Name: a decentralized (ex vivo) murine bladder model with the detrusor muscle removed for direct access to the suburothelium during bladder filling
Uploaded: 2019-11-28T12:30:00
Description: Watch this Scientific Journal Video about A Decentralized Ex Vivo Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling at JoVE.com

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK41315.

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...

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pannexin+1+channels+mediate+the+release+of+ATP+into+the+lumen+of+the+rat+urinary+bladder.">Pannexin 1 channels mediate the release of ATP into the lumen of the rat urinary bladder.</a> Journal of Physiology. 593, 1857-1871 (2015).</li><li>Collins, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OnabotulinumtoxinA+significantly+attenuates+bladder+afferent+nerve+firing+and+inhibits+ATP+release+from+the+urothelium.">OnabotulinumtoxinA significantly attenuates bladder afferent nerve firing and inhibits ATP release from the urothelium.</a> BJU International. 112, 1018-1026 (2013).</li><li>Daly, D. M., Nocchi, L., Liaskos, M., McKay, N. 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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...

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 DSM1,2,3. 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 pressure4,5; from cultured urothelial cells exposed to stretch6,7, hypotonicity-induced cell swelling7 or drag forces8; from isolated bladder wall strips upon receptor or nerve activation9,10,11,12,13,14; and in bladder lumen at end of bladder filling15,16,17,18,19. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CaCl2</td>
			<td>Fisher</td>
			<td>C79</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher</td>
			<td>D16</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Dissecting pins</td>
			<td>Fine Science Tools</td>
			<td>26002-20</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Infusion Pump</td>
			<td>Kent Scientific</td>
			<td>GenieTouch</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher</td>
			<td>P217</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher</td>
			<td>P284</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>SCHOTT ACEI</td>
			<td></td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus SZX7</td>
			<td></td>
			<td>Flexible to use any scope</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fisher</td>
			<td>M33</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>S671</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher</td>
			<td>S233</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Needles 25G</td>
			<td>Becton Dickinson</td>
			<td>305122</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Organ bath</td>
			<td>Custom made</td>
			<td></td>
			<td>Flexible source; We made it from Radnoti dissecting dish</td>
		</tr>
		<tr>
			<td>PE-20 tubing</td>
			<td>Intramedic</td>
			<td>427405</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Pressure transducer</td>
			<td>AD instrument</td>
			<td></td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>S&#38;T Forceps</td>
			<td>Fine Science Tools</td>
			<td>00632-11</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Software pressure-volume</td>
			<td>AD Instruments</td>
			<td>Power lab</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture Nylon, 6-0</td>
			<td>AD surgical</td>
			<td>S-N618R13</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Suture Silk, 6-0</td>
			<td>Deknatel via Braintree Scientific, Inc.</td>
			<td>07J1500190</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Syringes 1 ml</td>
			<td>Becton Dickinson</td>
			<td>309602</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Water circulator</td>
			<td>Baxter</td>
			<td>K-MOD 100</td>
			<td>Source flexible</td>
		</tr>
	</tbody>
</table>

a decentralized (ex vivo) murine bladder model with the detrusor muscle removed for direct access to the suburothelium during bladder filling

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.

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. <strong cl...

Watch this Scientific Journal Video about A Decentralized Ex Vivo Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling at JoVE.com

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

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.

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

Previous studies have established the release of chemical substances from flat bladder mucosa sheets affixed in Ussing chambers and exposed to changes in ...

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