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>

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 border="0" cellpadding="0" cellspacing="0" width="658">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Name of Material/ Equipment</td>
			<td style="width:123px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:180px;">Comments/Description</td>
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			<td height="17" style="height:17px;width:237px;">Equipment</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:180px;"></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Tissue Bath System with Reservoir</td>
			<td>Radnoti, LLC</td>
			<td style="width:119px;">159920</td>
			<td style="width:180px;">isolated tissue baths</td>
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		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Warm water recirculator pump</td>
			<td style="width:123px;">Kent Scientific Corporation&#160;</td>
			<td style="width:119px;">TPZ-749</td>
			<td style="width:180px;">to keep tissue baths to 37 C</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Computer</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:180px;"></td>
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		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Data Acquisiton System</td>
			<td style="width:123px;">DataQ Instruments</td>
			<td style="width:119px;">DI-710-UH</td>
			<td style="width:180px;">To view, record and analyze data</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Transbridge Transducer Amplifier</td>
			<td style="width:123px;">World Precision Instruments</td>
			<td style="width:119px;">SYS-TBM4M</td>
			<td style="width:180px;">Transducer amplifier</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Grass stimulator</td>
			<td style="width:123px;">Grass Technologies</td>
			<td>Model S88</td>
			<td style="width:180px;">Stimulator</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Anesthesia System</td>
			<td style="width:123px;">Kent Scientific Corporation&#160;</td>
			<td>ACV-1205S</td>
			<td style="width:180px;">To anesthetesize the animal</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:237px;"><a name="RANGE!A10">Anesthetizing Box</a></td>
			<td style="width:123px;">Harvard Apparatus</td>
			<td style="width:119px;">500116</td>
			<td style="width:180px;">To anesthetesize the animal</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Anesthesia Masks</td>
			<td style="width:123px;">Kent Scientific Corporation&#160;</td>
			<td style="width:119px;">AC-09508</td>
			<td style="width:180px;">To anesthetesize the animal</td>
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		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Materials and surgical instruments</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:180px;"></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:237px;">sylgard</td>
			<td>Dow Corning Corp</td>
			<td>184 SIL ELAST KIT</td>
			<td style="width:180px;">To pin, dissect &#38; cut tissue</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Petri Dish</td>
			<td>Corning</td>
			<td>3160-152</td>
			<td style="width:180px;">To dissect/cut tissue</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:237px;">Insect Pins</td>
			<td style="width:123px;">ENTOMORAVIA Austerlitz Insect Pins</td>
			<td style="width:119px;">Size 5</td>
			<td style="width:180px;">To pin tissue</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Bench Pad</td>
			<td style="width:123px;">VWR International</td>
			<td style="width:119px;">56617-014</td>
			<td style="width:180px;">Absorbent bench underpads</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Rat surgical Kit</td>
			<td style="width:123px;">Kent Scientific Corporation&#160;</td>
			<td style="width:119px;">INSRATKIT</td>
			<td style="width:180px;">To remove and dissect tissue</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">2 Dumont #3 Forceps</td>
			<td style="width:123px;">Kent Scientific Corporation&#160;</td>
			<td style="width:119px;">INS500064</td>
			<td style="width:180px;">To remove and dissect tissue</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Tissue Forceps</td>
			<td style="width:123px;">Kent Scientific Corporation&#160;</td>
			<td style="width:119px;">INS500092</td>
			<td style="width:180px;">To remove and dissect tissue</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Scalpel</td>
			<td style="width:123px;">Kent Scientific Corporation&#160;</td>
			<td style="width:119px;">INS500236</td>
			<td style="width:180px;">To remove and dissect tissue</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Scalpel blade</td>
			<td style="width:123px;">Kent Scientific Corporation&#160;</td>
			<td style="width:119px;">INS500239</td>
			<td style="width:180px;">To remove and dissect tissue</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Professional Clipper&#160;</td>
			<td style="width:123px;">Braintree Scientific, Inc.</td>
			<td style="width:119px;">CLP-223&#160;45</td>
			<td style="width:180px;">To remove fur</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Suture Thread</td>
			<td style="width:123px;">Fine Science Tools</td>
			<td style="width:119px;">18020-50</td>
			<td style="width:180px;">Tie tissue</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Tissue Clips</td>
			<td>Radnoti, LLC</td>
			<td style="width:119px;">158802</td>
			<td style="width:180px;">Attach tissue to rod/transducer</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">1g weight</td>
			<td style="width:123px;">&#160;Mettler Toledo</td>
			<td>11119525</td>
			<td style="width:180px;">For transducer calibration</td>
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		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Chemicals</td>
			<td style="width:123px;"></td>
			<td></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="170">
			<td height="170" style="height:170px;width:237px;">Krebs Solution:&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Sodium Chloride 
			Potassium Chloride 
			Monobasic Potassium Phosphate 
			Magnesium Sulfate 
			Dextrose 
			Sodium Bicarbonate 
			Calcium Chloride 
			Magnesium Chloride</td>
			<td style="width:123px;">Sigma&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
			Fisher 
			Fisher 
			Fisher 
			Fisher 
			Sigma 
			EMD 
			Baker</td>
			<td style="width:119px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; S7653 
			P217-500 
			P285-3 
			M65-500 
			D16-500 
			S5761 
			CX0130-2 
			2444</td>
			<td style="width:180px;">To prepare Krebs solution</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Isoflurane</td>
			<td style="width:123px;">Henry Schein</td>
			<td style="width:119px;">029405</td>
			<td style="width:180px;">To anesthetesize the animal</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">&#160;Oxygen tank</td>
			<td style="width:123px;">Matheson Tri Gas</td>
			<td style="width:119px;">ox251</td>
			<td style="width:180px;">To use with anesthesia system</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Carbogen Tank (95% Oxygen; 5% Carbon Dioxide)&#160;</td>
			<td style="width:123px;">Matheson Tri Gas</td>
			<td style="width:119px;">Moxn00hn36D</td>
			<td style="width:180px;">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...

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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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26.0K Views.  University of Pittsburgh School of Medicine. 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, and pathology.