This study was supported by NIH R37 DK54824 and R01 DK57284 grants to LB.

All procedures described here are approved by the IACUC committee at University of Pittsburgh.
1. Solutions
<ol>
	<li>Prepare Krebs solution according to the recipe. Composition in mM: NaCl 118, KCl 4.7, CaCl2 1.9, MgSO4 1.2, NaHCO3 24.9, KH2PO4 1.2, dextrose 11.7.</li>
	<li>Aerate Krebs with 95% O2, 5% CO2 and place it in a 37 &#186;C water bath to be used throughout the experiment. Place aside ~200 ml of aerated Krebs solution at room temperature to be used for tissue dissection.</li>
	<li>Measure pH (~7.4) and osmolarity (~ 300 mOsm) of aerated Krebs.</li>
</ol>
2. Experimental Set-up (Schematic Figure 1A)
<ol>
	<li>Fill aerated (95% O2, 5% CO2) chambers with 10 ml Krebs.</li>
	<li>Start the circulating water pump to heat the chambers to 37 &#176;C; turn on the necessary equipment: amplifier(s), stimulator(s) and recording software.</li>
	<li>Calibrate transducers with a 1 g weight.</li>
</ol>
3. Tissue (Figure 1B)
Remove the bladder from an adult na&#239;ve female Sprague Dawley rat (200-250 g; ~10-12 weeks old) following these steps:
<ol>
	<li>Prepare dissection area and necessary instruments: electric razors, forceps with teeth, scalpel blade, dissecting scissors, microscissors, two dissecting forceps (authors prefer Dumont forceps #3), tissue clips (or silk suture), a Sylgard coated dissection dish with Krebs and tissue dissection pins.</li>
	<li>Anesthetize the animal with isoflurane inhalation (4% in O2) in the induction chamber. Use veterinary ointment on eyes to prevent dryness while under anesthesia. Continuously monitor the level of anesthesia by observing the respiration rate, response to external stimuli, and loss of rear limb withdraw reflex.</li>
	<li>When the animal is anesthetized shave the lower abdominal area. Expose the pelvic organs via a midline abdominal incision. Identify the bladder and urethra. Remove the bladder by cutting at the bladder neck close to proximal urethra. Place tissue immediately in the Sylgard coated dish filled with aerated Krebs solution.</li>
	<li>If needed, remove additional tissue at this time: urethra, pieces of gastrointestinal (GI) tract and/or prostate, etc.</li>
	<li>Sacrifice the animal using IACUC approved methods (e.g., anesthetic overdose or CO2 asphyxiation followed by a secondary method).</li>
	<li>Insert tissue dissecting pins through the bladder dome, neck, and ureters, to stabilize the tissue for further dissection. Do not stretch the tissue. Remove fat, connective tissue, proximal urethra, and ureters if present.</li>
	<li>Open the bladder from the base to dome to create a flat sheet, serosa side down/luminal side up (Figure 1B). Place dissecting pins on each corner of the tissue. Remove bladder dome and neck tissue.</li>
	<li>If the purpose of the experiment is to determine the contribution of the mucosa (urothelium and lamina propria &#8212; see diagram Figure 1C) to the smooth muscle contraction, compare the properties of detrusor strips with and without the mucosa attached. For this, prior to cutting the tissue in strips, carefully remove the mucosal layer using iris spring scissors and fine forceps under a dissecting microscope. At the end of the experiment, fix the strips for H&#38;E staining to confirm complete removal of the mucosa. Note that this procedure is easier in mouse bladder than in rat bladder.</li>
	<li>Cut the tissue lengthwise from base to dome into strips of ~2 x 8 mm (Figure 1B). Tie or attach a tissue clip to both ends of each strip. 
	NOTE: One rat bladder can usually be cut into 4 strips but the number of strips can increase or decrease depending on the animal/bladder size.</li>
	<li>Transfer the strips to the experimental chambers. Attach one end of each strip to a force transducer, which measures the tissue contraction, and the other to a fixed glass/metal rod. 
	NOTE: Tissue chambers vary in size (0.2 ml to 20 ml or larger). Typical chambers for rodent bladders are 5-20 ml, which provide sufficient height for the strips to be completely submersed in solution. Some chambers come with built-in stimulation electrodes, others not. Care should be taken to ensure that all connections of the electrodes are in good condition, otherwise electrical field stimulation is not reliable.</li>
	<li>Apply a defined amount of force to each strip by gently stretching the tissue until baseline tension reaches 1 g (~10 mN). Initially the tissue tends to relax which is recorded as a decrease in baseline tension. Wash tissue approximately every 15 min using the warm aerated Krebs and adjust the baseline tension to 1 g after each wash. Allow tissue to equilibrate for ~1-2 hr or until baseline tension is stable (i.e. no further tissue relaxation).</li>
	<li>Test tissue viability by adding KCl (80 mM) directly to the bath for ~5 min, or until a plateau response is reached. Responses to high concentrations of KCl can also be repeated during the experiment or at the end of the experiment and used for normalizing responses to other drugs or between strips (see normalization under data analysis section).</li>
	<li>Wash tissue multiple times (3-5x) with the warm aerated Krebs to allow the tissue to return to pre-treatment conditions.</li>
</ol>
4. Stimulation Protocols
<ol>
	<li>To investigate the effects of pathology on spontaneous myogenic activity or smooth muscle tone, use smooth muscle strips from different animal models such as SCI, BOO, or neonates. Figure 2 illustrates the use of this method to investigate changes in bladder spontaneous activity during development and after SCI. In addition, pharmacological agents can be used to modulate spontaneous activity. Figure 3 illustrates the effect of the KCNQ channel modulators, flupirtine and XE991, on spontaneous activity and smooth muscle tone.</li>
	<li>For pharmacological smooth muscle stimulation construct concentration response curves by adding compounds from concentrated stock solutions directly to the bath at defined time intervals. Use drug and vehicle in parallel strips to account for vehicle and time effects.
	<ol>
		<li>Make stock solutions of desired test compounds at 1000x the final working concentration. For carbachol (CCh), a muscarinic receptor agonist, prepare following stocks: 10-5 M, 3 x 10-5 M, 10-4 M, 3 x 10-4 M, 10-3 M, 3 x 10-3 M, 10-2 M. Final concentrations in the bath is 10-8 M to 10-5 M (Figures 4C, D). For neuromedin B (NMB), a bombesin receptor subtype 1 agonist, prepare following stocks: 10-8 M, 10-7 M, 10-6 M, 10-5 M, 10-4 M, 10-3 M and final concentrations in the bath are 10-11 M to 10-6 M. Both CCh and NMB are expected to increase tissue contractility.</li>
		<li>For a 10 ml tissue bath, add 10 &#181;l of each CCh stock solution as soon as the response reaches a plateau (Figure 4C, D). In parallel strips add equal amounts of the vehicle (water). Similarly, add 10 &#181;l of each neuromedin B stock solution every ~5 min. 
		NOTE: Observe the excitatory effect of NMB and CCh on smooth muscle tone in strips from different species in Figure 4.</li>
		<li>Investigate the relaxation properties of the smooth muscles in pre-contracted tissue with an excitatory agent, usually CCh or KCl.</li>
		<li>To block an agonist response, pretreat the tissue for 10-20 min with the antagonist to allow tissue penetration, prior to agonist stimulation.</li>
	</ol>
	</li>
	<li>For neural stimulation of the smooth muscle, also called electric field stimulation (EFS) follow steps 1 to 3.13 and continue as described below. EFS is intended to selectively activate nerves versus smooth muscle. Parameters for stimulation should be carefully chosen to avoid direct smooth muscle stimulation.
	<ol>
		<li>Establish stimulation parameters: type of stimulus (single pulses or trains), duration (pulse duration and train duration), frequency and intensity, as described in the steps below and illustrated in Figures 5A, B.
		<ol>
			<li>For single pulse stimulation, set pulse duration, inter-stimulus interval and number of stimuli desired. Usual stimulation duration parameters are single pulses of 0.05-0.3 msec duration delivered at desired intervals (Figure 5A). Follow step 4.3.1.4 for stimulus intensity.</li>
			<li>For train stimulation, set the train duration and inter train interval. Typical values for bladder tissue are 3-10 sec delivered at least 1 min apart (Figure 5B). If tissue fatigue occurs (i.e. EFS contractions decrease during control period), increase the inter train interval.</li>
			<li>Establish the frequency of train stimuli (number of pulses in a train &#8212; Figure 5B). Run a frequency response curve ranging from 0.5-50 Hz. Typical frequencies for bladder are 10-20 Hz, which give reproducible and stable contractions mediated by ATP and ACh. Observe the frequency dependent responses to EFS stimulation in mouse bladder strips in Figure 5 demonstrating how this method can be used to assess the contribution of cholinergic and purinergic mechanisms to neurotransmission.</li>
			<li>Establish intensity of the stimulus: systematically increase the intensity (voltage) of the stimulus until the amplitude of the contraction reaches a plateau (if using trains keep the frequency constant).</li>
			<li>Set the intensity of the stimulus depending on the aim of the experiment. If the aim is to increase the neurally-evoked contractions, then use submaximal intensity such that the amplitude of contraction is ~50% of maximal contraction. If the aim is to decrease the neurally-evoked contractions, then set the intensity to ~80% of maximal amplitude to avoid tissue fatigue.</li>
		</ol>
		</li>
		<li>Once stimulation parameters (duration, frequency and intensity) are established, allow ~20-30 min for EFS- evoked contractions to stabilize prior to drug testing. 
		NOTE: To verify the selectivity of EFS for neural transmission, block neural transmission with the Na+ channel blocker, tetrodotoxin (TTX; 0.5-1 &#181;M). Perform this step at the beginning of the experiment, as TTX washes off relatively easy. In addition, perform this at the end of the experiment (see step 4.3.5. below).</li>
		<li>Prepare stock solutions at 1,000x the final working concentrations for: alpha,beta-methylene ATP (ABMA; a purinergic receptor activator and desensitizer) 10-2 M, atropine (a muscarinic receptor antagonist) 10-3 M (Figure 5C). Observe other examples in Figure 6. The 5HT4 receptor agonist, cisapride (3 x 10-6 M, 10-6 M, 3 x 10-5 M, 10-5 M, 3 x 10-4 M, 10-4 M, 3 x 10-3 M, 10-3 M),&#160;increases tissue contractility and SB-203186 (3 x 10-3 M), a 5HT4 receptor antagonist, reverses cisapride&#39;s effects.</li>
		<li>To test the effects of ABMA and atropine on EFS (Figure 5C), perform two control frequency response curves. Add 10 &#181;l of 10-2 M ABMA to the bath for a final concentration of 10 &#181;M. This will contract the tissue due to direct stimulation of purinergic receptors in the smooth muscle. After the response returns to baseline, repeat frequency response curves. Add 10 &#181;l of 10-3 M atropine for a final concentration of 1 &#181;M. After ~10 min (needed for the atropine to block muscarinic receptors), repeat frequency response curves. In parallel strips add 10 &#181;l of the vehicle, water, at each step. 
		NOTE: For other examples in Figure 6, add 10 &#181;l of each cisapride stock solution at defined time intervals (~ every 15 min; see discussion), followed by 10 &#181;l of SB-203186 stock solution directly to the bath and monitor their effect on EFS-induced contraction. In parallel strips add 10 &#181;l of the vehicle, DMSO. Observe the effects of cisapride, a 5HT4 receptor agonist, on EFS-evoked contractions in human bladder and ileum tissues in Figure 6. Additionally, observe the effect of DMSO, the vehicle for cisapride, on EFS-evoked contractions in human bladder and ileum tissues.</li>
		<li>At the end of the EFS protocol verify the selectivity of EFS by blocking neural transmission with the Na+ channel blocker, tetrodotoxin (TTX; 0.5-1 &#181;M). If TTX resistant contractions are still present, it is recommended to adjust the duration and intensity of the stimulus in subsequent experiments.</li>
	</ol>
	</li>
	<li>For determining the effects of drugs on pre or post-synaptic sites (Figure 7A) follow steps 1 to 4.3.2. Establish reproducible responses to carbachol and EFS, then add drug X.</li>
	<li>At the end of the experiment, unclip or untie the strips, blot them gently on a piece of tissue paper to eliminate extra fluid and measure each strip&#8217;s weight using a balance. Also measure the tissue length using a caliper to determine cross sectional area. This information is used for normalization of data (see section 5.4).</li>
</ol>
5. Data Analysis
Analyze data using adequate software (e.g., Windaq, LabChart).
<ol>
	<li>For spontaneous activity, select a window of at least 30 sec before and at the peak of the drug induced response and measure amplitude and frequency of myogenic activity (Figure 3).
	<ol>
		<li>Use fast Fourier transformation analysis to determine the spectrum of frequencies contributing to contractile responses and whether there are differences between different parts of the bladder (e.g., dome vs. neck) or with development, pathology, and drugs8.</li>
	</ol>
	</li>
	<li>For effects on smooth muscle tone select a window of at least 10-30 sec before and at the peak of the drug induced response and measure amplitude of contraction.</li>
	<li>For effects on neurally-evoked contractions measure amplitude, duration and area under the curve of contractions (at least 3) before and at the peak of the drug-induced response. 
	NOTE: It is necessary to measure both the amplitude and area under the curve of EFS-induced contractions because purinergic and cholinergic components have different kinetics. The purinergic component is fast and transient (ATP activates purinergic ionotropic channels such as P2X1 that allow fast influx of calcium, then they desensitize), thus contributing more to the peak amplitude response and less to the area under the curve. The cholinergic component is slower and sustained (ACh activates metabotropic muscarinic receptors, which require more time to activate intracellular pathways that ultimately activate ion channels that depolarize the smooth muscle to induce a contraction). Thus, the muscarinic component is captured better by measuring the area under the curve.</li>
	<li>Normalize the data to be able to compare results across strips and pharmacological treatments. The parameter chosen for normalization should not be influenced by the test compounds, pathological condition studied or experimental design. Among these parameters, use strip weight, cross sectional area, KCl responses (Figure 4B), % of the maximal response (Figure 7B) or % of the maximal response to another contractile agent (e.g., CCh) or relaxing agents (e.g., papaverine).</li>
</ol>

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<li>Andersson, K. E., Wein, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmacology+of+the+lower+urinary+tract%3A+basis+for+current+and+future+treatments+of+urinary+incontinence%5BTitle%5D)+AND+%22Pharmacological+reviews%22%5BJournal%5D)">Pharmacology of the lower urinary tract: basis for current and future treatments of urinary incontinence.</a> Pharmacological reviews. 56, 581-631 (2004).</li>
<li>D’Agostino, G., Condino, A. M., Gallinari, P., Franceschetti, G. P., Tonini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+prejunctional+serotonin+receptors+modulating+%5B3H%5Dacetylcholine+release+in+the+human+detrusor%5BTitle%5D)+AND+%22The+Journal+of+pharmacology+and+experimental+therapeutics%22%5BJournal%5D)">Characterization of prejunctional serotonin receptors modulating [3H]acetylcholine release in the human detrusor.</a> The Journal of pharmacology and experimental therapeutics. 316, 129-135 (2006).</li>
<li>Hawthorn, M. H., Chapple, C. R., Cock, M., Chess-Williams, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Urothelium-derived+inhibitory+factor%28s%29+influences+on+detrusor+muscle+contractility+in+vitro%5BTitle%5D)+AND+%22British+journal+of+pharmacology%22%5BJournal%5D)">Urothelium-derived inhibitory factor(s) influences on detrusor muscle contractility in vitro.</a> British journal of pharmacology. 129, 416-419 (2000).</li>
<li>Chaiyaprasithi, B., Mang, C. F., Kilbinger, H., Hohenfellner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+human+detrusor+contraction+by+a+urothelium+derived+factor%5BTitle%5D)+AND+%22J+Urol%22%5BJournal%5D)">Inhibition of human detrusor contraction by a urothelium derived factor.</a> J Urol. 170, 1897-1900 (2003).</li>
<li>Testa, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+different+5-hydroxytryptamine+receptor+subtype+antagonists+on+the+micturition+reflex+in+rats%5BTitle%5D)+AND+%22BJU+international%22%5BJournal%5D)">Effect of different 5-hydroxytryptamine receptor subtype antagonists on the micturition reflex in rats.</a> BJU international. 87, 256-264 (2001).</li>
<li>Craggs, M. D., Rushton, D. N., Stephenson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+putative+non-cholinergic+mechanism+in+urinary+bladders+of+New+but+not+Old+World+primates%5BTitle%5D)+AND+%22J+Urol%22%5BJournal%5D)">A putative non-cholinergic mechanism in urinary bladders of New but not Old World primates.</a> J Urol. 136, 1348-1350 (1986).</li>
<li>Fry, C. H., Bayliss, M., Young, J. S., Hussain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+age+and+bladder+dysfunction+on+the+contractile+properties+of+isolated+human+detrusor+smooth+muscle%5BTitle%5D)+AND+%22BJU+international%22%5BJournal%5D)">Influence of age and bladder dysfunction on the contractile properties of isolated human detrusor smooth muscle.</a> BJU international. 108, 91-96 (2011).</li>
<li>Kennedy, C., Tasker, P. N., Gallacher, G., Westfall, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+atropine-+and+P2X1+receptor+antagonist-resistant%2C+neurogenic+contractions+of+the+urinary+bladder%5BTitle%5D)+AND+%22The+Journal+of+neuroscience+%3A+the+official+journal+of+the+Society+for+Neuroscience%22%5BJournal%5D)">Identification of atropine- and P2X1 receptor antagonist-resistant, neurogenic contractions of the urinary bladder.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 27, 845-851 (2007).</li>
<li>Levin, R. M., Danek, M., Whitbeck, C., Haugaard, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+ethanol+on+the+response+of+the+rat+urinary+bladder+to+in+vitro+ischemia%3A+protective+effect+of+alpha-lipoic+acid%5BTitle%5D)+AND+%22Molecular+and+cellular+biochemistry%22%5BJournal%5D)">Effect of ethanol on the response of the rat urinary bladder to in vitro ischemia: protective effect of alpha-lipoic acid.</a> Molecular and cellular biochemistry. 271, 133-138 (2005).</li>
<li>Malysz, J., Afeli, S. A., Provence, A., Petkov, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ethanol-mediated+relaxation+of+guinea+pig+urinary+bladder+smooth+muscle%3A+Involvement+of+BK+and+L-type+Ca2%2B+channels%5BTitle%5D)+AND+%22American+journal+of+physiology.+Cell+physiology%22%5BJournal%5D)">Ethanol-mediated relaxation of guinea pig urinary bladder smooth muscle: Involvement of BK and L-type Ca2+ channels.</a> American journal of physiology. Cell physiology. 306, 45-58 (2013).</li>
<li>Longhurst, P. A., Briscoe, J. A., Rosenberg, D. J., Leggett, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+cyclic+nucleotides+in+guinea-pig+bladder+contractility%5BTitle%5D)+AND+%22British+journal+of+pharmacology%22%5BJournal%5D)">The role of cyclic nucleotides in guinea-pig bladder contractility.</a> British journal of pharmacology. 121, 1665-1672 (1997).</li>
<li>Takahashi, R., Yunoki, T., Naito, S., Yoshimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+effects+of+botulinum+neurotoxin+A+on+bladder+contractile+responses+to+activation+of+efferent+nerves%2C+smooth+muscles+and+afferent+nerves+in+rats%5BTitle%5D)+AND+%22J+Urol%22%5BJournal%5D)">Differential effects of botulinum neurotoxin A on bladder contractile responses to activation of efferent nerves, smooth muscles and afferent nerves in rats.</a> J Urol. 188, 1993-1999 (2012).</li>
<li>Sadananda, P., Chess-Williams, R., Burcher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contractile+properties+of+the+pig+bladder+mucosa+in+response+to+neurokinin+A%3A+a+role+for+myofibroblasts%5BTitle%5D)+AND+%22British+journal+of+pharmacology%22%5BJournal%5D)">Contractile properties of the pig bladder mucosa in response to neurokinin A: a role for myofibroblasts.</a> British journal of pharmacology. 153, 1465-1473 (2008).</li>
<li>Liu, G., Daneshgari, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alterations+in+neurogenically+mediated+contractile+responses+of+urinary+bladder+in+rats+with+diabetes%5BTitle%5D)+AND+%22American+journal+of+physiology.+Renal+physiology%22%5BJournal%5D)">Alterations in neurogenically mediated contractile responses of urinary bladder in rats with diabetes.</a> American journal of physiology. Renal physiology. 288, 1220-1226 (2005).</li>
</ol>

The authors declare that they have no competing financial interests.

In this paper we described a simple in vitro smooth muscle contractility method that can be used to address a number of important scientific questions related to bladder physiology and pathology, as well as aiding the discovery of new drugs to treat bladder dysfunctions. We have illustrated the use of this method for assessing developmental, pathological and pharmacological properties of bladder smooth muscle contractility (Figures 2-4), neurotransmission modulation (Figures 5-7A</strong...

The bladder smooth muscle relaxes to allow urine storage, and contracts to elicit urine elimination. Relaxation is mediated by intrinsic smooth muscle properties and by tonic release of norepinephrine (NE) from the sympathetic nerves, which activates beta adrenergic receptors (&#946;3AR in human) in the detrusor. Voiding is achieved by inhibiting the sympathetic input and activating the parasympathetic nerves that release ACh/ATP to contract the bladder smooth muscle1. Numerous pathological conditions, including brain and/or spinal cord injury, neurodegenerative diseases, diabetes, bladder outlet obstruction or interstitial cystitis, can profoundly alter bladder function, with severe impact on the patient&#8217;s quality of life2. These conditions alter the contractility of the smooth muscle by affecting one or more components of the bladder: the smooth muscle, the afferent or efferent nerves and/or the mucosa.
Several in vivo and in vitro methods to study bladder function have been developed. In vivo, cystometry is the primary measurement of bladder function. Though this is an intact preparation that allows collection of information under close to physiological conditions, there are a number of circumstances in which the use of smooth muscle strips is preferred. These include situations when surgical and/or pharmacological manipulations would affect the survival and stability of the in vivo preparation, or when the studies require the use of the human tissue or expensive chemicals. This method also facilitates an examination of the effects of drugs, age and pathology on each component of the bladder, i.e. smooth muscle, mucosa, afferent and efferent nerves.
Bladder strips have been employed over the years by many groups to answer a number of scientific questions. They were used to evaluate changes in myogenic spontaneous activity induced by pathology. This activity is believed to contribute to the urgency and frequency symptoms of overactive bladder (OAB), and is therefore a target for drugs being developed for OAB3-9. Bladder strips were also used to investigate myogenic and neuronal factors that modulate smooth muscle tone with the aim of discovering ion channels and/or receptors and/or intracellular pathways that could be targeted to induce either relaxation or contraction of the smooth muscle3,10-13. Other studies have focused on the nature of neurotransmission, including transmitters and receptors involved and changes induced by pathology14,15. In addition, the method has been used for comparisons between tissues from different species16-18, between organs19-21, and evaluation of drug structure-activity relationships22-24. An extension of this method has been used to measure the effect of drugs on transmitter release from efferent nerves25. Furthermore, a variety of tissues (bladder, urethra, gastrointestinal tract, GI) harvested from animals or humans (from surgeries or organ donor tissue approved for research) and from a variety of animal models including spinal cord injury (SCI), bladder outlet obstruction (BOO), or interstitial cystitis (IC) can be investigated using this technique.
In this paper we illustrate the use of this method along with necessary experimental protocols, to address several scientific questions mentioned above.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Tissue Bath System with Reservoir</td><td>Radnoti, LLC</td><td>159920</td><td>isolated tissue baths</td></tr><tr><td>Warm water recirculator pump</td><td>Kent Scientific Corporation&amp;nbsp;</td><td>TPZ-749</td><td>to keep tissue baths to 37 &amp;#176;C</td></tr><tr><td>Computer</td><td></td><td></td><td></td></tr><tr><td>Data Acquisiton System</td><td>DataQ Instruments</td><td>DI-710-UH</td><td>To view, record and analyze data</td></tr><tr><td>Transbridge Transducer Amplifier</td><td>World Precision Instruments</td><td>SYS-TBM4M</td><td>Transducer amplifier</td></tr><tr><td>Grass stimulator</td><td>Grass Technologies</td><td>Model S88</td><td>Stimulator</td></tr><tr><td>Anesthesia System</td><td>Kent Scientific Corporation&amp;nbsp;</td><td>ACV-1205S</td><td>To anesthetesize the animal</td></tr><tr><td>Anesthetizing Box</td><td>Harvard Apparatus</td><td>500116</td><td>To anesthetesize the animal</td></tr><tr><td>Anesthesia Masks</td><td>Kent Scientific Corporation&amp;nbsp;</td><td>AC-09508</td><td>To anesthetesize the animal</td></tr><tr><td colspan="4">&lt;strong&gt;Materials and Surgical Instruments&lt;/strong&gt;</td></tr><tr><td>Sylgard</td><td>Dow Corning Corp</td><td>184 SIL ELAST KIT</td><td>To pin, dissect, &amp;amp; cut tissue</td></tr><tr><td>Petri Dish</td><td>Corning</td><td>3160-152</td><td>To dissect/cut tissue</td></tr><tr><td>Insect Pins</td><td>ENTOMORAVIA Austerlitz Insect Pins</td><td>Size 5</td><td>To pin tissue</td></tr><tr><td>Bench Pad</td><td>VWR International</td><td>56617-014</td><td>Absorbent bench underpads</td></tr><tr><td>Rat surgical Kit</td><td>Kent Scientific Corporation&amp;nbsp;</td><td>INSRATKIT</td><td>To remove and dissect tissue</td></tr><tr><td>2 Dumont #3 Forceps</td><td>Kent Scientific Corporation&amp;nbsp;</td><td>INS500064</td><td>To remove and dissect tissue</td></tr><tr><td>Tissue Forceps</td><td>Kent Scientific Corporation&amp;nbsp;</td><td>INS500092</td><td>To remove and dissect tissue</td></tr><tr><td>Scalpel</td><td>Kent Scientific Corporation&amp;nbsp;</td><td>INS500236</td><td>To remove and dissect tissue</td></tr><tr><td>Scalpel blade</td><td>Kent Scientific Corporation&amp;nbsp;</td><td>INS500239</td><td>To remove and dissect tissue</td></tr><tr><td>Professional Clipper&amp;nbsp;</td><td>Braintree Scientific, Inc.</td><td>CLP-223&amp;nbsp;45</td><td>To remove fur</td></tr><tr><td>Suture Thread</td><td>Fine Science Tools</td><td>18020-50</td><td>Tie tissue</td></tr><tr><td>Tissue Clips</td><td>Radnoti, LLC</td><td>158802</td><td>Attach tissue to rod/transducer</td></tr><tr><td>1 g weight</td><td>&amp;nbsp;Mettler Toledo</td><td>11119525</td><td>For transducer calibration</td></tr><tr><td colspan="4">&lt;strong&gt;Chemicals&lt;/strong&gt;</td></tr><tr><td>Krebs Solution:&lt;br /&gt;Sodium chloride&lt;br/&gt; Potassium chloride&lt;br/&gt; Monobasic potassium phosphate&lt;br/&gt; Magnesium sulfate&lt;br/&gt; Dextrose&lt;br/&gt; Sodium bicarbonate&lt;br/&gt; Calcium chloride&lt;br/&gt; Magnesium chloride</td><td>&amp;nbsp;&lt;br /&gt;Sigma&lt;br/&gt; Fisher&lt;br/&gt; Fisher&lt;br/&gt; Fisher&lt;br/&gt; Fisher&lt;br/&gt; Sigma&lt;br/&gt; EMD&lt;br/&gt; Baker</td><td>&amp;nbsp; &lt;br /&gt;S7653&lt;br/&gt; P217-500&lt;br/&gt; P285-3&lt;br/&gt; M65-500&lt;br/&gt; D16-500&lt;br/&gt; S5761&lt;br/&gt; CX0130-2&lt;br/&gt; 2444</td><td>To prepare Krebs solution</td></tr><tr><td>Isoflurane</td><td>Henry Schein</td><td>029405</td><td>To anesthetesize the animal</td></tr><tr><td>&amp;nbsp;Oxygen tank</td><td>Matheson Tri Gas</td><td>ox251</td><td>To use with anesthesia system</td></tr><tr><td>Carbogen Tank (95% Oxygen; 5% Carbon Dioxide)&amp;nbsp;</td><td>Matheson Tri Gas</td><td>Moxn00hn36D</td><td>To aerate Krebs solutions</td></tr></tbody></table>

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.

Spontaneous Myogenic Activity 
Spontaneous myogenic activity is an important smooth muscle characteristic that undergoes changes with postnatal development6-9 and pathology (e.g., SCI, BOO)3-5. Because this activity is believed to contribute to the symptoms of overactive bladder (OAB)2, an evaluation of receptors, intracellular pathways and pharmacological agents that modulate it, is of high interest for developing effective treatments...

Watch this Scientific Journal Video about Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology at JoVE.com

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

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 ...

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Research

JoVE Journal

Medicine

26.2K Views.  University of Pittsburgh School of Medicine.  Dit handschrift bevat een eenvoudige, maar krachtige, in vitro methode voor het evalueren van de gladde spieren contractiliteit in reactie op farmacologische middelen of zenuwstimulatie. Belangrijkste toepassingen zijn het testen van geneesmiddelen en begrip weefsel fysiologie, farmacologie en pathologie. 

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

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. ...

Manual Muscle Testing:  A Method of Measuring Extremity Muscle Strength Applied to Critically Ill Patients 

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, and persistent nerve and muscle impairments after hospital discharge.1-6 Using an explicit protocol with a structured approach to training and quality assurance of research staff, manual muscle testing (MMT) is a highly reliable method for assessing strength, using a standardized clinical examination, for patients following ARDS, and can be completed with mechanically ventilated patients who can tolerate sitting upright in bed and are able to follow two-step commands. 7, 8 
This video demonstrates a protocol for MMT, which has been taught to &ge;43 research staff who have performed &gt;800 assessments on &gt;280 ARDS survivors. Modifications for the bedridden patient are included. Each muscle is tested with specific techniques for positioning, stabilization, resistance, and palpation for each score of the 6-point ordinal Medical Research Council scale.7,9-11 Three upper and three lower extremity muscles are graded in this protocol: shoulder abduction, elbow flexion, wrist extension, hip flexion, knee extension, and ankle dorsiflexion. These muscles were chosen based on the standard approach for evaluating patients for ICU-acquired weakness used in prior publications. 1,2.

Manual Muscle Testing:  A Method of Measuring Extremity Muscle Strength Applied to Critically Ill Patients

Survivors of acute respiratory distress syndrome (ARDS) and critical illness frequently develop long-lasting muscle weakness. Manual muscle testing (MMT) is a standardized clinical examination commonly used to measure strength of peripheral skeletal muscle groups. This video demonstrates MMT using the 6-point Medical Research Council scale.

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, ...

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

Bioengineering

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 ...

Contractility Measurements of Human Uterine Smooth Muscle to Aid Drug Development

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We describe methods to measure ex vivo myometrium contractility. This assay can be used to investigate factors and molecules involved in the modulation of myometrial contraction and to determine their excitatory or inhibitory actions, and hence their therapeutic potential in vivo. Biopsies are obtained from women undergoing cesarean section delivery with informed consent. Fine strips of myometrium are dissected, clipped and attached to a force transducer within 1 mL organ baths superfused with physiological saline solution at 37 &#176;C. Strips develop spontaneous contractions within 2&#8211;3 h under set tension and remain stable for many hours (&#62;6 h). Strips can also be stimulated to contract such as by the endogenous hormones, oxytocin and vasopressin, which cause concentration-dependent modulation of contraction frequency, force and duration, to more closely resemble contractions in labor. Hence, the effect of known and novel drug leads can be tested on spontaneous and agonist-induced contractions.
This protocol specifically details how this assay can be used to determine the potency of known and novel agents by measuring their effects on various parameters of human myometrial contraction. We use the oxytocin- and V1a receptor antagonists, atosiban and SR49059 as examples of known compounds which inhibit oxytocin- and vasopressin-induced contractions, and demonstrate how this method can be used to complement and validate pharmacological data obtained from cell-based assays to aid drug development. The effects of novel agonists in comparison to oxytocin and vasopressin can also be characterized. Whilst we use the example of the oxytocin/ vasopressin system, this method can also be used to study other receptors and ion channels that play a role in uterine contraction and relaxation to advance the understanding of human uterine physiology and pathophysiology.

This article describes experimental protocols to study ex vivo contractions of human myometrium and their application in drug discovery. This technique is used to improve the understanding of myometrial physiology and pathophysiology as well as to validate pharmacological data from novel research probes or drug leads.

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We ...

Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting in a significant economic and quality of life burden. Transgenic and other mouse models have been developed to recreate various aspects of this multifactorial disease; however, methods to accurately quantitate urinary dysfunction and the effectiveness of new therapeutic options are lacking. Here, we describe a method that can be used to measure bladder volume and detrusor wall thickness, urinary velocity, void volume and void duration, and urethral diameter. This would allow for the evaluation of disease progression and treatment efficacy over time. Mice were anesthetized with isoflurane, and the bladder was visualized by ultrasound. For non-contrast imaging, a 3D image was taken of the bladder to calculate volume and evaluate shape; the bladder wall thickness was measured from this image. For contrast-enhanced imaging, a catheter was placed through the dome of the bladder using a 27-gauge needle connected to a syringe by PE50 tubing. A bolus of 0.5 mL of contrast was infused into the bladder until a urination event occurred. Urethral diameter was determined at the point of the Doppler velocity sample window during the first voiding event. Velocity was measured for each subsequent event yielding a flow rate. In conclusion, high frequency ultrasound proved to be an effective method for assessing bladder and urethral measurements during urinary function in mice. This technique may be useful in the assessment of novel therapies for BPH/LUTS in an experimental setting.

We describe the use of high frequency ultrasound with contrast imaging as a method to measure bladder volume, bladder wall thickness, urine velocity, void volume, void duration, and urethral diameter. This strategy can be used to assess voiding dysfunction and treatment efficacy in various mouse models of lower urinary tract dysfunction (LUTD).

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting ...

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.

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.

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

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Biology

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. Therefore, accurate measurement of urothelial ATP release in a physiological setting is an important first step in studying the mechanisms that control purinergic signaling in the urinary bladder. Existing techniques to study mechanically evoked urothelial ATP release utilize cultured cells plated on flexible supports or bladder tissue pinned into Ussing chambers; however, each of these techniques does not fully emulate conditions in the intact bladder. Therefore, an experimental setup was developed to directly measure ATP concentrations in the lumen of the rodent urinary bladder.
In this setup, the bladders of anesthetized rodents are perfused through catheters in both the dome of the bladder and via the external urethral orifice. Pressure in the bladder is increased by capping the urethral catheter while perfusing sterile fluid into the bladder through the dome. Measurement of intravesical pressure is achieved using a pressure transducer attached to the bladder dome catheter, akin to the setup used for cystometry. Once the desired pressure is reached, the urethral catheter's cap is removed, and fluid collected for ATP quantification by luciferin-luciferase assay. Through this experimental setup, the mechanisms controlling both mechanical and chemical stimulation of urothelial ATP release can be interrogated by including various agonists or antagonists into the perfusate or by comparing results between wildtype and genetically modified animals.

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

This protocol describes the procedure for measuring ATP concentrations in the lumen of the bladder in an anesthetized rodent.

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. ...

Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents

Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, fresh, and isolated DSM cells presents an important technical challenge whose achievement provides optimal cells for subsequent functional and molecular studies. The method developed and elaborated herein, successfully used by our group for over a decade, describes dissection of human urinary bladder specimens obtained from open bladder surgeries followed by an enzymatic two-step treatment of DSM pieces and mechanical trituration to obtain freshly isolated DSM cells. The initial step involves dissection to separate the DSM layer (also known as muscularis propria) from mucosa (urothelium, lamina propria, and muscularis mucosa) and the adjacent connective, vascular, and adipose tissues present. The DSM is then cut into pieces (2-3 mm x 4-6 mm) in nominal Ca2+-containing dissection/digestion solution (DS). DSM pieces are next transferred to and sequentially treated separately with DS containing papain and collagenase at ~37 &#176;C for 30-45 min per step. Following washes with DS containing enzyme-free bovine serum and trituration with a fire-polished pipette, the pieces release single DSM cells. Freshly isolated DSM cells are ideally suited for patch-clamp electrophysiological and pharmacological characterizations of ion channels. Specifically, we show that the TRPM4 channel blocker 9-phenanthrol reduces voltage-step evoked cation currents recorded with the amphotericin-B perforated patch-clamp approach. DSM cells can also be studied by other techniques such as single cell RT-PCR, microarray analysis, immunocytochemistry, in situ proximity ligation assay, and Ca2+ imaging. The main advantage of utilizing single DSM cells is that the observations made relate directly to single cell characteristics revealed. Studies of freshly isolated human DSM cells have provided important insights characterizing the properties of various ion channels including cation-permeable in the urinary bladder and will continue as a gold standard in elucidating DSM cellular properties and regulatory mechanisms.

We describe a method for preparation of the single freshly isolated detrusor smooth muscle cells from human urinary bladder specimens employing a two-step enzymatic procedure. The obtained viable DSM cells can be studied by various single cell techniques including the described amphotericin-B patch-clamp electrophysiology to reveal physiological and pharmacological properties.

Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, ...

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

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