Corneal neurotization maintains corneal epithelial integrity and restores nerve-derived peptides in a rat model of neurotrophic keratopathy by Kira Antonyshyn A thesis submitted in conformity with the requirements for the degree of Master of Science Institute of Medical Science University of Toronto ii Corneal neurotization maintains corneal epithelial integrity and restores nerve-derived peptides in a rat model of neurotrophic keratopathy Kira Antonyshyn Master of Science Institute of Medical Science University of Toronto 2019 Abstract PURPOSE: Investigate effect of corneal neurotization on epithelial integrity and restoration of nerve-derived peptides in denervated rat corneas. METHODS: Three rat corneal conditions were assessed (n=45): denervation, denervation + neurotization, normal innervation. Corneal epithelial and stromal thicknesses were evaluated using Hematoxylin and Eosin staining. Corneal ulceration and perforation were assessed under a Wood’s lamp and normal light, respectively. Immunohistochemistry and western blot were used to evaluate Substance P (SP) and calcitonin gene-related peptide (CGRP) presence. RESULTS: Where significance is p<0.05, central epithelium was thinner in the denervated compared to the denervated + neurotized and normally innervated corneas. The denervated stroma was thinner than the normally innervated, but not the denervated + neurotized stroma. Neurotization protected the denervated cornea from ulceration and iii perforation. Neurotization restored the denervated cornea with SP and CGRP, co- localized with axons innervating the cornea. CONCLUSION: Corneal neurotization restores epithelial integrity and nerve-derived peptides in the denervated cornea. iv Table of Contents ABSTRACT ......................................................................................................................................... II CONTRIBUTIONS ............................................................................................................................ VIII LIST OF ABBREVIATIONS ................................................................................................................... IX LIST OF FIGURES ................................................................................................................................ X LIST OF TABLES ............................................................................................................................... XII 1.1 PREAMBLE ...................................................................................................................................2 1.2 THESIS ORGANIZATION .................................................................................................................3 1.3 CORNEAL ANATOMY AND PHYSIOLOGY ........................................................................................4 I - INTRODUCTION..........................................................................................................................4 II - CORNEAL ANATOMY ...............................................................................................................4 III - CORNEAL INNERVATION ..................................................................................................... 12 ........................................................................................................................................................ 15 IV - CORNEAL EPITHELIAL MAINTENANCE AND REPAIR ..................................................... 16 V - LIMBAL STEM CELLS ............................................................................................................ 19 VI - SCHWANN CELLS IN THE CORNEA .................................................................................... 22 1.4 NEUROTROPHIC KERATOPATHY .................................................................................................. 24 I. INTRODUCTION......................................................................................................................... 24 II. ETIOLOGY, DIAGNOSIS, PROGNOSIS .................................................................................. 24 III - MOLECULAR BASIS OF DISEASE ....................................................................................... 26 IV - ANIMAL MODELS OF NEUROTROPHIC KERATOPATHY ................................................. 28 1.5 TREATMENTS FOR NEUROTROPHIC KERATOPATHY AND CORNEAL NEUROTIZATION .................... 32 I – INTRODUCTION ....................................................................................................................... 32 II - MANAGEMENT AND TREATMENT OF NEUROTROPHIC KERATOPATHY ...................... 32 III - CORNEAL NEUROTIZATION AND OTHER METHODS OF RE-INNERVATION................. 34 IV - ANIMAL MODELS OF CORNEAL REINNERVATION AND NEUROTIZATION ................... 38 V - WOUND HEALING AFTER CORNEAL NEUROTIZATION .................................................... 41 1.6 THESIS AIMS AND HYPOTHESIS ................................................................................................... 43 v AIM 1 .............................................................................................................................................. 43 AIM 2 .............................................................................................................................................. 43 AIM 3 .............................................................................................................................................. 44 2.1 INTRODUCTION .......................................................................................................................... 46 2.2 MATERIALS AND METHODS ........................................................................................................ 48 I – ANIMALS ..................................................................................................................................... 48 III – STERILE SURGERIES ................................................................................................................. 50 IV – CORNEAL HARVEST .................................................................................................................. 53 V – EVALUATION OF CORNEAL EPITHELIAL AND STROMAL THICKNESS ............................................. 53 VI – EVALUATION OF CORNEAL ULCERATION, SCARRING AND PERFORATION ................................... 53 VII - IMMUNOHISTOCHEMISTRY ......................................................................................................... 54 VIII - WESTERN BLOT....................................................................................................................... 55 IX – STATISTICS .............................................................................................................................. 55 2.3 RESULTS ..................................................................................................................................... 56 I - CORNEAL NEUROTIZATION PREVENTS CENTRAL EPITHELIAL THINNING IN THE DENERVATED CORNEAL MODEL OF NK .................................................................................................................. 56 II - CORNEAL NEUROTIZATION PROTECTS THE DENERVATED CORNEA FROM ULCERATION, SCARRING AND PERFORATION .......................................................................................................................... 58 III - CORNEAL NEUROTIZATION RESTORES SP AND CGRP IN THE DENERVATED CORNEA ................. 60 ........................................................................................................................................................ 62 ........................................................................................................................................................ 62 2.4 DISCUSSION ............................................................................................................................... 63 3.1 – SUMMARY OF RESULTS ............................................................................................................ 69 3.2 – FUTURE DIRECTIONS ................................................................................................................ 69 FUTURE DIRECTION 1 – CORNEAL EPITHELIUM .................................................................... 70 FUTURE DIRECTION 2 – TROPHIC SUPPORT .......................................................................... 70 FUTURE DIRECTION 3 – SCHWANN CELLS ............................................................................. 74 FUTURE DIRECTION 4 – LIMBAL STEM CELLS ....................................................................... 79 COLLABORATIONS FOR FUTURE DIRECTIONS ..................................................................... 88 3.3 – CONCLUDING REMARKS ........................................................................................................... 88 REFERENCES .................................................................................................................................... 90 vi Acknowledgements I would like to acknowledge my primary supervisor, Dr. Gregory Borschel, and my co- supervisor, Dr. Tessa Gordon. Thank you for challenging me and teaching me the fundamental skills to value and to pursue research in the future. Thank you for teaching me how to turn failures into learning opportunities that result in successes. Your continuous guidance and encouragement