A Decentralized (Ex Vivo) Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling

Summary

The detrusor-free bladder model enables direct access to the suburothelium to study local mechanisms for regulation of biologically active mediator availability in suburothelium/lamina propria during storage and voiding of urine. The preparation closely resembles filling of an intact bladder and allows pressure-volume studies to be performed without systemic influences.

Abstract

Previous studies have established the release of chemical substances from flat bladder mucosa sheets affixed in Ussing chambers and exposed to changes in hydrostatic pressure or mechanical stretch and from cultured urothelial cells upon hydrostatic pressure changes, stretch, cell swelling, or drag forces, and in bladder lumen at end of filling. Such findings led to the assumption that these mediators are also released in suburothelium (SubU)/lamina propria (LP) during bladder filling, where they affect cells deep in the bladder wall to ultimately regulate bladder excitability. There are at least two obvious limitations in such studies: 1) none of these approaches provide direct information about the presence of mediators in SubU/LP, and 2) the stimuli used are not physiological and do not recapitulate authentic filling of the bladder. Here, we discuss a procedure that enables direct access to the suburothelial surface of the bladder mucosa in the course of bladder filling. The murine detrusor-free preparation we created closely resembles filling of the intact bladder and allows pressure-volume studies to be performed on the bladder in the absence of confounding signaling from spinal reflexes and detrusor smooth muscle. Using the novel detrusor-free bladder model, we recently demonstrated that intravesical measurements of mediators cannot be used as a proxy to what has been released or present in the SubU/LP during bladder filling. The model enables examination of urothelium-derived signaling molecules that are released, generated by metabolism and/or transported into the SubU/LP during the course of bladder filling to transmit information to neurons and smooth muscle of the bladder and regulate its excitability during continence and micturition.

Introduction

The purpose of this model is to enable direct access to the submucosal side of the bladder mucosa during different phases of bladder filling.

The bladder must refrain from premature contraction during filling and empty when critical volume and pressure are reached. Abnormal continence or voiding of urine are frequently associated with abnormal excitability of the detrusor smooth muscle (DSM) in the course of bladder filling. Excitability of DSM is determined by factors intrinsic to the smooth muscle cells and by influences generated by different cell types within the bladder wall. The wall of the urinary bladder consists of urothelium (muco....

Protocol

All procedures involving animals described in this manuscript were conducted according the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Institutional Animal Use and Care Committee at the University of Nevada.

NOTE: The model presented here consists of the removal of the detrusor muscle while the urothelium and SubU/LP remain intact (Figure 1B) to enable investigators direct access to SubU/LP in the course of bladder filling.

1. Dissection of the Detrusor-free Bladder Preparation

1. Place the....

Results

The wall of murine detrusor-free bladder preparation is intact and contains all layers except the DSM and serosa. Proof-of-principle studies demonstrated that the DSM-free bladder wall includes urothelium and SubU/LP while the tunica muscularis and the serosa are absent (Figure 2)¹³.

Filling of the detrusor-free bladder approximates normal bladder filling.

Discussion

The bladder has two functions: storage and voiding of urine. Normal operation of these functions requires proper mechanical sensing of intraluminal volume and pressure and transduction of signals through cells in the bladder wall to regulate detrusor muscle excitability. The bladder mucosa (urothelium) is believed to regulate bladder excitability by releasing a variety of signaling molecules in the SubU/LP that affect numerous cell types in the bladder wall. Currently, most attempts at characterization of urothelium-deri.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CaCl2	Fisher	C79	Source flexible
Dextrose	Fisher	D16	Source flexible
Dissecting pins	Fine Science Tools	26002-20	Source flexible
Infusion Pump	Kent Scientific	GenieTouch	Source flexible
KCl	Fisher	P217	Source flexible
KH2PO4	Fisher	P284	Source flexible
Light source	SCHOTT ACEI		Source flexible
Microscope	Olympus SZX7		Flexible to use any scope
MgCl2	Fisher	M33	Source flexible
NaCl	Fisher	S671	Source flexible
NaHCO3	Fisher	S233	Source flexible
Needles 25G	Becton Dickinson	305122	Source flexible
Organ bath	Custom made		Flexible source; We made it from Radnoti dissecting dish
PE-20 tubing	Intramedic	427405	Source flexible
Pressure transducer	AD instrument		Source flexible
S&T Forceps	Fine Science Tools	00632-11	Source flexible
Software pressure-volume	AD Instruments	Power lab
Suture Nylon, 6-0	AD surgical	S-N618R13	Source flexible
Suture Silk, 6-0	Deknatel via Braintree Scientific, Inc.	07J1500190	Source flexible
Syringes 1 mL	Becton Dickinson	309602	Source flexible
Vannas Spring Scissors	Fine Science Tools	15000-08	Source flexible
Water circulator	Baxter	K-MOD 100	Source flexible