of Medicine, 1995 Jan 5; 332: 6-11. Czeisler CA, Ede MC, Regestein QR, Kisch ES, Fang VS, Ehrlich EN. Episodic 24hour cortisol secretory patterns in patients awaiting elective cardiac surgery. The Journal of Clinical Endocrinology and Metabolism, 1976 Feb; 42: 273-283. Lucas RJ, Freedman MS, Muñoz M, Garcia-Fernández JM, Foster RG. Regulation of the mammalian pineal by non-rod, non-cone, ocular photoreceptors. Science, 1999 Apr 16; 284: 505-507. Foster RG. Bright blue times. Nature, 2005 Feb 17; 433: 698-699. Lockley SW, Skene DJ, Tabandeh H, Bird AC, Defrance R, Arendt J. Relationship between melatonin rhythms and visual loss in the blind. The Journal of Clinical Endocrinology and Metabolism, 1997; 82: 3763–3770. Lockley SW, Skene DJ, Butler LJ, Arendt J. Sleep and activity rhythms are related to circadian phase in the blind. Sleep, 1999; 22: 616–623. Keeler CE. Le réflexe irien a la lumière chez la souris à rétine sans batonnets. Comptes rendus des séances de la Société de biologie et de ses filiales, 1927; 96:10. Abstract in Zentralblatt für die Gesamte Ophthalmologie und ihre Grenzgebiete, 1927; 18: 252. Keeler CE. Iris movements in blind mice. The American Journal of Physiology, 1927; 81: 107-112. Zaidi FH. Novel Photoreceptor Cells, Pupillometry and Electrodiagnosis in Orbital, Vitreo-retinal and Refractive disorders. Imperial College London, the University of London: PhD thesis. 2008. Dacey DM, Liao HW, Peterson BB, Robinson FR, Smith VC, Pokorny J, Yau KW, Gamlin PD. Melanopsin-expressing ganglion cells in primate retina signal colour and irradiance and project to the LGN. Nature, 2005 Feb 17; 433: 749-754. Evans K, Fryer A, Inglehearn C, Duvall-Young J, Whittaker JL, Gregory CY, Butler R, Ebenezer N, Hunt DM, Bhattacharya S (1994). Genetic linkage of cone-rod retinal dystrophy to chromosome 19q and evidence for segregation distortion. Nature Genetics, 1994; 6: 210–213. Evans K, Duvall-Young J, Fitzke FW, Arden GB, Bhattacharya SS, Bird AC. Chromosome 19q cone-rod retinal dystrophy. Ocular phenotype. Archives of Ophthalmology, 1995; 113: 195–201. Cowey A, Weiskrantz L. Contour discrimination in rats after frontal and striate cortical ablations. Brain Research, 1971 Jul 23; 30: 241-252. Weiskrantz L, Cowey A. Effects of striate cortex removals on visual discrimination. Brain Research 1971 Aug 20; 31: 376.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Non-Rod Non-Cone Photoreception in Humans: Roles in Vision and Disease

233

[20] Weiskrantz L. Blindsight: A Case Study and Implications. Oxford University Press. 1986. [21] Schwartz SH. Visual Perception: a Clinical Orientation. 3rd edition. USA: McGrawHill. 2004. [22] Cowey, A. The 30th Sir Frederick Bartlett lecture. Fact, artefact, and myth about blindsight. The Quarterly Journal of Experimental Psychology. A, Human Experimental Psychology, 2004; 57: 577–609. [23] Gooley JJ, Lu J, Fischer D, Saper CB. A broad role for melanopsin in nonvisual photoreception. The Journal of Neuroscience, 2003; 23: 7093–7106. [24] Hattar S, Kumar M, Park A, Tong P, Tung J, Yau KW, Berson DM. Central projections of melanopsin-expressing retinal ganglion cells in the mouse. The Journal of Comparative Neurology, 2006; 497: 326–349. [25] Perrin F, Peigneux P, Fuchs S, Verhaeghe S, Laureys S, Middleton B, Degueldre C, Del Fiore G, Vandewalle G, Balteau E, Poirrier R, Moreau V, Luxen A, Maquet P, Dijk DJ. Nonvisual responses to light exposure in the human brain during the circadian night. Current Biology, 2004; 14: 1842–1846. [26] Vandewalle G, Balteau E, Phillips C, Degueldre C, Moreau V, Sterpenich V, Albouy G, Darsaud A, Desseilles M, Dang-Vu TT, Peigneux P, Luxen A, Dijk DJ, Maquet P. Daytime light exposure dynamically enhances brain responses. Current Biology, 2006; 16: 1616–1621. [27] Vandewalle G, Gais S, Schabus M, Balteau E, Carrier J, Darsaud A, Sterpenich V, Albouy G, Dijk D, Maquet P. Wavelength-dependent modulation of brain responses to a working memory task by daytime light exposure. Cerebral Cortex, 2007; 17: 2788– 2795. [28] Lockley SW, Brainard GC, Czeisler CA. High sensitivity of the human circadian melatonin rhythm to resetting by short wavelength light. The Journal of Clinical Endocrinology and Metabolism, 2003; 88: 4502–4505. [29] Lockley SW, Evans EE, Scheer FA, Brainard GC, Czeisler CA, Aeschbach D. Shortwavelength sensitivity for the direct effects of light on alertness, vigilance, and the waking electroencephalogram in humans. Sleep, 2006: 29:161–168. [30] Cajochen C, Munch M, Kobialka S, Krauchi K, Steiner R, Oelhafen P, Orgul S, WirzJustice A. High sensitivity of human melatonin, alertness, thermoregulation, and heart rate to short wavelength light. The Journal of Clinical Endocrinology and Metabolism, 2005; 90: 1311–1316. [31] Foster RG, Hankins MW. Circadian vision. Current Biology 2007; 17: R746–R751. [32] Lockley SW, Gooley JJ. Circadian photoreception: Spotlight on the brain. Current Biology, 2006; 16: R795–R797. [33] Zaidi FH, Bremner FD, Gregory-Evans K, Cocker KD, Moseley MJ. Subretinal membranes are associated with abnormal degrees of pupil "evasion": an index of clinical macular dysfunction. The British Journal of Ophthalmology, 2006; 90: 11151118. [34] Lucas RJ, Douglas RH, Foster RG. Characterization of an ocular photopigment capable of driving pupillary constriction in mice. Nature Neuroscience 2001 Jun; 4: 621-626.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

234

Farhan Husain Zaidi

[35] Lockley SW, Skene DJ, James K, Thapan K, Wright J, Arendt J. Melatonin administration can entrain the free-running circadian system of blind subjects. The Journal of Endocrinology 2000; 164: R1–R6. [36] Sack RL, Brandes RW, Kendall AR, Lewy AJ. Entrainment of free-running circadian rhythms by melatonin in blind people. The New England Journal of Medicine, 2000; 343: 1070–1077. [37] MacLaren RE, Pearson RA, MacNeil A, Douglas RH, Salt TE, Akimoto M, Swaroop A, Sowden JC, Ali RR. Retinal repair by transplantation of photoreceptor precursors. Nature, 2006 Nov 9; 444: 203-207. [38] MacLaren RE, Pearson RA. Stem cell therapy and the retina. Eye. 2007 Oct; 21: 13521359. [39] Buch PK, MacLaren RE, Ali RR. Neuroprotective gene therapy for the treatment of inherited retinal degeneration. Current Gene Therapy, 2007 Dec; 7: 434-445. [40] Bainbridge JW, Smith AJ, Barker SS, Robbie S, Henderson R, Balaggan K, Viswanathan A, Holder GE, Stockman A, Tyler N, Petersen-Jones S, Bhattacharya SS, Thrasher AJ, Fitzke FW, Carter BJ, Rubin GS, Moore AT, Ali RR. Effect of gene therapy on visual function in Leber's congenital amaurosis. The New England Journal of Medicine, 2008 May 22; 358: 2231-2239. [41] Diabetic Retinopathy Clinical Research Network. A randomized trial comparing intravitreal triamcinolone acetonide and focal/grid photocoagulation for diabetic macular edema. Ophthalmology, 2008 Sep; 115: 1447-1449. [42] Kook D, Wolf A, Kreutzer T, Neubauer A, Strauss R, Ulbig M, Kampik A, Haritoglou C. Long-term effect of intravitreal bevacizumab (avastin) in patients with chronic diffuse diabetic macular edema. Retina, 2008 Oct; 28: 1053-1060. [43] Gupta B, Grewal J, Adewoyin T, Pelosini L, Williamson TH. Diurnal variation of macular oedema in CRVO: prospective study. Graefe’s Archive for Clinical and Experimental Ophthalmology, 2008 Dec 4 [Electronic publication ahead of print]. [44] Diabetic Retinopathy Clinical Research Network. Optical coherence tomography measurements and analysis methods in optical coherence tomography studies of diabetic macular edema. Ophthalmology, 2008 Aug; 115: 1366-1371. [45] Hong S, Seong GJ, Hong YJ. Long-term intraocular pressure fluctuation and progressive visual field deterioration in patients with glaucoma and low intraocular pressures after a triple procedure. Archives of Ophthalmology, 2007 Aug; 125: 10101013. [46] Caprioli J. Intraocular pressure fluctuation: an independent risk factor for glaucoma? Archives of Ophthalmology, 2007 Aug; 125: 1124-1125. [47] Orzalesi N, Fogagnolo P, Rossetti L. Intraocular pressure fluctuations in glaucoma. Archives of Ophthalmology, 2008 May; 126: 745. [48] Nouri-Mahdavi K, Medeiros FA, Weinreb RN. Fluctuation of intraocular pressure as a predictor of visual field progression. Archives of Ophthalmology, 2008 Aug; 126: 1168-1169 and 1170; also 2008 Oct; 126: 1456.

Non-Rod Non-Cone Photoreception in Humans: Roles in Vision and Disease

235

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

[49] Miglior S. Long-term intraocular pressure fluctuations and progressive visual field deterioration in patients with glaucoma: which comes first? Archives of Ophthalmology 2008 Nov; 126: 1609 and 1609-1610. [50] Pandi-Perumal SR, Trakht I, Srinivasan V, Spence DW, Maestroni GJ, Zisapel N, Cardinali DP. Physiological effects of melatonin: role of melatonin receptors and signal transduction pathways. Progress in Neurobiology 2008 Jul; 85: 335-353. [51] Mainster MA. Violet and blue light blocking intraocular lenses: photoprotection versus photoreception. British Journal of Ophthalmology, 2006 Jun; 90: 784-792.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

In: Retinal Degeneration: Causes, Diagnosis and Treatment ISBN 978-1-60741-007-2 Editor: Robert B. Catlin © 2009 Nova Science Publishers, Inc.

Chapter 12

In Vivo Visualization of Photoreceptor Layer and Lipofuscin Accumulation in Stargardt’s Disease / Fundus Flavimaculatus by Optical Coherence Tomography Giuseppe Querques∗1,2, Domenico Martinelli3, Lea Querques1,2, Gisèle Soubrane1 and Eric H Souied1 1

Department of Ophthalmology, Hopital Intercommunal de Creteil, University Paris XII, France 2 Department of Ophthalmology, Ospedali Riuniti, University of Foggia, Italy 3 Department of Hygiene, Ospedali Riuniti, University of Foggia, Italy

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Abstract Purpose: Retinal flecks are commonly observed in both Stargardt’s disease (STGD) and fundus flavimaculatus (FFM). The aim of our study was to determine the precise localisation of these flecks within the retinal layers using Stratus optical coherence tomography (OCT3, Humphrey-Zeiss, San Leandro, California). Moreover we assessed photoreceptor (PR) morphology in patients with STGD and FFM using high definition OCT (HD-OCT, OCT 4000 Cirrus, Humphrey-Zeiss, San Leandro, California). Finally, we tried to investigate the relationship between PR layer morphology and localization of retinal flecks, as evaluated respectively by HD-OCT and OCT, and best corrected visual acuity (BCVA). Methods: This was a prospective observational case series. A complete ophthalmologic examination, including best corrected visual acuity (BCVA) and OCT (OCT3 and/or HD-OCT) was performed in 40 consecutive patients with STGD/FFM.

∗

Telephone: +33 (0)1 45 17 52 22; Fax: +33 (0)1 45 17 52 66; E mail : [email protected]

238

Giuseppe Querques, Domenico Martinelli, Lea Querques et al. Results: A total of 76 eyes were included in the study. Using OCT (OCT3 and/or HD-OCT), we observed hyperreflective deposits which we classified in two types: type 1 lesions located in the inner part of the retinal pigment epithelium layer and type 2 lesions located at the level of the outer nuclear layer. Moreover, HD-OCT was capable of visualizing regions of transverse PR loss in the foveal region. BCVA impairment showed a statistically significant correlation to the presence of complete loss of PR layer in the foveal region (p 98 % pure and remaining ~ 2 % constituents have not been defined but do contain immunoglobulins [45]. Serum provides nutritive support to the retina and albumin, a major component of the serum and vitreous, augments retinal precursor cell proliferation [46]. The present report discusses the role of critical components of R16 medium and compares the in vivo and in vitro development of retina. The effects of various molecules and compounds added to the medium, individually or in combination, to rescue photoreceptors of rd1 mouse model of Retinitis pigmentosa were also compared. The factors / nutrients included lens epithelium-derived growth factor (LEDGF), ciliary neurotrophic factor (CNTF), brain-derived growth factor (BDNF), nerve growth factor (NGF), basic fibroblast

266

Satpal Ahuja, Poonam Ahuja-Jensen, A. Romeo Caffé et al.

growth factor (bFGF), glutathione-S-transferases (GST) and antioxidants namely lutein, zeaxanthin, glutathione and α-lipoic acid. Table 1. Composition of Basal R16 culture medium (Invitrogen Life Technologies, Paisley, Scotland, UK) mg l-1

Ingredient

mg l-1

Ingredient (x 10-3 M)

Putescine

16.11

(x 10-3 M) Glucose (g l-1)

(0.18)

CaCl2.2H2O

188.74 (1.28)

MgSO4.7H2O

NaH2PO4.2H2O

95.38

(0.61)

Na2HPO4

NaCl (g l-1)

6.03

(103.0)

KCl

-4

(19.1) 168.27 (0.68)

31.95

(0.23) 320.34

(4.29)

104.12

(4.94)

-4

(x 10 M)

(x 10 M)

L-Alanine

2.01

(0.23)

L-Arginine

L-Asparagine H2O

3.38

(0.23)

L-Cystine Na2

L-Glycine

21.94

(2.92)

L-Histidine HCl.H2O

33.07

(1.58)

L-Isoleucine

71.63

(5.46)

L-Leucine

73.70

(5.62)

L-Lysine HCl

106.90

(5.85)

L-Methionine

21.25

(1.42)

L-Phenylalanine

45.67

(2.76)

L-Proline

7.78

(0.68)

L-Serine

30.72

(2.92)

L-Threonine

66.94

(5.62)

L-Tryptophan

11.26

(0.55)

L-Tyrosine

49.82

(2.75)

L-Valine

65.82

(5.62)

CDP Ethanolamine 1.28

10.0

(1.34)

2.56 -5

(x 10 M) D(+)-Mannose

38.33

CDP Choline -5

(x 10 M) (5.6)

D(+)-Galactose

-6

15.0

(8.3)

-6

(x 10 M)

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

3.443

(x 10 M)

L-Carnitine

2.0

(12.4)

Choline Chloride

6.07

(43.5)

Pyridoxal HCl

2.72

(13.4)

D-Ca Pentothenate

2.75

(5.77)

FeSO4.7H2O

0.19

(0.68)

Fe (NO3)3.9H2O

0.068

(0.17)

ZnSO4.7H2O

0.20

(0.70)

Folic Acid

3.0

(6.79)

i-Inositol

8.78

(48.7)

Nicotinamide

2.71

(22.2)

Hypoxanthine

0.92

(6.75)

Riboflavin

0.28

(0.74)

Thymidine

0.162

(0.67)

CDP-cytidine 5´-diphospho-, NaHCO3, 2.7 g l-1, Sodium Phenol Red 5.0 mg l-1

Serum-Free Retinal Explant Culture System and Comparative Rescue Effects…

267

Material and Methods Animals and Tissues

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

All mice were treated in accordance with the ARVO Statement for the use of Animals in Ophthalmic and Vision Research and the European Communities Council Directive No. 86/609/EEC. The Swedish National Animal Care and Ethics Committee approved the experiments. Wild type control (wt) mice of the C3H strain and congenic homozygous (rd1/rd1) retinal degeneration 1 (rd1) mice were used for the studies. The day of birth was considered as postnatal day 0 (PN0). Pups, PN7 and younger were sacrificed by decapitation and older mice were sacrificed by asphyxiation with dry ice.

Figure 1. Aseptic dissection of retina after proteinase K treatment of the eye: Using a dissecting microscope (Olympus SZX9, Olympus in Europe, Hamburg, Germany) retina was aseptically dissected from an eye placed in the R16 medium in a Petri dish. By cutting a little behind the limbus (step 1), the anterior segment, lens and vitreous body were removed as one unit (step 2). Sclera and choroids were peeled off carefully by using the # 5 forceps with rounded edges and the neural retina was left intact with the RPE attached to it (steps 3, 4). With Venessa iridoectomy scissors, four cuts were made on the retina in a manner perpendicular to its periphery. The retina (with the attached RPE) was gently lifted by holding the remnants of the cloquets canal and then flat mounted (step 5), with the photoreceptor side facing downwards, on a piece of GN4 Metricel Cellulose filter paper (pore size of 0.8 µm, Pall Gelman Sciences, Lund, Sweden) attached to a polyamide grid (Monodur PA56N, AB Derma, Gråbo, Sweden). The explants were then placed in a well of the six well culture dish plate containing 1.5 to 1.6 ml R16 complete culture medium and incubated at 37ºC, 100 % humidity and 5 % CO2 in air. The spent medium was replaced by fresh R16 complete medium. The explants were discarded if the medium turned yellow in any of the wells. Ready-made membrane inserts of Metricel cellulose or nitrocellulose or mixed cellulose esters with pore size between 0.4 µm and 0.8 µm are used for culturing retinal explants. The six well plates were covered with loose fitting plastic lids. These instead can be desirably covered with a gas permeable sealing film. Reproduced from Reference 127

For morphological and immunohistochemical analyses, eyes were collected both from wt and rd1 mice at the age of PN2, -7, -14, -21 and -28. After enucleation, the eyes were fixed in

268

Satpal Ahuja, Poonam Ahuja-Jensen, A. Romeo Caffé et al.

cold 4 % paraformaldehyde in Sorensen’s phosphate buffer (pH 7.4) for 1-2 hours, rinsed and cryoprotected in Sorensen’s buffer containing increasing concentrations of sucrose. The 8 µm sections were obtained on a cryotome and stored at -20°C until used. For biochemical studies, eyes were enucleated and the anterior segment, vitreous body, sclera and choroids were dissected out in cold dissecting medium and retinas with attached retinal pigment epithelium (RPE) were removed (Figure 1) and frozen at -80°C until analyzed.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Culture method After sacrificing the animals, the heads were removed and cleaned with 70 % ethanol. The eyes were enucleated aseptically and incubated in R16 medium supplemented with 0.12 % proteinase K (ICN Biomedicals Inc., Aurora, Ohio, USA) at 37°C for 15 minutes. This step was incorporated to facilitate the separation of the neural retina with attached RPE from the adjoining mesenchymal cell layers. The proteinase K was inactivated by placing the eyes in excess of 10 % FCS in R16 medium to dilute out proteinase K activity with excess of proteins. This was followed by dissection and initiation of culturing as described in Figure 1. The isolated retinas with attached RPE were placed with the photoreceptor side down onto nitrocellulose membranes of 0.8µm pore size attached to a grid or cell culture inserts of 0.4 µm pore size for 6-well plates. Currently the latter are used. The retinal explants were incubated for various periods of time in 1.5 to 1.6 ml of serum-free R16 complete medium at 37°C. Ready-made membrane inserts of mixed cellulose esters are now used for culturing retinal explants. During incubation the plates were covered with a loose fitting plastic cover. However, a gas permeable microplate sealing film should be preferred as it allows uniform flow of gases. Composition of culture medium A chemically defined (Table 1), custom-made serum free R16 culture medium (Invitrogen Life Technologies, Paisley, Scotland, UK) supplemented with a number of nutrients (Table 2) was designated as R16 complete medium and used to culture the mouse retinal explants collected at different ages after birth. The R16 complete medium is made up of salts, sodium bicarbonate, trace elements, CDP-ethanolamine, amino acids (except neurotoxic glutamate and aspartate), BSA, transferrins, sugars, hormones namely insulin, steroids, T3 and vitamins like nicotinamide, biotin, riboflavin, folic acid and vitamin C (Table 2). The test substances LEDGF (10 ng ml-1), α-GST (10 ng ml-1), µ-GST (10 ng ml-1) CNTF, BDNF (each 10 ng ml-1 individually and in combination), NGF, bFGF (10 ng ml-1 individually or in combination) and antioxidants, namely reduced glutathione (GSH), α-lipoic acid, lutein and zeaxanthin (individually or in combination [8]) were added to the R16 complete medium in required concentrations. Where applicable, the untreated controls received equivalent amounts of vehicle. On alternating days, the spent medium was replaced with the same volume of fresh medium. LEDGF was a gift from Professor T. Shinohara, Department of Ophthalmology, University of Nebraska Medical Center, Omaha, USA). GST (Oxford Medical Research, Oxford, Michigan, USA), GSH (Sigma-Aldrich Inc.), lutein and zeaxanthin (Extrasynthese, Genay, France) and α-lipoic acid (Fluka Chemie GmbH, Buche, Switzerland) were of commercial origin.

Serum-Free Retinal Explant Culture System and Comparative Rescue Effects…

269

Table 2. Concentration of components added to the R16 culture medium to form complete R16 Medium with and without fetal calf serum (FCS) -------------------------------------------------------------------------------------------------------------------Ingredient

Concentration for 100 ml complete R16

Basal R16

-

BSA

0.2% Added 100 µL each of the following to each 100 ml of the medium

Transferrin

10 µg ml-1

Progesterone

6.3 ng ml-1

Insulin

0.2 µg ml-1

T3

2.0 ng ml-1

Corticosterone

20.0 ng ml-1

Thiamine HCL

2.77 µg ml-1

Vitamin B12

0.31 µg ml-1

Thioctic acid

0.045 µg ml-1

L-Cysteine

7.09 µg ml-1

Glutathione

1.0 µg ml-1

Sodium Pyruvate

50 µg ml-1

Ethanolamine

1 µL ml-1

Biotin

0.1 mg ml-1

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Added 200 µL each of the following to each 100 ml of the medium Retinol

0.1 µg ml-1

Retinyl Acetate

0.1 µg ml-1

DL-Tocopherol

1.0 µg ml-1

Tocopherol Acetate

1.0 µg ml-1

Linoleic Acid

1.0 µg ml-1

Linolenic Acid

1.0 µg ml-1

Added 1 ml each of the following to each 100 ml of the medium Glutamine

25 µg ml-1

Vitamin C

100 µg ml-1

______________________________________________________________________ Added 12.3 ml Millipore water without FCS to get serum free medium or added 5.3 Millipore water with 10.0 ml of 10.0 % FCS to get complete R16 medium with serum; Micronutrients

270

Satpal Ahuja, Poonam Ahuja-Jensen, A. Romeo Caffé et al. Na2SeO3.5H2O 7.9 mg, MnCl2.4H2O 1.0 mg, CuSO4.5H2O 2.5 mg dissolved in 1 100 ml and used 10 µl dl-1 medium

Retina Organ Culture and In Vivo Treatment

For in vitro studies, retinas from homozygous rd1 and congenic wt control mice of the C3H strain at PN2, -7, -11 and -21 were used for culturing up to the age of PN28 implying that in case of PN2, -7, -11 and -21 retinas were cultured for 26, 21, 17 and 7 days in vitro (div) respectively. To test the rescue effects of LEDGF, GST, NGF, bFGF, CNTF and BDNF, retinas were obtained at PN2 and PN7 only and for antioxidant combinations, the retinas were taken at PN5 and cultured for div13. After completion of culture period, the explants attached to the nitrocellulose membrane or other membrane inserts were fixed in 4 % paraformaldehyde for one hour, and processed as described above. For in vivo studies, the antioxidant combination was administered orally to PN3 mice for two weeks and retinas were collected at PN17. Similar studies were conducted with individual antioxidants.

Examination of Retinas For routine morphology and cell counting studies, sections were stained with hematoxylin and eosin (H & E). Vertical columns of cells in the centre of the explants were chosen for counting the number of rows of nuclei in the outer nuclear layer (ONL). For retinas obtained from mice treated in vivo with antioxidants, the rows of nuclei in the central, mid- and far-peripheral regions were counted. The number of sections in each category was taken randomly and 4-5 explants were counted in each category. Details of the immunohistochemical procedures used to localize rhodopsin, arrestin, interphotoreceptor retinol binding protein (IRBP), calbindin, parvalbumin, calretinin, GST and green cone specific proteins have been provided previously [24, 6, 8, 40 and 31].

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Quantification of Malondialdehyde, GSH, Glutathione Reductase and –Peroxidase The activities of glutathione peroxidase (GPx) [47] and glutathione reductase (GR) [48] in PN2, -7, -14, -21 and -28 retinal extracts [see references 2, 15, 16 for preparation of retinal extracts] of wt and rd1 mice were measured spectrophotometrically by monitoring the oxidation of NADPH at 340 nm. The levels of malondialdehyde [49] and GSH [50] in the above mentioned retinal extracts were determined by high-pressure liquid chromatography.

Lipid and Antioxidant(s) Pigment Extraction from Wolfberry Fruits The retinal macula contains the carotenoids zeaxanthin, lutein and meso-zeaxanthin [51] which originate from pigments of dietary fruits and plants. Wolfberry (Lycium barbarum L.) fruits are commonly used in China as source of antioxidants and were therefore analyzed. The ripe wolfberry fruits were pulverized in a porcelain pestle and mortar with excess of

Serum-Free Retinal Explant Culture System and Comparative Rescue Effects…

271

liquid nitrogen. This was done until the berries were converted in to a sticky powder with small intact kernels. The powdered berries were repeatedly extracted with a total of 20 times volume of chloroform : methanol (2 : 1, v / v). The pooled extracts were dried at 70°C in the presence of gaseous nitrogen. The dried extract representing pigment plus lipids were weighed. A dilute solution of the dried extract was prepared in chloroform and the λ max wavelength absorption spectrum of the same was measured by UV-Vis Spectrophotometer (Hitachi U-2001, Japan) and compared with the spectra reported in the literature for purified retinal zeaxanthin and lutein [52, 53 and 54].

Statistical Analysis Statistical analyses of the data was performed by using StatView Software (version 5.0, SAS Institute Inc., Cary, NC, USA) and one-way analysis of variance and Fisher’s protected least significant differences post hoc comparisons.

Results

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Comparative Histology of In Vivo and In Vitro Retinas Retinal Morphology and Cellular Localization of IRBP, Arrestin, Rhodopsin and Opsin in In Vivo and In Vitro Retinas H&E staining (Figure 2 a, e, i and m) showed that PN2+div26, -7+div21 (data not shown), -11+div17 and -21+div7 retinal explants and PN28 in vivo retinas had similar pattern of lamination (Figure 2 a). The strong staining for IRBP (Figure 2 b), arrestin (Figure 2 c) and rhodopsin (Figure 2 d) by photoreceptor segments (when present in the explants) in PN28 in vivo retina as compared to PN2+div26 (Figure 2 f-h), -11+div17 (Figure 2 j-l) and -21+div7 (Figure 2 n-p) explants indicated expression of these proteins during in vitro culture. Pigment laden cells traversing the subretinal space were observed in PN21+div7 explants (Figure 2 n) after immunostaining for IRBP. In PN2+div26 explants (Figure 2 h), the immunoreactivity for rhodopsin was limited to cell bodies of the ONL since characteristic photoreceptor segments do not appear to be formed. The number of photoreceptor rows in the ONL of PN28 in vivo and of PN2+div26 (Figure 2 e), -11+div17 (Figure 2 i) and -21+div7 (Figure 2 m) retinal explants were 13.6±0.2, 7.9±0.2, 8.0±0.3 and 7.4±0.3, respectively. The PN7+div21 explants (histological picture not shown) exhibited 8.1±0.3 rows of photoreceptor nuclei.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

272

Satpal Ahuja, Poonam Ahuja-Jensen, A. Romeo Caffé et al.

Figure 2. Comparison of the morphology (a, e, i and m) and expression of IRBP (b, f, j and n), arrestin (c, g, k and o) and rhodopsin (d, h, l and p) proteins by PN28 (a-d) in vivo retina and by PN2 (e-h), -11 (i-l) and -21 (m-p) retinal explants cultured in vitro respectively for div26, div17 and div7 in a serum free R16 complete medium.S, photoreceptor segments; ONL, INL, outer- and inner nuclear layers; Scale bar = 50 µm. (Copyright Permission Reference 24)

Serum-Free Retinal Explant Culture System and Comparative Rescue Effects…

273

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Comparison of H&E stained sections from PN2+div26 (Figure 3 A, C) and PN7+div21 (Figure 3 B, D) retinas of wt (Figure 3 A, B) and rd1 (Figure 3 C, D) mice, showed preservation of the retinal cellular characteristics of each genotype. However, the numbers of photoreceptor rows in the retinal explants were fewer than in corresponding in vivo retinas. Similar sections of PN2+div26 (Figure 3 E, G) and PN7+div21 (Figure 3 F, H) retinas of wt (Figure 3 E, F) and rd1 (Figure 3 G, H) mice, respectively showed clear immunostaining of opsin in the photoreceptor cells present in the ONL of both genotypes. The inner nuclear layer (INL) as well as the retinal ganglion cell (RGC) profiles in the ganglion cell layer (GCL) could also be appreciated (Figure 3 E-H).

Figure 3. Retinal morphology as observed by H&E (A, B, C and D) and spatial immunolocalization (E, F, G and H) of opsin in the sections of PN2 (A, C, E and G) and PN7 (B, D, F and H) wt (A, B, E and F) and rd1 (C, D, G and H) retinas respectively cultured for div26 and div21 days confirmed the preservation of histology and biosynthetic activities of cultured explants. GCL, ganglion cell layer; IPL, inner plexiform layer; INL and ONL, inner- and outer nuclear layers, Scale bar = 25 μm.

In Vivo and In Vitro Retinal Expression of Calbindin, Parvalbumin, Calretinin and Green Cone Specific Proteins In in vivo retinas (Figure 4 a), horizontal cells, cells present in the GCL, in the inner part of the INL, and the three lamini formed by neurites occurring in the inner plexiform layer

274

Satpal Ahuja, Poonam Ahuja-Jensen, A. Romeo Caffé et al.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

(IPL) showed strong labeling for calbindin, a neuronal marker. Although similar elements were stained in cultured explants, the immunoreactivity was less organized as shown in PN2+div26 explants (Figure 4 b). Calbindin stained profiles sprouting into the ONL were also observed in the explants. Parvalbumin immunostaining along both sides of the IPL was similar in the PN28 in vivo retina (Figure 4 c) and in PN2+div26 explants (Figure 4 d). However, in the latter the number of stained profiles was lower and there was no staining for axonal sprouts. Similarly, the number of calretinin immunostained profiles was lower in the PN2+div26 explants (Figure 3 f) as compared to PN28 in vivo retinas (Figure 4 e). Green cone specific protein was observed both in the in vivo PN28 retina (Figure 5 a) and PN21+div7 (Figure 5 d) in vitro explants but was sporadic in PN11+div17 (Figure 5 c) explants and was absent in PN2+div26 (Figure 5 b) explants.

Figure 4. Spatial immunolocalization of calbindin (a, b), parvalbumin (c, d) and calretinin (e, f) proteins in PN28 (a, c and e) in vivo retinas and PN2+div26 (b, d and f) in vitro retinal explants was similar. Three lamini were formed by neurites present in the inner plexiform layer (IPL). Scale bar = 50 µm. (Copyright Permission Reference 24)

In Vivo Retinal Expression and Localization of Α-GST and µ-GST in Rd1 and Wt Retinas The PN2 (Figure 6 a, b) and PN7 (Figure 6 c, d) in vivo retinas of both genotypes showed α-GST immunostaining in the GCL (arrow). The PN7 retinas additionally showed a weak immunoreaction in the presumptive horizontal cell bodies and their processes.

Serum-Free Retinal Explant Culture System and Comparative Rescue Effects…

275

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Figure 5. Spatial immunolocalization of green cone specific proteins in PN28 in vivo retina (a) and PN2+div26 (b) PN11+div17 (c) and PN21+div7 (d) in vitro retinal explants was similar. Scale bar = 50 µm. (Copyright Permission Reference 24)

In PN14 rd1 retinas (Figure 6 e) immunoreactivity in the GCL was decreased and was localized in the Mueller cell endfeet, at the level of outer plexiform layer (OPL) (*) and possibly in the astrocytes (arrow). In PN14 wt (Figure 6 f) retinas immunostaining was observed only in the GCL and OPL. In PN21 (Figure 6 g, h) and PN28 (Figure 6 i, j) retinas of both genotypes the immunoreactivity in the inner retina was restricted to the presumptive Mueller cell endfeet (arrow) and to the OPL (*). Distinct immunostaining in the Mueller cell radial endfeet, descending processes and in the horizontally oriented fibers in the OPL (*) was confirmed at higher magnification of the boxed area (Figure 6 j) [Reference 7 for details]. The PN2 (Figure 6 a, b) and PN7 (Figure 6 c, d) in vivo retinas of both genotypes showed µ-GST immunostaining in the GCL (arrows, Figure 6 a-d). The PN14 (Figure 6 e, f), -21 (Figure 6 g, h) and -28 (Figure 6 i, j) stages of both genotypes expressed µ-GST in Mueller cell endfeet (arrows) and in the OPL (*). Distinct immunostaining in the Mueller cell radial endfeet, descending processes and in the horizontally oriented fibers in the OPL (*) was confirmed at higher magnification of boxed the area (Figure 6 j) [Reference 7 for details]. These observations were further confirmed in colocalization studies of µ-GST with glutamine synthetase and neurofilament protein [7]. All these results indicated that in vivo and in vitro retinas of different ages and cultured for up to the in vitro age of PN28 showed development of cell layers and presence of marker proteins characteristic of each genotype and stage of development. However, the extent of in vitro development of these features was lower than that observed in in vivo.

276

Satpal Ahuja, Poonam Ahuja-Jensen, A. Romeo Caffé et al.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Α-GST (Figure 6 Lanes 1 and 2)

Figure 6. Spatial immunolocalization of α-GST (a-j, lanes1 and 2) and µ-GST (a-j, lanes 3 and 4) in rd1 (a, c, e, g and i) and wt (b, d, f, h and j) retinas at PN2 (a, b), -7 (c, d), -14 (e, f), -21 (g, h) and -28 (i, j) in vivo retinas was similar [see reference 4 for the details]. GCL, ganglion cell layer, IPL, OPL, innerand outer- plexiform layer, INL, ONL, inner- and outer- nuclear layers, RPE, retinal pigment epithelium. Scale bar = 50 µm. (Copyright permission Reference 7) µ-GST (Figure 6 Lanes 3 and 4)

Comparative Levels of GSH, Gpx, GR and Malondialdehyde Indicated Oxidative Stress in Rd1 Retinas Elevation of GSH and malondialdehyde and deficiency of GPx in PN2 rd1 retinas and elevation of malondialdehyde [Reference 5, Figure 1 d] in PN28 wt retinas suggested oxidative stress at two different stages of development. The PN2 rd1 retina is unable to

Serum-Free Retinal Explant Culture System and Comparative Rescue Effects…

277

counteract the oxidative stress in spite of higher GSH but the PN28 wt retina possibly takes care of the oxidative stress. GSH decreased with age in both genotypes [Reference 5, Figure 1 a]. In rd1 retinas GPx increased with age but was not sufficient to counteract the oxidative stress and in wt retinas it was almost constant after the PN2 stage [Reference 5, Figure 1 b]. GR increased with age in rd1 retinas but was almost unchanged in wt retinas [Reference 5, Figure 1 c]. Such results suggest the need to provide therapeutic help at an early age of development, even before the development of retinal lamination.

Wolfberry Fruits Are a Rich Source of Retinal Pigments The retinal macula contains the carotenoids such as zeaxanthin, lutein and mesozeaxanthin [51] which originate from pigmented dietary fruits. The mean content of pigments plus lipids in the wolfberry fruits as extracted by chloroform and methanol mixture (2 : 1, v / v) was 4.10 %. The wavelength absorption spectra (Table 3) of the diluted extracts showed λ max peak in the UV light absorption range at 389 nm with proportional absorbance of 0.125. Additionally five λ max peaks in the visible light absorption range were observed at 407, 432, 458, 488 and 509 nm. Table 3. Proportional absorption spectrum of the pigments extracted from ripe wolfberry (Lycium barbarum L.) fruits

Peak

λ max (nm)

Absorbance

Q-ratio

Absorption in the UV light region I

389

0.125

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Absorption in the visible light region II

407

0.160

III

432

0.219 (0.019)

IV

458

0.259 (0.059)

V

488

0.210 (0.010)

VI

509

0.101

0.322 {32.2}

0.170 {17.0}

The proportional absorbance of these peaks was 0.160, 0.219, 0.259, 0.210 and 0.101, respectively. The λ max peaks at 432 (III), 458 (IV) and 488 (V) nm showed higher absorption. If 0.200 is considered as the minimum baseline, the absorbance of residual peaks then measures 0.019, 0.059 and 0.010, respectively. The height ratio of two cis peaks to that of the main peak is referred to as Q-ratio and indicates the fine structure of the carotenoids [53, 54]. The percent ratio of peak III / IV and peak V / IV were 32.2 and 17.0, respectively.

278

Satpal Ahuja, Poonam Ahuja-Jensen, A. Romeo Caffé et al.

The wavelength absorption spectrum of the wolfberry extract appeared to be similar to that of the retinal carotenoids zeaxanthin and lutein. This was confirmed when these spectra were compared with those of purified retinal lutein and zeaxanthin reported in the literature [52, 53 and 54]. There was a weak absorption in the UV range which along with the characteristic visible light spectrum suggests the presence of all-trans and 9-, 9-cis isomers of zeaxanthin and lutein. All-trans isomers absorb strongly in the visible region between 400 and 500 nm. The visible spectrum of zeaxanthin and lutein - derivatives resembles that of β-carotene [52, 53 and 54]. Q-ratio of the cis peaks, around the highest λ max (nm), was calculated after considering 0.200 absorbance value as the common base line. The residual absorbance values given in parenthesis were then used to calculate the Q-ratio. [53]. Q-ratios given in parenthesis in percent.

Rescue of Rd1 Photoreceptors by LEDGF, GST, CNTF + BDNF, NGF + Bfgf and Antioxidant Supplementation In Vitro Supplementation of LEDGF Did Not Affect the Expression of Opsin and Arrestin in Rd1 and Wt Explants LEDGF supplementation did not affect the expression of opsin and arrestin [Reference 6, Figure 1 a-d] in PN2+div26 [Reference 6, Figure 1 a, b] and PN7+div21 [Reference 6, Figure 1 c, d] rd1 explants. Irrespective of the treatment, the wt (data not shown) and rd1 explants showed opsin and arrestin immunoreactivity in the photoreceptor somata and in their segment-like structures when present.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

LEDGF Supplementation Rescued the Photoreceptors in Rd1 Retina H&E staining of sections of PN2 and PN7 rd1 retinal explants showed that the LEDGF supplementation for div26 and div21, respectively, increased the number of photoreceptor rows in the ONL but did not do so in wt retinal explants [Reference 6, Figure 1]. This was confirmed by quantitative analysis [Reference 6, Figure 2]. The expression of opsin and arrestin [Reference 6, Figure 2 a-d] in PN2+div26 [Reference 6, Figure 2 a] and PN7+div21 [Reference 6, Figure 2 c] untreated rd1 explants was similar to that of LEDGF treated PN2+div26 [Reference 6, Figure 2 b] and PN7+div21 rd1 explants [Reference 6, Figure 2 d]. LEDGF supplementation of PN2+div26 and PN7+div21 [Reference 6, Figure 2] explants of rd1 mice, respectively showed a significant increase in the number of photoreceptor rows. Photoreceptor rows in the PN2 and PN7 control mice [Reference 6, Figure 2] were inherently higher and were not influenced by the LEDGF supplementation.

Serum-Free Retinal Explant Culture System and Comparative Rescue Effects…

279

Both Α-GST and µ-GST Rescued the Rd1 Photoreceptors In both genotypes, the level of α-GST was higher than that of µ-GST and α-, µ-GST decreased with age, especially in rd1 retinas. The highest levels of α-GST were seen in PN2 and PN7 retinas of both genotypes, particularly in rd1 retinas (Figure 7 A). This corresponded with the lower level of GPx and the higher levels of GSH and malondialdehyde [Reference 5, Figure 1] in such retinas. Deficiency of GST observed at an early stage in the rd1 retina was confirmed by a better rescue of photoreceptor after supplementation with GST (Figure 7 C), LEDGF [Reference 6, Figure 2] or antioxidant combination [Reference 8, Figures 2 and 4] provided to in vivo and in vitro rd1 mice retinas at PN2/3 stage. The untreated (Figure 8 c) PN2+div26 rd1 explants had 2 rows of photoreceptors compared to 5 such rows shown by the corresponding PN2+div26 (Figure 8 a, b) explants treated with α-GST or µ-GST. As compared to PN2+div26 rd1 explants (Figure 8 a, b), the PN7+div21 rd1 explants (Figure 8 g, h) were less responsive to α-GST or µ-GST treatments. The corresponding PN2+div26 or PN7+div21 wt explants were not affected by α-GST or µGST treatment.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

The Rd1 Retina Showed Oxidatively Damaged DNA [Reference 5 for Details] PN11 rd1 mouse retinal sections immunostained with an antibody to 8-hydroxy deoxyguanosine [Reference 8, Figure 1 A] and incubated with Texas Red labeled avidin [Reference 8, Figure 1 C] showed colabeling of cells in the ONL [Reference 8, Figure 1 B] and indicated localization of oxidized and damaged DNA. The PN11 wt control retinal section [Reference 8, Figure 1 F] lacked any reactions with avidin, whereas retinal sections of PN9, -11 and -13 [Reference 8, Figure 1 D and E] rd1 mice showed increasing number of avidin positive cells in the ONL. A few positive cells were seen in the INL (arrow heads) of sections of PN9 and PN11 rd1 mouse retinas. Retinal sections obtained from control mice and from those treated with a combination of antioxidants [Reference 8, Figure 3 A and B] were processed for terminal deoxynucleotidyltransferase mediated dUTP nick end labeling (TUNEL) assay to show the localization of dying cells. Sections were also stained with Texas Red avidin to show damaged DNA [Reference 8, Figure 3 C and D]. Superimposed images [Reference 8, Figure 3 E and F] showing colocalization of the two markers in photoreceptors confirmed oxidatively damaged DNA in dying cells (white arrows). Avidin positive cells (blue arrows) and TUNEL positive cells (yellow arrows) not showing colocalization indicated different stages of cell death. The number of avidin and TUNEL positive cells was much lower in the ONL of retinas treated with a combination of antioxidants and this was supported by actual counts of photoreceptor rows [8].

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

280

Satpal Ahuja, Poonam Ahuja-Jensen, A. Romeo Caffé et al.

Figure 7. In both genotypes the level of α-GST (A) was higher than that of µ-GST (B) and both α-GST and µ-GST decreased with age after attaining a peak at PN7. This was particularly so in rd1 retinas. Both α-GST and µ-GST rescued the rd1 photoreceptors (C) when added to the culture medium and the rescue effect of α-GST was higher than that of µ-GST (C) when supplemented to PN7 explants. Both αGST and µ-GST had similar rescue effect on PN2 explants but it was higher than that on PN7 explants. (Copyright Permission Reference 7)

Serum-Free Retinal Explant Culture System and Comparative Rescue Effects…

281

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Figure 8. H&E staining showed the rescue of photoreceptors by GST supplementation. As compared to the untreated control (c, f, i and l), the supplementation of α-GST (a, d, g and j) or µ-GST (b, e, h and k) into the culture medium for PN2+div26 (a, b, d and e) and PN7+div21 (g, h, j and k), rd1 (a, b, g and h) and wt (d, e, j and k) retinal explants showed rescue of rd1 photoreceptors. Scale bar = 25 µm. (Copyright Permission Reference 7)

A Combination of Antioxidants Protected the Photoreceptors in Rd1 Retina The PN5 rd1 retinal explants [Reference 8, Figure 2 C] treated in vitro with a combination of GSH, α-lipoic acid, lutein and zeaxanthin for div13 had significantly larger (P