University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2011 Inflammation-Induced Plasticity of Micturition Reflex aP thways Lauren Arms University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Recommended Citation Arms, Lauren, "Inflammation-Induced Plasticity of Micturition Reflex aP thways" (2011). Graduate College Dissertations and Theses. 9. https://scholarworks.uvm.edu/graddis/9 This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. INFLAMMATION-INDUCED PLASTICITY OF MICTURITION REFLEX PATHWAYS A Dissertation Presented by Lauren Arms to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Neuroscience January, 2011 Accepted by the Faculty ofthe Graduate College, The University ofVermont, in partial fulfillment ofthe requirements for the degree ofDoctor ofPhilosophy, specializing in Neuroscience. Dissertation Examination Committee: Advisor Q~JfiJ~ Deborah H. Damon, Ph.D. Chairperson Dean, Graduate College Date: December 7,2010 Abstract Although a seemingly basic and simple behavior, micturition necessitates precise integration and coordination of multiple divisions of the nervous system: visceral sensory, somatic motor, sympathetic, parasympathetic, as well as voluntary control from higher brain/ brainstem centers. When coordination of this circuitry falters, the consequences can be devastating and include severely decreased quality of life and substantial economic burden. This dissertation project investigates the potential role(s) of inflammatory mediators in bladder sensory physiology with the long term goal of elucidating potential targets for intervention. The overall hypothesis is that inflammatory-induced changes in the urinary bladder or afferent projections ultimately lead to dysfunctional micturition symptoms. Using a rodent model of cyclophosphamide (CYP)-induced bladder inflammation, we examined the expression and function of the chemokine/ receptor pair, CXCL12/CXCR4, and the activated (phosphorylated) form of ubiquitous signaling molecule, AKT using a multidisciplinary approach that includes: immunohistochemistry, protein and transcript quantification techniques, and in vivo bladder physiology studies combined with pharmacological tools. Peripheral chemokine levels are elevated in patients with various chronic pelvic inflammatory/ pain syndromes including interstitial cystitis/bladder pain syndrome (IC/BPS) and are implicated in numerous inflammatory and mechanical pain models in rodents. However studies had not previously shown a direct functional role for chemokine signaling in micturition. We hypothesized that CXCL12 and CXCR4 would increase in the urinary bladder with CYP-induced bladder inflammation and that CXCR4 receptor blockade with AMD3100 would reduce CYP-induced bladder hyperreflexia. ELISA, immunohistochemical and qRT-PCR experiments demonstrate duration- dependent increases in CXCL12 and CXCR4 protein and transcript expression in specific tissue compartments of the urinary bladder, mainly the urothelium. In vivo studies provide evidence of a role for chemokine signaling in the mediation of micturition function. Intravesical infusion with AMD3100, a CXCR4 receptor antagonist, significantly reduced CYP-induced bladder hyperreflexia as evidenced by increased bladder capacity, intercontraction interval and decreased voiding frequency. AKT is a putative cellular survival signal, however, recent studies also implicate the signaling molecule in the induction and maintenance of pain processes, development of long term plasticity (e.g. LTP and central sensitization) and visceral inflammation. Functional studies addressing the contribution of pAKT in micturition have not been performed. We hypothesized that increasing pAKT levels would contribute to CYP- induced bladder hyperreflexia. Western blot and immunohistochemical studies demonstrated that phosphorylation of AKT increases in the whole urinary bladder with CYP-induced bladder inflammation in a tissue compartment- and time-dependent manner. Intravesical infusion with inhibitors of AKT phosphorylation, AKT Inhibitor IV and deguelin, significantly improved symptoms of CYP-induced bladder hyperreflexia suggesting a functional role for pAKT in bladder physiology. These studies demonstrate the functional capacity of inflammatory mediators and inflammatory associated signaling pathways in micturition reflex pathways. Chemokine signaling via the CXCR4 receptor and upstream activators of AKT may provide therapeutic targets with respect to inflammatory-induced bladder sensory physiology dysfunction. Citations Material from this dissertation has been published in the following form: Arms, L., B.M. Girard, M.A. Vizzard (2010). Expression and function of CXCL12/CXCR4 in rat urinary bladder with cyclophosphamide-induced cystitis. American Journal of Physiology: Renal Physiology, 298: F589-F600. ii Dedication For my mother, Susan Arms. She is the epitome of strength and determination; she is my inspiration. iii Acknowledgements First and foremost I thank my parents, Susan and Roger Arms, for the solid foundation they have worked tirelessly to create and maintain in all aspects of my life. Their encouragement throughout the years has enabled me to believe that I can achieve anything. I owe the success of my academic career and happiness in life to their unyielding and selfless support. Graduate school has been an incredible learning and growing experience. I am immensely thankful to have worked with my advisor, Dr. Margaret Vizzard, who dedicated so much of her valuable time to me and my project. While working with Margaret, I gained invaluable skills that will help me undoubtedly throughout my career and personal life. Her unwavering kind and positive demeanor in the face of all situations, combined with her professional and personal ambitions make her an exceptional role model and a cultivating mentor. I would also like to thank members of the Vizzard lab, Susan Malley, Abbey Peterson, Kimberly Corrow and Dr. Beatrice Girard for all they have taught me, their willingness to always lend a helping hand, and their sincere friendship. I have had the fortunate opportunity to work with incredibly intelligent as well as caring and supportive faculty at UVM. My committee members, Dr. Deborah Damon, Dr. Cynthia Forehand, Dr. Gary Mawe and Dr. George Osol have offered valuable insight and taken sincere interest to help me achieve my goals, both present and future. I would also like to thank Dr. Jean Szilva, Dr. Ellen Cornbrooks, Laura Merriam, M.S and Leslie Hoffman, M.S. for making my teaching experiences at UVM incredibly fun, educational, motivational and memorable. My teaching experiences were truly rewarding and special, and I know that I will draw on these experiences for inspiration throughout my career. I extend a special thank you to Andre Zdunczyk for his continued love, support and devotion. He finds a way to keep my feet on positive ground during all the ups and downs experienced while chasing my dreams. iv Table of Contents Citations ........................................................................................................................ ii Dedication .................................................................................................................... iii Acknowledgements ...................................................................................................... iv List of Figures .............................................................................................................. ix List of Tables .................................................................................................................x Abbreviations ............................................................................................................... xi Chapter 1: Comprehensive Literature Review.............................................................1 I. Introduction ...................................................................................................................... 1 A. Anatomy of the Lower Urinary Tract ........................................................................ 3 B. Neural Control of Micturition .................................................................................... 7 C. Awakening of c-fiber Afferents ................................................................................ 13 D. Cystometry: Evaluation of Bladder Function in Humans and Animals ..................... 13 II. Micturition in Injury or Disease ...................................................................................... 16 A. Spinal Cord Injury ................................................................................................... 17 B. Multiple Sclerosis ................................................................................................... 18 C. Parkinson’s Disease ................................................................................................ 18 D. Interstitial Cystitis/ Painful Bladder Syndrome (IC/BPS) .......................................... 19 III. Inflammation and IC/BPS ...........................................................................................