WWaasshhiinnggttoonn UUnniivveerrssiittyy iinn SStt.. LLoouuiiss WWaasshhiinnggttoonn UUnniivveerrssiittyy OOppeenn SScchhoollaarrsshhiipp Arts & Sciences Electronic Theses and Arts & Sciences Dissertations Spring 5-15-2013 UUrriinnee TTrroouubbllee:: AA MMoolleeccuullaarr aanndd AAnnaattoommiiccaall EExxaammiinnaattiioonn ooff BBllaaddddeerr PPaaiinn Lara Wiley Crock Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Biology Commons RReeccoommmmeennddeedd CCiittaattiioonn Crock, Lara Wiley, "Urine Trouble: A Molecular and Anatomical Examination of Bladder Pain" (2013). Arts & Sciences Electronic Theses and Dissertations. 132. https://openscholarship.wustl.edu/art_sci_etds/132 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology & Biomedical Sciences Neurosciences Dissertation Examination Committee: Robert W. Gereau, Chair Yu-Qing Cao Michael C. Crowder Scott J. Hultgren Indira U. Mysorekar Gina Story Urine Trouble: A Molecular and Anatomical Examination of Bladder Pain by Lara Wiley Crock A dissertation presented to the Graduate School of Arts and Sciences of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2013 St. Louis, Missouri Copyright by Lara Wiley Crock May 2013 TABLE OF CONTENTS LIST OF FIGURES ......................................................................................................................... v ACKNOWLEDGEMENTS ............................................................................................................. vii ABSTRACT OF THE DISSERTATION .......................................................................………..….. ix CHAPTER 1: Introduction to the Role of Metabotropic Glutamate Receptor 5 (mGluR5) in Bladder Pain ............................................................................................................................... 1 Assessment of Bladder Pain in Rodents ........................................................................... 4 Structure and Innervation of the Bladder …………………………………………………...... 6 Role of the Urothelium in IC/PBS …………………………………….....……………………. 7 Urothelium Injury Models ……………….....…………………………………………………… 8 UPEC Injury Model ……………………………………………………………………... 8 PS Injury Model …………………………………………………………………………. 9 LPS Injury Model ……………………………………………………………………..… 9 Sensory Innervation of the Bladder ……………………………….………………………….. 9 Visceral Pain is Different than Somatic Pain …………………………………..…………… 10 mGluR5 Regulates Inflammatory Pain Signaling ……………………........………………. 11 mGluR5 and the Pain Neuroaxis ………………………………………........…………….... 13 mGluR5 and ERK ………………………………………………………….....……………….. 16 References …………………...…………………………………………….....……………….. 18 CHAPTER 2: Protamine Sulfate Induced Bladder Injury Protects from Distention Induced Bladder Pain ……………………………………………………………………………………………….……… 31 Abstract ………………………………...…………………………………………………….... 32   ii Introduction …………………………………………………………………………………..… 33 Materials and Methods ……………………………………………………………………….. 34 Results …………………………………………………………….……………………………. 36 Discussion ……………………………………………………………………………………... 47 References ………………………………………………………...………………………….. 50 CHAPTER 3: Metabotropic Glutamate Receptor 5 (mGluR5) Regulates Bladder Nociception .. 54 Abstract ………………………………………………………………………………………… 55 Background …...……………………………………………………………………………….. 56 Results ……………………………………………………………………………………….… 57 Discussion ……………………………………………………………………………………... 67 Methods ……………………………………………………………………...………………... 72 References …………………………………………………………...……………………….. 75 CHAPTER 4: Amygdala mGluR5 in the modulation of visceral pain ………...……………..……. 83 Abstract …………………………………………………………………………………...…… 84 Introduction …………………………………………………………………………...……….. 85 Methods ………………………..………………………………………………...……………. 87 Results ………………………………………………………………...………………………. 95 Discussion ……………………………………………………………...……………………. 109 References ……………………………………………………………...…………………… 111   iii CHAPTER 5: Discussion and Future Directions ……………………………………………….... 118 Overview ……………………………………………………………….…………………... 119 Differential injury to the bladder results in distinct evoked-responses to bladder distention …..…………………………………...………………………………………….. 119 mGluR5 is necessary for the full expression of bladder pain ………………………… 124 mGluR5 in the CeA modulates distention-induced bladder pain …………….………. 125 Targeting mGuR5 to treat IC/PBS? …………………………………………….……….. 129 References ……………………………………………………………………………….... 131 CURRICULUM VITAE ……………………………………………………………………………… 135   iv LIST OF FIGURES CHAPTER 1: FIGURE 1.1: Proposed etiologic cascade leading to IC/PBS ………………………….….. 4 FIGURE 1.2: Illustration of in vivo urinary bladder distention VMR setup ......................... 6 FIGURE 1.3: Summary of bladder injury models ………………....………………………… 8 FIGURE 1.4: A simplified model of the pain neuroaxis and mGluR5 expression ……… 14 CHAPTER 2: FIGURE 2.1: Protamine sulfate injury is analgesic to bladder distention ………......….……. 38 FIGURE 2.2: Pyuria is not induced by protamine sulfate treatment …………………...…….. 39 FIGURE 2.3: Noxious distention induces hematuria in protamine sulfate-injured animals ... 40 FIGURE 2.4: Histologic analysis shows protamine sulfate injury protects against distention- induced damage …………………………………………………………………….…………...… 42 FIGURE 2.5: Global gene expression profiles after UPEC or PS injury reflect nociception trends …………………………………………………………………………………………….…. 44 TABLE 2.1: Inflammatory genes are differentially regulated by PS- or UPEC-mediated injuries ………………………………..……………………………………………………………. 46 CHAPTER 3: FIGURE 3.1: mGluR5 is necessary for the full expression of non-inflammatory bladder nociception ………..…………………………………………………………………………… 58 FIGURE 3.2: The selective mGluR5 antagonist, fenobam, is analgesic in a distention- induced bladder pain model …………………………………………..…………………….. 60 FIGURE 3.3: mGluR5 KO mice have an increased intermicturition interval ………….… 61 FIGURE 3.4: Fenobam treatment increases the intermicturition interval ……………….. 63 FIGURE 3.5: UPEC infection results in changes in bladder histology and an increased VMR …………………………………………………………………………………………..… 65 FIGURE 3.6: Treatment with an mGluR5 antagonist, fenobam, is analgesic in a UPEC infection-induced inflammatory bladder pain model ………………………………….....….66   v CHAPTER 4: FIGURE 4.1: Intra-amygdala DHPG results in hyperalgesia to bladder distention …….. 96 FIGURE 4.2: Intra-amygdala MPEP is analgesic during bladder distention ….………… 98 FIGURE 4.3: Lentivirus-mediated conditional disruption of mGluR5 in the central amygdala is analgesic ………………….. …………………..………………...……………. 101 FIGURE 4.4: Bladder distention induces spinal cord ERK phosphorylation …………... 103 FIGURE 4.5: Genetic disruption of mGluR5 in the right amygdala reduces bladder distention-induced ERK phosphorylation …………………………………………..……… 104 FIGURE 4.6: Optogenetic stimulation of the right central amygdala induces bladder hyperalgesia ……………………………………………………….…………………………. 106 FIGURE 4.7: Optogenetic stimulation of the right amygdala increases the VMR to bladder distention ……………………………………………………………………………. 102 FIGURE 4.8: Optogenetic activation of the CeA without bladder distention does not alter ERK phosphorylation in the spinal cord …………………………………………………… 104     vi ACKNOWLEDGMENTS I would like to thank my mentor Rob Gereau for his support. I feel incredibly lucky to have a boss who supports his students as a mentor and as a friend. I am in constant awe of his seemingly endless breadth and depth of knowledge, creativity, enthusiasm and sense of humor. Rob is truly inspiring, and I feel honored to be a student in his lab. I want to thank Henry Lai and Chang-Shen Qiu for introducing me to this project and for their guidance throughout. My experience in the Gereau lab would not have been the same without the friendship and support from Sherri Vogt. The lab would not function without her, and I would not be where I am without her help. Gina Story has not only been a mentor, but a friend and confidant. She is always available to talk about science or life and I am grateful for her support. To my current and past lab members, thank you for everything. To Ben Kolber, thank you for being a fantastic collaborator and mentor. I know that Ducane is lucky to have you, and the lab is not the same without you or your sense of humor. Steve Davidson, thank you for your generosity with your time and mentorship. I appreciate the humor that you bring to the lab as well as your wealth of ideas and knowledge. Dan O’Brian, I appreciate your upbeat personality and kindness. You are always willing to lend a hand and you made the lab a welcoming place. Dani Brenner, thank you for always striving to make me laugh and for reading part of my thesis. Judy Golden, thank you for great scientific discussions and your support. Mena Morales, Ben Alter and Mike Montana thank you for expecting the best from me and for challenging me to be a better scientist. Kristina Stemler, thank you for being a friend as well as the best collaborator a person could ask for. Our chance meeting has become a fruitful collaboration. I hope that our scientific paths continue to cross so we can work together again. Thank you Indira Mysorekar for your warmth, kindness and support. I feel incredibly lucky to have found you and Kristi. Many thanks to my committee: Michael C. Crowder, Scott J. Hultgren, Yu-Qing Cao, Gina Story, and Indira Mysorekar. Their critical input, encouragement, guidance and support has shaped my project positively and helped me become a better scientist. Thank you to Jeff Gordon, Brian Sullivan, Daniel Goldberg, Wayne Yokoyama, the MSTP   vii program and staff as well as Karen Winters for their support. I know that at any other MSTP program in the country I would have had to give up on my dreams as the result of chronic pain. Thank you for believing in me, and for both allowing me to take an extended medical leave to recover and for allowing me to re-join the MSTP program. That experience demonstrated the importance of good doctors and inspired me to research pain. To my friends both near and far, I love you and I feel lucky to know such amazing individuals. Especially to Julie, Meredith, Jen, Alissa, Heather, Jamie, Patty, Amanda and the knitters: Thank you for always cheering me on. Thank you to Vito De Pinto, Martha Hansen, Samuel Silverstein and Indra Sethy-Coraci for showing me that science is fun and inspiring me to pursue this path. I cannot express the gratitude I have for my family for their endless support and encouragement. My parents’ kindness, love and understanding are beyond compare. You taught me to work hard for what I want, and to never give up. Thank you for never giving up on me. My mom’s strength and boundless energy has been a continued source of inspiration. To my sisters, Vanessa and Terry, you continually inspire me to try harder. Thank you for always being there for me. A special thank you to Nick, my partner and best friend for the past 8 years. Thank you for being here for me during the good and bad. You keep me positive and strong and you believed in me even when I didn’t believe in myself. I don’t know where I would be without the support you provide, I love you. A special thank you to Ralph and Kay Weidlich for the love and overwhelming support they have given me. Most of the work presented in this thesis was funded by grants from the National Institute of Health. Including an F30 DK089969 (Lara Crock), T32-A1007172 (Kristina Stemler) DK082315 (Rob Gereau), DK082315-02S1 (Henry Lai), NS48602 (Rob Gereau), K99/R00 Pathway to independence award, DK080643 (Indira Mysorekar), the Alafi Neuroimaging Laboratory, the Hope Center for Neurological Disorders, and NIH Neuroscience Blueprint Center Core Grant P30 NS057105 to Washington University.     viii