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Understanding and Preventing Visual Loss in Commotio Retinae UNDERSTANDING AND PREVENTING VISUAL LOSS IN COMMOTIO RETINAE By RICHARD JAMES BLANCH A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Clinical and Experimental Medicine College of Medical and Dental Sciences University of Birmingham August 2013 I University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Commotio retinae describes retinal opacification following trauma and affected 16% of British soldiers suffering major trauma. The macula was affected in 55% of soldiers and 31% of civilian cases, permanently reducing vision to less than 6/9 in 26% of cases, associated with photoreceptor degeneration. In an experimental rat model, commotio retinae was more readily induced by high velocity ballistic injury (20m/s) than low velocity weight drop (2-7m/s). In rats, after experimentally induced commotio retinae, photoreceptors died by a combination of necrosis (central to the impact site) and apoptosis (peripheral to it), demonstrated by morphological changes on electron micrographs and TUNEL staining. Photoreceptor death after commotio retinae was associated with reduced ERG a- wave amplitude. Apoptosis occurred through the intrinsic pathway, mediated by caspase 9 but not involving any of the classical executioner caspases (3, 6 and 7). Inhibition of caspase 9 reduced photoreceptor death and improved retinal function, assessed by a-wave amplitude. Clinical studies suggested a protective effect of female gender after commotio retinae, but progesterone treatment increased photoreceptor death after ballistic injury in the experimental model. II Acknowledgements A great many people have helped with the research and the preparation of this thesis. Thanks go to my supervisor Prof Rob Scott for making me look at ocular blast injury in the first place and for encouraging, supporting and advising me at every stage of the project. Equal thanks go to Prof Ann Logan, my primary supervisor, for welcoming me into her research group, giving me the skills and support I needed to be able to make the laboratory side of the project work and supporting me throughout. I am also immensely indebted to Zubair Ahmed, without whose advice and teaching I couldn’t have done research into apoptosis and to Prof Martin Berry for his research advice and for his endless patience in editing and redrafting manuscipts. Thanks also go to Prof Attila Sik, Theresa Morris and Paul Stanley for teaching me to do electron microscopy and get publishable images. Thanks to David Snead for showing me how to look at electron micrographs and, along with Sean James, helping me do the first pilot study and giving me continued and invaluable support from their laboratories. Thanks to Peter Nightingale and Jon Bishop for statistical advice. Thanks to Peter Good and Antonio Calcagni for electrophysiology advice. Thanks to Ana Maria Gonzalez for help and advice in learning laboratory techniques. Final thanks go to Andy Thewles, Lisa Hill, Hannah Botfield, Ben Mead, Sarina Kundi, Vasanthy Vigneswara, Jenna O’Neill and Peter Morgan-Warren for good company in the write-up room, advice and assistance with laboratory work and anaesthetic assistance in BMSU. III Funding Ministry of Defence, UK Drummond Foundation, UK Sir Ian Fraser Foundation, Blind Veterans UK IV Illustrations Figure 1.1.1. Cross-sectional diagram of a human eye. 1 Figure 1.1.2. Fundus photograph. 3 Figure 1.1.3. Diagram of the retinal cell types. 4 Figure 1.2. Birmingham Eye Trauma Terminology System. 8 Figure 1.4.1.2.-4. Rat, rabbit and pig eye cross-sections. 14 Figure 1.4.2. Diagram of caspase-dependent apoptosis. 19 Figure 1.5.1. Typical rat ERG. 47 Figure 2.1.3. Diagram of a left lateral canthotomy. 52 Figure 3.5. Birmingham Eye Trauma Terminology System. 70 Figure 4.5. Electron microscopic results. 80 Figure 5.5.1.-2 Fundus and OCT images. 87 Figure 5.5.3 Final VA against initial VA. 88 Figure 6.4.1.-2. Weight drop apparatus. 95 Figure 6.5.2. Electron microscopic results. 100 Figure 6.5.3.1.-2. Fundoscopy after injury. 103 Figure 6.5.3.3. Electron microscopic results. 104 Figure 6.5.4.1.-2. Fundoscopy 1d after injury. 108 Figure 6.5.4.3. Light and electron microscopic results. 109 Figure 6.5.5.1. Fundoscopy after injury. 112 Figure 6.5.5.2.-3. Light and electron microscopic results. 113 Figure 6.6.3.1. Diagram of experimental apparatus. 116 Figure 6.6.3.2. and .5.2 Head holder and ballistic injury apparatus. 117 Figure 6.6.5. Mean velocity at different distances from target. 122 Figure 6.6.6.1.-2. Fundoscopy immediately after injury. 124 Figure 6.6.6.3. Electron microscopy results. 125 Figure 6.6.7.1. Fundoscopy 2 d after injury. 128 Figure 6.6.7.2. Gross pathology results. 129 V Figure 6.6.7.3. Light microscopic results. 130 Figure 6.6.7.4. Electron microscopy results. 131 Figure 6.6.8.1. Cell count diagram. 134 Figure 6.6.8.2. and .3. Fundoscopy and light microscopy 2 wk after injury. 135 Figure 6.6.8.4. Photoreceptor survival by distance from impact. 136 Figure 6.6.9.1. Diagram of counting on retinal whole mounts. 138 Figure 6.6.9.2. Mean number of surviving photoreceptors. 139 Figure 6.6.10. TUNEL staining. 141 Figure 7.4.3.1. ERG trace obtained with poor technique. 148 Figure 7.4.3.2. Normal rat ERG. 149 Figure 7.5.4.1. ERG results after ballistic injury. 152 Figure 8.4.1. Initiator caspase western blots. 157 Figure 8.4.2. Executioner caspase western blots. 158 Figure 8.5.1. Caspase 9 in immunohistochemistry. 161 Figure 8.5.2. Activated caspase 6 in immunohistochemistry. 162 Figure 8.6. Caspase capture assay results. 165 Figure 8.7.1. ONL thickness measurement. 168 Figure 8.7.2. XBIR3 results. 170 Figure 8.8. C6DN results. 175 Figure 9.1.1. Progesterone treatment structural results. 186 Figure 9.1.2. Progesterone ERG results. 187 Figure 9.1.3. Progesterone ELISA results 188 Figure 9.2. Progesterone ELISA results 191 VI Tables Table 1.2.1 . Distribution of PBI reported in the literature 10 Table 1.4.2.1. Animal models of retinal injury induced by blunt ocular trauma. 26 Table 1.4.3.2. Animal models used to study retinal injury responses. 29 Table 1.4.4. Choice of species and models for ocular trauma research. 45 Table 3.5.1. Closed globe injuries. 72 Table 3.5.2. Correlation of final VA with OTS. 73 Table 6.4. Measurements to calculate velocity and kinetic energy 96 Table 6.5.2.4. Results 101 Table 6.5.3.3. Results 105 Table 6.5.4.3. Results 110 Table 6.5.5.5. Results 114 Table 6.6.4.3. Results 120 Table 6.6.5.3. Mean BB velocity at different distances from target. 122 Table 7.5.3. ERG recording protocol. 151 Table 8.7.4. Scotopic and photopic ERG recording protocol. 169 VII Abbreviations ADB Antibody diluting buffer Apaf-1 Apoptosis activating factor 1 BB Ball bearing BDNF Brain derived neurotrophic factor bVAD-fmk Biotinylated valine-alanine-aspartate-fluoromethyl ketone C6DN Caspase 6 dominant negative protein CF Counting fingers CNTF Ciliary neurotrophic factor d Day/days df Degrees of freedom ERG Electroretinogram HM Hand movements hr Hour/hours HtrA2 High temperature requirement protein 2 ILM Inner limiting membrane INL Inner nuclear layer IPL Inner plexiform layer IS Inner segments IUGR Intra-uterine growth retardation LE Left eye min Minute/minutes MOMP Mitochondrial outer membrane permiabilisation MPTP Mitochondrial permeability transition pores NPL No perception of light OLM Outer limiting membrane ONL Outer nuclear layer OPL Outer plexiform layer VIII OS Outer segments OTS Ocular trauma score PB Phosphate buffer PBI Primary blast injury PBS Phosphate-buffered saline Pen1 Penetratin 1 PL Perception of light PTEN Phosphatase and tensin homolog RD Retinal detachment RE Right eye RGC Retinal ganglion cell RIP1 Receptor interacting protein kinase 1 RNFL Retinal nerve fibre layer RPE Retinal pigment epithelium SDS Sodium dodecyl sulphate sec Second/seconds smac Second mitochondrial-derived activator of caspases SOCS3 Suppressor of cytokine signalling TBI Traumatic Brain Injury TBS Tris-buffered saline TBST TBS with Tween TSC1 Tuberous sclerosis complex 1 VA Visual acuity XBIR3 X-linked inhibitor of apoptosis (IAP)-baculoviral IAP repeat 3 domain XIAP X-linked inhibitor of apoptosis IX Contents 1. Introduction .................................................................................................................................... 1 1.1. Structure and Function of the Eye .......................................................................................... 1 1.2. Ocular Trauma ......................................................................................................................... 6 1.2.1. Blast Injuries .................................................................................................................... 8 1.3. Commotio Retinae ................................................................................................................ 10 1.3.1. Clinical Picture ..............................................................................................................
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