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 (mucosa), suburothelium (SubU)/lamina propria (LP), detrusor smooth muscle (DSM) and serosa (Figure 1A). The urothelium consists of umbrella cells (i.e., the outermost layer of the urothelium), intermediate cells, and basal cells (i.e., the innermost layer of the urothelium). Various types of cells, including interstitial cells, fibroblasts, afferent nerve terminals, small blood vessels, and immune cells reside in the SubU/LP. It is widely assumed that the bladder urothelium is a sensory organ that initiates reflex micturition and continence by releasing mediators into the submucosa that affect cells in the SubU/ LP and the DSM^1,2,3. For the most part, such assumptions are based on studies that have demonstrated release of mediators: from pieces of mucosa exposed to changes in hydrostatic pressure^4,5; from cultured urothelial cells exposed to stretch^6,7, hypotonicity-induced cell swelling⁷ or drag forces⁸; from isolated bladder wall strips upon receptor or nerve activation^{9,10,11,12,13,14}; and in bladder lumen at end of bladder filling^{15,16,17,18,19}. While such studies were instrumental to demonstrate release of mediators upon mechanical stimulation of bladder wall segments or cultured urothelial cells, they need to be supported by direct evidence for release of mediators in the submucosa that is elicited by physiological stimuli that reproduce bladder filling. This is a challenging task given that the SubU/LP is located deep in the bladder wall hampering the straightforward access to the vicinity of SubU/LP during bladder filling.

Here, we illustrate a decentralized (ex vivo) murine bladder model with the detrusor muscle removed¹³ that was developed to facilitate studies on local mechanisms of mechanotransduction that participate in the signaling between the bladder urothelium, DSM and other cell types in the bladder wall. This approach is superior to using flat bladder wall sheets, bladder wall strips or cultured urothelial cells because it allows direct measurements in the vicinity of SubU/LP of urothelium-derived mediators that are released or formed in response to physiological pressures and volumes in the bladder and avoids potential phenotypic changes in cell culture. It can be used to measure availability, release, metabolism and transurothelial transport of mediators in SubU/LP at different stages of bladder filling (Figure 1B). The preparation can also be used to examine urothelial signaling and mechanotransduction in models of overactive and underactive bladder syndromes.

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 isolated bladder in a dissecting dish filled with cold (10 °C) and oxygenated with 5% CO₂/95 % O₂ Krebs bicarbonate solution (KBS) with the following composition (mM): 118.5 NaCl, 4.2 KCl, 1.2 MgCl₂, 23.8 NaHCO₃, 1.2 KH₂PO₄, 11.0 dextrose, 1.8 CaCl₂ (pH 7.4)¹³.
2. Pin a small portion of the dome of the isolated bladder to a Sylgard-covered dissecting dish filled with KBS. Make sure that the pin goes through a piece of the serosa or the outermost edge of the detrusor muscle far from the innermost edge of the muscle that faces the SubU/LP.
3. Using a microscope, identify the urethra and ureters and pin each of them to the bottom of the dissecting dish.
4. Remove the excess adipose and connective tissues so that the entire main body of the bladder, the urethra and both ureters are displayed.
5. Tie the ureters with 6-0 nylon sutures. Then, pin the open ends of ureters towards the bottom of the dissecting dish to secure the preparation.
6. Using fine-tip forceps, gently pull a piece of the serosa at the corner between the ureter and the bladder body.
7. Adjust the light of the microscope to increase transparency and distinguish the margin of the submucosa underneath the detrusor muscle.
8. Start cutting (not peeling!) the bladder wall with fine-tip scissors along the inner surface of the detrusor muscle layer while gently pulling the cut segment away from the preparation. At all times, ensure that the lateral edge of mucosa can be seen and avoid touching it.
9. Remove the detrusor muscle entirely by turning around the dissecting dish so that the position of the preparation is comfortable to continue dissecting out the detrusor muscle.
10. Leave a small piece of detrusor muscle on the top of the bladder dome to ensure ability to immobilize the preparation during the remaining steps of the protocol.
11. Make a double loop of 6-0 nylon thread, place it around the neck of the bladder preparation, and leave the loop loose.
12. Add a second double loop of 6-0 silk thread, place it around the neck of the bladder preparation, and leave the loop lose. Having two sutures prevents leaks around the sutures.
13. Cut about 2 cm of 20 PE tubing (catheter), flare up the tip by moving slowly the tip close to a flame.
14. Fill the catheter with warm (37 °C) oxygenated KBS.
15. Insert the catheter in the orifice of the bladder urethra and gently push the catheter until the catheter tip reaches approximately the middle of the bladder.
16. Tie the suture around the catheter and the surrounding tissue of the bladder neck.
17. Slowly fill the bladder with about 50-100 μL of warm (37 °C) oxygenated KBS, lift it briefly (<10 s) above the surface of KBS, and monitor for leaks at the sutures and bladder body.
18. If no leak is observed, the preparation is ready for the experiment. If a leak around the suture is observed, remove the suture and replace it. If a leak from a hole in the bladder body is noticed, discard the preparation.

2. Filling of the Denuded Bladder Preparation

1. Perfuse KBS (37 °C) into a 3 mL chamber of a water (37 °C) jacketed organ dish with a Sylgard bottom.
2. Adjust the oxygen and suction lines.
3. Place the denuded bladder preparation in the chamber.
4. Secure the catheter to the side of the chamber so that the preparation does not float above the surface of the perfusion solution.
5. Connect the bladder catheter to a longer PE20 tubing (infusion line) connected to the three way stopcock using same size fitting.
6. Make sure that the lines between the infusion pump, the pressure transducer and the bladder are open.
7. Fill the infusion syringe with fresh, warm (37 °C) and oxygenated KBS.
8. Adjust the pump parameters: type/volume of syringe (i.e., 1 mL), operation (i.e. Infuse), flow (i.e., constant), and flow rate (i.e., 15 μL/min).
9. Press the Start button on the syringe pump to fill the bladder.
10. Monitor filling volume and intravesical pressure during bladder filling.

3. Detection of Mediators in the SubU/LP Aspect of the Denuded Bladder Preparation

1. Collect aliquots of the bath solution into ice-cold microcentrifuge tubes or high-performance liquid chromatography (HPLC) inserts.
2. Prepare and process the samples according to the appropriate detection application. In the case of detecting purine availability, process the samples by HPLC with fluorescence detection^13,18.

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