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 to SubU/LP in the course of bladder filling.
1. Dissection of the Detrusor-free Bladder Preparation
<ol>
	<li>Place the isolated bladder in a dissecting dish filled with cold (10 &#176;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.</li>
	<li>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.</li>
	<li>Using a microscope, identify the urethra and ureters and pin each of them to the bottom of the dissecting dish.</li>
	<li>Remove the excess adipose and connective tissues so that the entire main body of the bladder, the urethra and both ureters are displayed.</li>
	<li>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.</li>
	<li>Using fine-tip forceps, gently pull a piece of the serosa at the corner between the ureter and the bladder body.</li>
	<li>Adjust the light of the microscope to increase transparency and distinguish the margin of the submucosa underneath the detrusor muscle.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Make a double loop of 6-0 nylon thread, place it around the neck of the bladder preparation, and leave the loop loose.</li>
	<li>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.</li>
	<li>Cut about 2 cm of 20 PE tubing (catheter), flare up the tip by moving slowly the tip close to a flame.</li>
	<li>Fill the catheter with warm (37 &#176;C) oxygenated KBS.</li>
	<li>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.</li>
	<li>Tie the suture around the catheter and the surrounding tissue of the bladder neck.</li>
	<li>Slowly fill the bladder with about 50-100 &#956;L of warm (37 &#176;C) oxygenated KBS, lift it briefly (&#60;10 s) above the surface of KBS, and monitor for leaks at the sutures and bladder body.</li>
	<li>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.</li>
</ol>
2. Filling of the Denuded Bladder Preparation
<ol>
	<li>Perfuse KBS (37 &#176;C) into a 3 mL chamber of a water (37 &#176;C) jacketed organ dish with a Sylgard bottom.</li>
	<li>Adjust the oxygen and suction lines.</li>
	<li>Place the denuded bladder preparation in the chamber.</li>
	<li>Secure the catheter to the side of the chamber so that the preparation does not float above the surface of the perfusion solution.</li>
	<li>Connect the bladder catheter to a longer PE20 tubing (infusion line) connected to the three way stopcock using same size fitting.</li>
	<li>Make sure that the lines between the infusion pump, the pressure transducer and the bladder are open.</li>
	<li>Fill the infusion syringe with fresh, warm (37 &#176;C) and oxygenated KBS.</li>
	<li>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 &#956;L/min).</li>
	<li>Press the Start button on the syringe pump to fill the bladder.</li>
	<li>Monitor filling volume and intravesical pressure during bladder filling.</li>
</ol>
3. Detection of Mediators in the SubU/LP Aspect of the Denuded Bladder Preparation
<ol>
	<li>Collect aliquots of the bath solution into ice-cold microcentrifuge tubes or high-performance liquid chromatography (HPLC) inserts.</li>
	<li>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 detection13,18.</li>
</ol>

<ol>
	<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. 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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. 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Parts of this work was previously published in the Journal of Physiology (PMCID: PMC6418748; DOI:10.1113/JP27692413). Permission has been granted by Wiley and Sons, Inc. for the use of materials from this publication. The authors have no financial or other conflicts to disclose.

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><tbody><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</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>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</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>MgCl&lt;sub&gt;2&lt;/sub&gt;</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>NaHCO&lt;sub&gt;3&lt;/sub&gt;</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&amp;amp;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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7.1K Views.  University of Nevada Reno School of Medicine. Ons blaasmodel met een verwijderde detrusorspier is een nieuwe experimentele benadering om tegelijkertijd de beschikbaarheid van urotheliale bemiddelaars in de lamiapropria en blaaslumen tijdens het vullen van de blaas te bestuderen. Dit model kan nieuwe inzichten bieden in de signaleringsmechanismen tussen urothelium, suburothelium en detrusorspier die blaasexcitability reguleren tijdens het vullen. Begin met het plaatsen van de intacte murine blaas monster in een ontleden schotel onder een ...

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

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 ...

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 ...

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 ...

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites have been the gold standards for conditional or inducible gene targeting. To provide faster and more efficient experimental models, an ex vivo organoid culture system is developed using adenovirus Cre and normal urothelial cells carrying multiple loxP alleles of the tumor suppressors Trp53, Pten, and Rb1. Normal urothelial cells are enzymatically disassociated from four bladders of triple floxed mice (Trp53f/f: Ptenf/f: Rb1f/f). The urothelial cells are transduced ex vivo with adenovirus-Cre driven by a CMV promoter (Ad5CMVCre). The transduced bladder organoids are cultured, propagated, and characterized in vitro and in vivo. PCR is used to confirm gene deletions in Trp53, Pten, and Rb1. Immunofluorescence (IF) staining of organoids demonstrates positive expression of urothelial lineage markers (CK5 and p63). The organoids are injected subcutaneously into host mice for tumor expansion and serial passages. The immunohistochemistry (IHC) of xenografts exhibits positive expression of CK7, CK5, and p63 and negative expression of CK8 and Uroplakin 3. In summary, adenovirus-mediated gene deletion from mouse urothelial cells engineered with loxP sites is an efficient method to rapidly test the tumorigenic potential of defined genetic alterations.

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

Cancer Research

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 ...

Establishing a Murine Superficial Bladder Cancer Model via an Intravesical Cell Administration Technique

This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. After anesthetizing female C57BL/6J mice in a supine position, a 24 G intravenous needle is inserted into the bladder to evacuate its contents. A 34 G dispensing needle is then introduced through the catheter, rotated five times to create a focal injury to the bladder dome mucosa, and subsequently removed. The MB49 cell suspension is aspirated and connected to a 30 G dispensing needle, which is inserted into the bladder via the catheter. Tumor cells are injected submucosally into the bladder under pressure. This technique results in minimal trauma to the mice, a high tumor take rate, and a fixed tumor location. It is characterized by simplicity and excellent reproducibility. This model provides an ideal experimental platform for developing intravesical therapies for bladder cancer, facilitating the advancement and optimization of treatment strategies for this malignancy.

This protocol describes a unique method for constructing an orthotopic model of superficial bladder cancer.

This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. ...

Detrusor Underactivity Model in Rats by Conus Medullaris Transection

The goal of the presented protocol was to establish a detrusor underactivity (DU) model in the rat through conus medullaris transection. Laminectomy was performed in a total of 40 female Wistar rats (control group: 10 rats; test group: 30 rats) weighing 200&#8211;220 g, and the conus medullaris was transected at the L4&#8210;L5 level in the test group. All the rats were housed and fed under the same environmental conditions for six weeks. In the test group, urine voiding was performed twice daily for six weeks, and mean residual urine volume was recorded. A cystometrogram was performed in both groups. Maximum cystometric capacity (MCC), detrusor opening pressure (DOP), and compliance of the bladder were recorded and calculated. The test group showed significant urinary retention after the surgery, both during and after the spinal shock. However, no abnormality was observed in the control group. When compared to the control group, the MCC and compliance of bladder in the test group was significantly higher than that of the test group (3.24 &#177; 2.261 mL versus 1.04 &#177; 0.571 mL; 0.43 &#177; 0.578 mL/cmH2O versus 0.032 &#177; 0.016 mL/cmH2O), whereas DOP in the test group was lower than control (20.28 &#177; 14.022 cmH2O versus 35 &#177; 13.258 cmH2O). This method of establishing an animal model of DU by the conus medullaris transection offers an excellent opportunity to understand DU&#8217;s pathophysiology in a better manner.

We present a method for establishing a detrusor underactivity model by conus medullaris transection in rats. Detrusor underactivity was successfully stimulated in these animals. The model can be used for studying urinary tract function.

The goal of the presented protocol was to establish a detrusor underactivity (DU) model in the rat through conus medullaris transection. Laminectomy was ...

Neuroscience

A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury

We describe the implementation of spinal cord injury in mice to elicit detrusor-sphincter dyssynergia, a functional bladder outlet obstruction, and subsequent bladder wall remodeling. To facilitate assessment of the cellular composition of the bladder wall in non-injured control and spinal cord injured mice, we developed an optimized dissociation protocol that supports high cell viability and enables the detection of discrete subpopulations by flow cytometry.
Spinal cord injury is created by complete transection of the thoracic spinal cord. At the time of tissue harvest, the animal is perfused with phosphate-buffered saline under deep anesthesia and bladders are harvested into Tyrode&#8217;s buffer. Tissues are minced prior to incubation in digestion buffer that has been optimized based on the collagen content of mouse bladder as determined by interrogation of publicly available gene expression databases. Following generation of a single cell suspension, material is analyzed by flow cytometry for assessment of cell viability, cell number and specific subpopulations. We demonstrate that the method yields cell populations with greater than 90% viability, and robust representation of cells of mesenchymal and epithelial origin. This method will enable accurate downstream analysis of discrete cell types in mouse bladder and potentially other organs.

The goal of this protocol is to apply an optimized tissue dissociation protocol to a mouse model of spinal cord injury and validate the approach for single cell analysis by flow cytometry.

We describe the implementation of spinal cord injury in mice to elicit detrusor-sphincter dyssynergia, a functional bladder outlet obstruction, and ...

Biology

Unilateral Ureteral Obstruction Murine Model: A Surgical Procedure to Obstruct the Urinary Flow from a Kidney in Mouse Models

Source: Yanai, K. et al. <a href="https://www.jove.com/video/61383" target="_blank">Quantitative real-time PCR evaluation of microRNA expressions in mouse kidney with unilateral ureteral obstruction.</a> J. Vis. Exp. (2020)
This video demonstrates the procedure to generate a unilateral ureteral obstruction mouse model. The procedure involves cutting one of the ureters of the mouse to obstruct urine flow.

Source: Yanai, K. et al. Quantitative real-time PCR evaluation of microRNA expressions in mouse kidney with unilateral ureteral obstruction. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Urinary Tract Cancer

In vivo studies

Detrusor Smooth Muscle Cell Isolation: An Enzymatic Digestion Procedure to Obtain DSM Cells from Human Urinary Bladder Specimens

Begin by examining the whole thickness urinary bladder specimen. Pin the specimen, mucosa facing upwards and serosa down, onto a silicone enantiomer-coated 150-millimeter round dish filled with ice-cold DS, and carefully remove all of the mucosa by sharp dissection.
Cut out several mucosa-free detrusor smooth muscle or DSM pieces. Place 3 to 6 DSM pieces into a tube with 1 to 2 milliliters of pre-warmed DS containing papain and dithiothreitol or DS-P. Incubate them at 37 degrees Celsius for 30 to 45 minutes, making sure to shake the tube every 10 to 15 minutes.
After the incubation, remove the DS-P from the tube and briefly wash the DSM pieces with ice-cold DS. Transfer the DS with DSM pieces to a new tube and remove the DS, leaving the DSM pieces at the bottom of the tube. Add 1 to 2 milliliters of DS containing collagenase type II or DS-C to the tube and incubate at 37 degrees Celsius with occasional shaking.
Discard the DS-C and wash the enzyme-treated DSM pieces 5 to 10 times with ice-cold DS. After the last wash, leave the DS inside the tube and gently triturate the pieces with a fire-polished Pasteur pipette to release single DSM cells.

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

Samenvatting

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