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Immunohistochemical Mapping of Corneal Sensory Innervation in Mice: Effects of Sjögren`S Syndrome Associated Dry Eye

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Immunohistochemical Mapping of Corneal Sensory Innervation in Mice: Effects of Sjögren`S Syndrome Associated Dry Eye IMMUNOHISTOCHEMICAL MAPPING OF CORNEAL SENSORY INNERVATION IN MICE: EFFECTS OF SJÖGREN`S SYNDROME ASSOCIATED DRY EYE By RENATA VELLOSO RAMOS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2016 © 2016 Renata Velloso Ramos To Viviane and Otto ACKNOWLEDGMENTS I would like to recognize and express my profound gratitude to my advisor Dr. Caryn Plummer for believing in me and giving me the opportunity of pursing my Master of Science degree and residency under her mentorship. I am greatly thankful for her guidance, encouragement, and friendship. I admire her remarkable ability to mentor, lead and teach while also being a caring friend; she will always be a professional model for me. I would like to extend my sincere gratitude to my co-advisor Dr. Rick Johnson for helping me in the development of critical thinking and commitment to high quality research. I am very honored to be able to benefit from his guidance, experience and friendship. I would like to thank Dr. Whitley, Dr. Brooks and Dr. Hamor for their intellectual guidance, support and enthusiasm in both research and clinical settings. I would like to express my gratitude to Dr. Cuong Nguyen, for the use of his mouse colony also offering his knowledge and support. I extend my appreciation to Dr. Dan Gibson and Dr. Jasenka Zubcevic for their continued input, interest and brilliant solutions along the way. Their encouragement helped me to transform moments worth of frustration into a successful endeavor. I would like to thank Victoria Dugan, Huy Nguyen, Alexandria Voigt and Dr. Arun Wanchoo for their patience and support in the lab. I extend my sincere appreciation to my dearest resident-mates, past and present, Dr. Bianca Martins, Dr. Caroline Monk, Dr. Sarah Czerwinski, Dr. Proietto and Dr. William Berkowski and to our remarkable technicians Holly Kitchen, Michelle Wilhelmy and Katherine Devine for their guidance, support and camaraderie over this journey. Each of them has taught me something that I will take for life. I am forever grateful for 4 the opportunity to train in the country’s premier ophthalmology residency and become part of the ophthalmology family at UF. I would like to thank UF College of Veterinary Medicine Graduate Office for their financial support, and Sally O’connell for her unrelenting help and positive attitudes. I would also like to express my gratitude to my family and friends. The distance that separated us in the last years has not prevented them to continue nurturing our love. I specially thank my parents, Gastão and Isabella Ramos, for supporting me not only during this time but also throughout all my life. Lastly, I would like to thank my partners in crime, Fernanda Ferreira and Achilles Vieira Neto, without their love, strength and unending support this thesis would not have been possible. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ............................................................................................. 9 ABSTRACT ................................................................................................................... 11 1 INTRODUCTION .................................................................................................... 13 2 LITERATURE REVIEW .......................................................................................... 15 The Cornea ............................................................................................................. 16 Anatomy and Physiology .................................................................................. 17 Innervation ........................................................................................................ 19 Neuropeptides ........................................................................................................ 21 CGRP ............................................................................................................... 21 Substance P ..................................................................................................... 22 Protein Gene Product 9.5 – PGP 9.5 ...................................................................... 23 Dry Eye Syndrome .................................................................................................. 24 Clinical Significance of Sjögren’s Syndrome .................................................... 26 Immunopathology ............................................................................................. 27 Epidemiology .................................................................................................... 28 Diagnostic, Treatment and Prognosis .............................................................. 29 C57BL/6.NOD-Aec1Aec2 Mouse Model .......................................................... 29 3 CHARACTERIZATION OF THE ARCHITECTURAL PATTERN OF CORNEAL INNERVATION IN C57BL/6.NOD-AEC1AEC2 MICE WITH SJÖGREN`S SYNDROME COMPARED TO WILD TYPE ........................................................... 32 Background ............................................................................................................. 32 Materials and Methods............................................................................................ 35 Animals............................................................................................................. 35 Antibodies ......................................................................................................... 35 Tissue Preparation and Immunofluorescence Staining .................................... 35 Imaging............................................................................................................. 36 Data Analysis ................................................................................................... 37 Results .................................................................................................................... 38 Stromal Nerve Architecture .............................................................................. 38 Epithelial Nerve Architecture ............................................................................ 39 PGP 9.5 and SP ............................................................................................... 39 Co-Localization of SP and CGRP ..................................................................... 40 Sjögren’s Syndrome-Related Changes in Nerve Density ................................. 40 Discussion .............................................................................................................. 41 6 Corneal Nerve Anatomy ................................................................................... 41 Methodological Considerations ........................................................................ 43 CGRP and SP .................................................................................................. 45 Disease-Related Changes in Corneal Nerve Density ....................................... 46 4 CONCLUSIONS ..................................................................................................... 55 LIST OF REFERENCES ............................................................................................... 57 BIOGRAPHICAL SKETCH ............................................................................................ 70 7 LIST OF FIGURES Figure page 3-1 Immunofluorescence imaging of wild type mouse cornea stained with PGP 9.5 ...................................................................................................................... 50 3-2 Subbasal nerves in a wild type mouse cornea with whorl-like structure or vortex, labeled with PGP 9.5 antibody ................................................................ 50 3-3 Representative images showing the expression of PGP 9.5-and-SubP positive nerves in the central and peripheral cornea .......................................... 51 3-4 Difference of corneal nerve density between the central and the peripheral zones .................................................................................................................. 52 3-5 Difference of corneal nerve density between the central and the peripheral zones .................................................................................................................. 52 3-6 Representative images showing the expression of CGRP-and-SubP positive nerves in the central and peripheral cornea ....................................................... 53 3-7 Difference of corneal nerve density between the C57BL/6.NOD-Aec1Aec2 and wild type mouse ........................................................................................... 53 3-8 Representative images showing decrease in the expression of PGP 9.5 positive nerves in normal mice and C57BL/6.NOD-Aec1Aec2 mice at the central and peripheral cornea ............................................................................. 54 8 LIST OF ABBREVIATIONS 5-HTT Serotonin-transported gene Aec Autoimmune exocrinopathy ANA Antinuclear antibody AOI Area of interest ARVO Association for Research in Vision and Ophthalmology CGRP Calcitonin gene-related peptide CO2 Carbon dioxide DSU Disk scanning unit
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