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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	<li>Apodaca, G., Balestreire, E., Birder, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+uroepithelial-associated+sensory+web.">The uroepithelial-associated sensory web.</a> Kidney International. 72, 1057-1064 (2007).</li><li>Fry, C. H., Vahabi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+the+Mucosa+in+Normal+and+Abnormal+Bladder+Function.">The Role of the Mucosa in Normal and Abnormal Bladder Function.</a> Basic and Clinical Pharmacology and Toxicology. , 57-62 (2016).</li><li>Merrill, L., Gonzalez, E. J., Girard, B. M., Vizzard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptors,+channels,+and+signalling+in+the+urothelial+sensory+system+in+the+bladder.">Receptors, channels, and signalling in the urothelial sensory system in the bladder.</a> Nature Reviewes Urology. 13, 193-204 (2016).</li><li>Ferguson, D. R., Kennedy, I., Burton, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+is+released+from+rabbit+urinary+bladder+epithelial+cells+by+hydrostatic+pressure+changes--a+possible+sensory+mechanism?.">ATP is released from rabbit urinary bladder epithelial cells by hydrostatic pressure changes--a possible sensory mechanism?.</a> Journal of Physiology. 505, 503-511 (1997).</li><li>Wang, E. C., 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=ATP+and+purinergic+receptor-dependent+membrane+traffic+in+bladder+umbrella+cells.">ATP and purinergic receptor-dependent membrane traffic in bladder umbrella cells.</a> Journal of Clinical Investigation. 115, 2412-2422 (2005).</li><li>Miyamoto, T., 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=Functional+role+for+Piezo1+in+stretch-evoked+Ca(2)(+)+influx+and+ATP+release+in+urothelial+cell+cultures.">Functional role for Piezo1 in stretch-evoked Ca(2)(+) influx and ATP release in urothelial cell cultures.</a> Journal of Biological Chemistry. 289, 16565-16575 (2014).</li><li>Mochizuki, T., 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=The+TRPV4+cation+channel+mediates+stretch-evoked+Ca2++influx+and+ATP+release+in+primary+urothelial+cell+cultures.">The TRPV4 cation channel mediates stretch-evoked Ca2+ influx and ATP release in primary urothelial cell cultures.</a> Journal of Biological Chemistry. 284, 21257-21264 (2009).</li><li>McLatchie, L. M., Fry, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+release+from+freshly+isolated+guinea-pig+bladder+urothelial+cells:+a+quantification+and+study+of+the+mechanisms+involved.">ATP release from freshly isolated guinea-pig bladder urothelial cells: a quantification and study of the mechanisms involved.</a> BJU International. 115, 987-993 (2015).</li><li>Birder, L. A., Apodaca, G., de Groat, W. C., Kanai, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenergic-+and+capsaicin-evoked+nitric+oxide+release+from+urothelium+and+afferent+nerves+in+urinary+bladder.">Adrenergic- and capsaicin-evoked nitric oxide release from urothelium and afferent nerves in urinary bladder.</a> American Journal of Physiology Renal Physiology. 275, F226-F229 (1998).</li><li>Birder, L. A., Kanai, A. J., de Groat, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DMSO:+effect+on+bladder+afferent+neurons+and+nitric+oxide+release.">DMSO: effect on bladder afferent neurons and nitric oxide release.</a> Journal of Urology. 158, 1989-1995 (1997).</li><li>Birder, L. A., 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=Vanilloid+receptor+expression+suggests+a+sensory+role+for+urinary+bladder+epithelial+cells.">Vanilloid receptor expression suggests a sensory role for urinary bladder epithelial cells.</a> Proceedings of the National Academy of Sciences U S A. 98, 13396-13401 (2001).</li><li>Birder, L. A., 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=Beta-adrenoceptor+agonists+stimulate+endothelial+nitric+oxide+synthase+in+rat+urinary+bladder+urothelial+cells.">Beta-adrenoceptor agonists stimulate endothelial nitric oxide synthase in rat urinary bladder urothelial cells.</a> Journal of Neuroscience. 22, 8063-8070 (2002).</li><li>Durnin, L., 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=An+ex+vivo+bladder+model+with+detrusor+smooth+muscle+removed+to+analyse+biologically+active+mediators+released+from+the+suburothelium.">An ex vivo bladder model with detrusor smooth muscle removed to analyse biologically active mediators released from the suburothelium.</a> Journal of Physiology. 597, 1467-1485 (2019).</li><li>Yoshida, 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=Non-neuronal+cholinergic+system+in+human+bladder+urothelium.">Non-neuronal cholinergic system in human bladder urothelium.</a> Urology. 67, 425-430 (2006).</li><li>Beckel, J. 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. G., Chapple, C., Grundy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+afferent+pathways+and+urothelial+function+in+the+male+mouse+bladder.">Age-related changes in afferent pathways and urothelial function in the male mouse bladder.</a> Journal of Physiology. 592, 537-549 (2014).</li><li>Durnin, L., Hayoz, S., Corrigan, R. D., Yanez, A., Koh, S. D., Mutafova-Yambolieva, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urothelial+purine+release+during+filling+of+murine+and+primate+bladders.">Urothelial purine release during filling of murine and primate bladders.</a> American Journal of Physiology Renal Physiology. 311, F708-F716 (2016).</li><li>Gonzalez, E. J., Heppner, T. J., Nelson, M. T., Vizzard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+signalling+underlies+transforming+growth+factor-beta-mediated+bladder+afferent+nerve+hyperexcitability.">Purinergic signalling underlies transforming growth factor-beta-mediated bladder afferent nerve hyperexcitability.</a> Journal of Physiology. 594, 3575-3588 (2016).</li></ol>

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 ...

Our bladder model with a removed detrusor muscle is a new experimental approach to studying simultaneously the availability of urothelial mediators in the lamia propria and bladder lumen during bladder filling. This model can provide new insights into the signaling mechanisms between urothelium, suburothelium, and detrusor muscle that regulate bladder excitability during filling. Begin by placing the intact murine bladder specimen into a dissecting dish under a dissection microscope containing 10 degrees celsius oxygenated KBS and cleaning out the connective and fat tissues around the bladder.Pin a small portion of the dome of the isolated bladder all the way through the outermost edge of the detrusor muscle far from the innermost edge of the muscle that faces the suburothelium lamia propria to a sylgard covered dissecting dish filled with KBS, and pin the ureters and bladder neck to display the intact bladder preparation. Using fine-tipped forceps, gently pull a piece of the serosa at the corner between the ureter and the bladder body and adjust the light of the microscope to increase the transparency and to distinguish the margin of the submucosa underneath the detrusor muscle. To avoid perforating the preparation wall, be sure to cut away the detrusor muscle only while the lateral edge of the bladder mucosa is clearly visible.Using fine-tipped scissors, begin cutting the bladder wall along the inner surface of the detrusor muscle layer while gently pulling the cut segment away from the preparation taking care that the lateral edge of the mucosa can be observed while avoiding touching the edge of the tissue. Leave a small piece of the detrusor muscle on top of the bladder dome to allow preparation to be immobilized during the remaining steps of the protocol and fill a two centimeter piece of 20 polyethylene tubing with 37 degree celsius oxygenated KBS. After inserting the catheter into the orifice of the bladder urethra, gently push the catheter until the catheter tip reaches the approximate middle of the bladder.Then secure the catheter in the surrounding tissue of the bladder neck with a double loop 6.0 nylon suture. After checking the tissue for leaks, place the denuded bladder preparation into a three milliliter chamber of 37 degree celsius water jacketed organ dish with a sylgard bottom and secure the catheter to the side of the chamber so that the preparation does not float above the surface of the perfusion solution. Connect the bladder catheter to a piece of polyethylene 20 tubing connected to a three-way stopcock and an infusion syringe filled with 37 degree celsius oxygenated KBS and start the infusion pump to fill the bladder with KBS taking care to monitor the filling volume in the intravesical pressure during the filling.To detect mediators within the suburothelium lamina propria aspect of the preparation, collect aliquots of the bath solution at various appropriate experimental time points and process the samples according to the planned downstream analysis. The denuded bladder preparation wall contains the urothelium and suburothelium but not detrusor smooth muscle. The pressure volume relationships are remarkably similar in the intact and denuded preparations.The suitability of the model for measuring urothelium-derived mediators that are released in the suburothelium lamina propria side during filling can be tested by measuring the release of purine mediators into the solution bathing the suburothelium lamina propria of the denuded preparation. As demonstrated, negligible amounts of purines can be detected in a bath containing an isolated bladder preparation with an intact detrusor muscle;whereas the amounts of these purines are significantly higher in samples collected from the bath containing a denuded bladder preparation. Notably, the distribution of purines and metabolites in samples collected from the lumen and the suburothelium lamina propria at the end of filling differ significantly.Addition of atheno ATP, a highly fluorescent analog of ATP, to the suburothelial side of the detrusor-free preparation results in a decrease in atheno ATP and an increase in the atheno ATP products, atheno ADP, atheno AMP, and atheno ADO. Likewise, the addition of atheno ATP to the lumen preparation results in a decrease in atheno ATP and an increase in atheno ADP, atheno AMP, and atheno ADO in the lumen. Further, the addition of atheno ATP to the suburothelium lamina propria side of the denuded preparation results in the appearance of atheno AMP and atheno ADO within the lumen.Likewise, adding atheno ATP to the lumen results in the appearance of atheno AMP and atheno ADO in the suburothelium lamina propria. Following this procedure, the availability of excitatory and inhibitory mediators can be measured at the lamina propria and in the bladder lumen at different stages of bladder filling. Bilateral transurethral transport of mediators and metabolism of mediators can also be studied using this model.Using this new bladder model will result in new understanding of intrinsic, local mechanosensitive signaling between the urothelium and detrusor smooth muscle that control bladder excitability in health and disease.

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Research

JoVE Journal

Medicine

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology,&#160;and pathology.

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...