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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Research

JoVE Journal

Medicine

26.1K vistas.  University of Pittsburgh School of Medicine.  Este manuscrito presenta una simple, pero potente, método in vitro para evaluar la contractilidad del músculo liso en respuesta a agentes farmacológicos o la estimulación nerviosa. Las principales aplicaciones son la detección de drogas y la comprensión fisiología del tejido, la farmacología y la patología. 

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.

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, ...

Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory1 and gastrointestinal tracts,2-6 CLE has also been explored in the urinary tract for bladder cancer diagnosis.7-10 Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature.7
The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use.11 Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent&mdash;most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile.12 Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy.10 Recent availability of a &lt; 1 mm imaging probe13 opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e. photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities14 that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides15 and antibodies for endoscopic imaging of disease processes with molecular specificity.

Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection.

Probe-based  confocal laser endomicroscopy (CLE) is an emerging optical imaging technology  that enables real-time in vivo microscopy of mucosal surfaces ...

A Modified Precipitation Method to Isolate Urinary Exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

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

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

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

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

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

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 ...

Microelectrode Impalement Method to Record Membrane Potential from a Cannulated Middle Cerebral Artery

Membrane potential (Vm) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ion channels and electrogenic pumps that regulate Vm in disease conditions could potentially alter Vm, vascular tone, and blood flow. Thus, a basic understanding of electrophysiology and the methods necessary to accurately record Vm in healthy and diseased states are essential. This method will allow modulating Vm using different pharmacological agents to restore Vm. Although there are several methods, each with its advantages and disadvantages, this article provides protocols to record Vm from cannulated resistance vessels such as the middle cerebral artery using the microelectrode impalement method. Middle cerebral arteries are allowed to gain myogenic tone in a myograph chamber, and the vessel wall is impaled using high resistance microelectrodes. The Vm signal is collected through an electrometer, digitized, and analyzed. This method provides an accurate reading of the Vm of a vessel wall without damaging the cells and without changing the membrane resistance.

The primary goal of this article is to provide details of how to record membrane potential (Vm) from the middle cerebral artery using the microelectrode impalement method. The cannulated middle cerebral artery is equilibrated to gain myogenic tone, and the vessel wall is impaled using high resistance microelectrodes.

Membrane potential (Vm) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

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 ...