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Retinal Transplantation in a Rodent Model of Retinitis Pigmentosa

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Retinal Transplantation in a Rodent Model of Retinitis Pigmentosa Retinal transplantation in a rodent model of Retinitis Pigmentosa Anthony S L Kwan Institnte of Ophthalmology University College London A dissertation submitted for the degree of Doctor of Medicine at the University of London 2003 ProQuest Number: 10014454 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10014454 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Table of Contents Preface 5 Acknowledgements 6 Abbreviations 8 List of figures 10 List of tables 13 Abstract 14 Chapters 1.0. Introduction 16 1.1. Retinitis pigmentosa (RP) 19 1.1.1. Morphological changes in the RP retina 21 1.1.2. Potential treatments for RP 27 1.2. A mouse model of retinitis pigmentosa 34 1.2.1. Origin of the retinal degeneration (rd) mouse 34 1.2.2. Morphological changes in rd phenotype 35 1.2.3. Visual function of the rd mouse 48 1.2.4. Experimental treatments in the rd mouse 53 1.3. Background of experimental retinal transplantation in rodent models 59 1.3.1. Donor cells 59 1.3.2. Technique 60 1.3.3. Behavioural tests 62 1.3.4. Electrophysiology 63 1.4. Experimental aims 66 2.0. Methodology and technique development 67 2.1. Animal source 67 2.2. Donor tissue preparation 68 2.3. Transplantation technique 70 2.4. Histology 75 2.4.1. Baseline study and mouse-to-mouse transplantation 75 2.4.2. Rat-to-mouse transplantation 80 2.4.3. Reagents and reactions 82 2.5. Light-dark preferential test (LDPT) 89 2.5.1. Pilot study 89 2.5.2. A computerised tracking system - Tru Scan™ 90 2.5.3. Experimental design and hypothesis testing 94 2.5.4. Statistical method and strategy 96 2.6. Retinal threshold measurement 98 3.0. Results 103 3.1. Light-dark preferential test results 103 3.1.1. Baseline study of normal and dystrophic mice 103 3.1.2. Effects of retinal transplantation 105 3.2. Retinal threshold measurement results 113 3.2.1. Baseline study of normal and dystrophic mice 113 3.2.2. Effects of retinal transplantation 122 3.3. Histology results 125 3.3.1. Baseline study of normal and dystrophic retina 125 3.3.2. Transplanted retina 139 4.0. Discussion 152 5.0. Learning Points 165 6.0. Conclusion 167 7.0. References 169 8.0. Appendix - Publications 186 Preface The studies described in this dissertation are my own work and were carried out in the Department of Pathology, Institute of Ophthalmology, University College London (UCL). None of these experiments are substantially the same as any that I have submitted for a degree, diploma, or other qualification at any other university. I further state that no part of my dissertation has already been, or is currently submitted for any such degree, diploma, or certificate. Acknowledgements There are so many people that I would like to thank. I sincerely apologise to anyone I unintentionally forget to mention. Firstly, I would like to thank my supervisor, Professor Ray Lund, for all his guidance from the very start and for being brave enough to take on a clinician like me who had no experience in laboratory work. His relaxed style eased me into laboratory science. Our countless conversations were full of little gems of knowledge. I very much appreciate all his time and effort over the last few years. Secondly my colleague and friend. Dr Yves Sauvé, has been most helpful in the electrophysiology experiments. He performed these experiments in his unassuming manner and helped me to understand the reasoning of the tests. Thirdly I would like to thank Dr. Pete Coffey from University of Sheffield. His experience in animal behavioural experiments is simply unsurpassed. Thanks to Shaomei Wang for her patience in explaining the technique in electron microscopy and teaching me Mandarin, Jean Lawrence for her constant support and help in proof reading, Sergej Girman for showing me how a scientist should work (the Russian way !), David Keegan for his company and witty comments during those long days of transplantation, Tim Pheby for the countless help around the lab, Toby Holmes for being Toby Holmes, and last but not least everyone from the Pathology Department for their help. Finally I would like to thank those who are closest to me, my parents for giving me a wonderful life and endless support in everything I do. They are thousands of miles away in lovely Vancouver but I can feel that they are with me in every step I take. To my wife, Julia, for putting up with my episodes of depression during my time in research and writing up and for giving us a wonderful son, Alex, thank you. To my wife, Julia, and my son, Alex Abbreviations ACAID anterior chamber associated immune deviation ANOVA analysis of variance AMD age related macular degen^ation PPDE P-subunit o f phosphodiesterase bFGF basic fibroblast growth factor cd/m^ candela/metre square CNS central nervous system CNTF ciliary neurotrophic factor cGMP cyclic guanosine monophosphate DAB diaminobenzidine DCT dark compartment time (in seconds) DNA deoxyribonucleic acid E embryonic ERG electroretinogram GCL ganglion cell layer GFAP glial fibrillary acid protein H2O2 hydrogen peroxide HBSS Hank’s balanced salt solution HRP streptavidin-hOTseradish peroxidase INL inner nuclear layer IPL inner plexiform layer LDPT light-dark preferential test mfERG multifocal electroretinogram mRNA messenger ribonucleic acid MHC major histocompatibility complex NGF nerve growth factor ONE outer nuclear layer/photoreceptor layer OPL outer plexiform layer PBS phosphate buffered saline PKC protein kinase C PLP periodate/lysine/paraformaldehyde fixative PLR pupillary light reflex PN postnatal PSTH post-stimulus time histogram RCS Royal College of Surgeons rd retinal degeneration RetNeT^“ Retinal Information Network RF receptive field RP retinitis pigmentosa RPE retinal pigment epithelium SC superior colliculus SIRP signal-regulatory protein TPL transplanted layer List of figures Figure 1 (A) Colour fundus photograph of a human with normal sight 20 (B) Colour fundus photograph of a patient with Retinitis Pigmentosa (C) Schematic diagram of Apoptosis Figure 2 (A) Colour fundus photograph of a 3-month-old normal mouse 44 (B) Colour fimdus photograph of a 3-month-old rd (retinal degeneration) mouse (C) Classical visual cliff apparatus Figure 3 (A) Schematic diagram of the subretinal injection method 73 (B) Subretinal transplant, example I (C) Subretinal transplant, example 2 Figure 4 (A) Subretinal ink injection in a normal mouse 74 (B) Subretinal graft in normal mouse Figure 5 (A) Tru Scan^“ apparatus - arena floor 93 (B) Tru Scan^“ apparatus - light and dark compartment (C) Tru Scan™ apparatus - unit with light source and computer Figure 6 (A) Retinal threshold measurement apparatus - Ganzfield Bowl 102 (B) Schematic diagram of the threshold recording method Figure 7 (A) Baseline cross-sectional study (RUN I) in LDPT 108 (B) Baseline cross-sectional study (RUN 2) in LDPT 109 Figure 8 Baseline l(xigitudinal study in LDPT 110 Figure 9 Pre- and post-transplantation LDPT results in sham and all transplant groups 111 Figure 10 Pre- and 2 weeks post-transplantation LDPT results in sham control, 112 histology-positive and histology-negative transplant groups Figure 11 Individual retinal threshold measuranent maps 114 Figure 12 Post-stimulus time histograms of retinal threshold measurements 124 fi’om a normal, a dystrophic rd and a rd mice (Subject XV) three weeks after transplantation Figure 13 (A) Cresyl violet staining of 7-day-old ncrnnal mouse retina 130 (B) Cresyl violet staining of 6-week-old normal mouse retina (C) Cresyl violet staining of 6-week-old rd mouse retina 10 Figure 14 (A) Rhodopsin labelling of 6-week-old normal mouse retina 131 (B) Rhodopsin labelling of edge o f subretinal transplant (C) Rhodopsin labelling of subretinal graft at high magnification Figure 15 (A) GFAP labelling of 6-week-old normal mouse retina 132 (B) GFAP labelling of 6-week-old rd mouse retina (C) GFAP labelling of subretinal transplant Figure 16 (A) RT-97 labelling of 6-week-old normal mouse retina 133 (B) RT-97 labelling of 6-week-old rd mouse retina (C) RT-97 labelling of subretinal transplant Figure 17 (A) PKC labelling of 6-week-old normal mouse retina 134 (B) PKC labelling of 6-week-old rd mouse retina (C) PKC labelling of subretinal transplant Figure 18 (A) Calbindin labelling o f 6-week-old normal mouse retina 135 (B) Calbindin labelling of 6-week-old rd mouse retina (C) Calbindin labelling of subretinal transplant Figure 19 (A) F4/80 labelling of 6-week-old normal mouse retina 136 (B) F4/80 labelling of 6-week-old rd mouse retina (C) Cresyl violet staining of subretinal fibrosis after transplantation Figure 20 (A) Parvalbumin labelling of 6-week-old normal mouse retina 137 (B) Parvalbumin labelling of 6-week-old rd mouse retina (C) Parvalbumin labelling of subretinal transplant Figure 21 (A) P84 labelling of 6-week-old normal mouse retina 138 (B) P84 labelling of 6-week-old rd mouse retina (C) P84 labelling of subretinal transplant Figure 22 (A) Cresyl violet staining o f subretinal transplant 147 (B) Cresyl violet staining of the edge of subretinal transplant (C) Cresyl
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