Phrenic and Cervical Afferents Following Spinal Cord Injury

PHRENIC AND CERVICAL AFFERENTS FOLLOWING SPINAL CORD INJURY

JAYAKRISHNAN NAIR

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2016

To my patients who inspired me to study the remarkable ability of the nervous system to change and adapt itself, and my family for their constant support in this quest.

ACKNOWLEDGMENTS

This work would not be possible without the support and encouragement from a

lot of people. First and foremost, I would like to thank my mentor Dr. David Fuller for

accepting me as a student when I was left stranded in the middle of my PhD. I also

thank him for having trust in my abilities and his constant encouragement and guidance to wade through some of my toughest times in the lab when experiments failed miserably for over a year. I was also fortunate to have a team of world expert on my

PhD supervisory committee to steer my thesis in the right direction without wandering off too much from what I set out to investigate. I am proud that my committee had an all- star lineup with experts like Dr. Paul Davenport, a world authority in respiratory afferents, Dr. Paul Reier, revered veteran in spinal cord physiology, Dr. Danny Martin and Dr. Emily Fox with years of clinical research expertise, and Dr. David Baekey for his insightful expertise in cardiorespiratory integrative physiology. I also would like to thank

Dr. Gordon Mitchell whose recent migration to University of Florida totally transformed the lab work/fun environment by bringing in high quality science and people along with him. I thank my colleagues in Fuller, Reier and Mitchell laboratories for setting up very high standards for designing and executing high quality research. Specifically, I would like to thank our lab manager, Amy Poirier for her prompt support during the entire period I worked in the lab. Her immense organizational skills help to provide an efficient lab environment for me and everyone else to work at optimal capacity. I would also like to thank Dr. Elisa Gonzalez-Rothi for teaching me surgical skills and with her help with

brainstem injections. I thank my friends and colleagues Brendan Doyle and Yasin

Seven for proofreading my manuscript and also for spending countless hours

discussing science just for fun; and, Drs. Sara Turner and Kristi Streeter for providing

informed constructive criticism on projects and for their help with various lab related works. I am extremely grateful to Marda Jorgenson for her one on one mentoring on optimizing minute nuisances of immuno-florescence technique to get the perfect staining. I also thank members of Reier lab Emma Denholtz and Dr. Danielle Meola for all the help with immunostaining involved in this project. I would also like to thank Dr.

Ron Mandel, Marissa Ciesla and Dr. Lynne Mercier for their extremely valuable help with the brainstem injection project. I also express my sincere gratitude to my good friend and a former member of Fuller lab, Dr. Milapjit Sandhu for his constant support and help during my initial days in the lab. I would like to acknowledge and thank my parents Gopalakrishnan Nair and Nirmala Nair along with my brother Harikrishnan Nair for having trust in my crazy dreams and pursuits and to provide all the support to allow me to reach where I am today. Last but not the least, I thank my wife Kavya

Kamalamma for motivating me to pursue a career in science and made all the sacrifices in her life to ensure that I succeed in that endeavor. She was the rock I anchored during all the troubled times. Finally, I want to thank my son Tanmay for providing an ideal distraction from all the madness associated with the graduate student life.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 9

LIST OF FIGURES ...... 10

ABSTRACT ...... 13

CHAPTER

1 LITERATURE REVIEW ON PHRENIC AFFERENTS ...... 15

Diaphragm Receptors ...... 15 Phrenic Nerve Axons ...... 16 Spinal Projection ...... 17 Supra Spinal Projections...... 20 Functional Role of Phrenic Afferents...... 28 Diaphragm Receptors and Its Function...... 29 Role of Phrenic Afferents Based on the Receptor Type ...... 29 Sympathetic Preganglionic fibers and Non-Diaphragmatic Afferents ...... 32 Phrenic Afferents Mediated Cervical Interneuronal Activity ...... 33 Phrenic Afferents Mediated Reflexes ...... 35 Role of phrenic afferents following spinal cord injury ...... 41

2 OUTLINE OF EXPERIMENTS ...... 43

Aim One ...... 45 Objective ...... 45 Rationale ...... 45 Hypothesis ...... 45 Experimental Design ...... 45 Aim Two ...... 46 Objective ...... 46 Rationale ...... 46 Hypothesis ...... 47 Experimental Design ...... 47 Aim Three ...... 47 Objective ...... 47 Rationale ...... 47 Hypothesis ...... 48 Experimental Design ...... 48

3 HISTOLOGICAL IDENTIFICATION OF PHRENIC AFFERENT PROJECTIONS TO THE SPINAL CORD USING CHOLERA TOXIN ...... 50

Introduction ...... 50 Materials and methods...... 51 Experimental animals ...... 51 Labeling techniques ...... 52 Tissue preparation ...... 55 Immunochemistry ...... 55 Microscopy ...... 56 Results ...... 57 Phrenic motoneurons, DRG soma, and phrenic afferent projections are labeled by the CT-β nerve labeling method...... 57 Pretreatment with C4 dorsal rhizotomy removes C4 afferent labeling...... 58 Afferent labeling after intrapleural or direct diaphragm application of CT-B. .... 58 Addition of a transynaptic retrograde tracer...... 59 Discussion ...... 60 Comparison to published histological studies of phrenic afferents...... 60 Use of CT-β to trace afferent neuronal projections ...... 62 Interpretational issues ...... 63 Summary and Conclusions ...... 63

4 PHRENIC AND MID-CERVICAL AFFERENT PLASTICITY FOLLOWING SPINAL CORD INJURY ...... 74

Introduction ...... 74 Materials and methods...... 76 Experimental animals ...... 76 Spinal hemisection ...... 77 Labeling technique ...... 77 Tissue preparation ...... 79 Immunofluorescence ...... 79 Microscopy ...... 81 Image analysis ...... 81 Results ...... 82 CT-β labeled phrenic afferents in spinal intact rats...... 82 Remodeling of myelinated CT-β labeled phrenic afferents following SCI ...... 82 CT-β labeled C4-DRG afferent projections and their plasticity following SCI ... 86 Group III and IV mid cervical afferent staining ...... 87 Discussion ...... 88 Evidence for the presence and functional relevance of phrenic afferents in the intact spinal cord ...... 89 Phrenic afferents and their functional relevance following SCI ...... 93 Limitations and caveats ...... 98 Summary and Conclusions ...... 99

5 USE OF AAV9 AS AN ANTEROGRADE BULBOSPINAL TRACER IN THE RESPIRATORY CONTROL SYSTEM ...... 119

Introduction ...... 119

Materials and methods...... 121 Experimental animals ...... 121 Surgical approach ...... 122 Recording brainstem inspiratory activity ...... 122 Spinal hemisection ...... 123 Intra-parenchymal vector delivery ...... 123 Postoperative care ...... 124 Tissue processing ...... 124 Experimental cohorts ...... 125 CT-β immunofluorescence ...... 126 Microscopy ...... 127 Results ...... 127 AAV9 vector dose-response trials ...... 127 Brainstem AAV9 micro-injection in spinal intact and SCI rats using the common rVRG coordinates ...... 128 Brainstem AAV9 micro-injection prior to SCI using the common rVRG coordinates...... 130 Brainstem AAV9 micro-injection in spinal intact and SCI rats using individually determined rVRG coordinates...... 131 Discussion ...... 134 Use of AAV9 as an anterograde neuronal tracer ...... 135 Comparison to published histological studies of brainstem rVRG projections 137 Interpretational issues and caveats ...... 139 Summary and conclusion ...... 139

6 CONCLUSIONS AND FUTURE DIRECTIONS ...... 157

Introduction ...... 157 Summary of Project #1: Dir