Urine Trouble: A Molecular and Anatomical Examination of Bladder Pain

148 Pages·2016·6.48 MB·English

by  Lara Wiley Crock

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Arts & Sciences Electronic Theses and
Arts & Sciences
Dissertations
Spring 5-15-2013
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Lara Wiley Crock
Washington University in St. Louis
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Crock, Lara Wiley, "Urine Trouble: A Molecular and Anatomical Examination of Bladder Pain" (2013). Arts &
Sciences Electronic Theses and Dissertations. 132.
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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

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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

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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

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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

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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

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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

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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.

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