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 K...

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.

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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 ...

bladder-smooth-muscle-strip-contractility-as-method-to-evaluate-lower

Research

JoVE Journal

Medicine

26.0K visualizações.  University of Pittsburgh School of Medicine.  Este manuscrito apresenta um simples, mas poderoso, método in vitro para a avaliação da contratilidade do músculo liso em resposta a agentes farmacológicos ou estimulação do nervo. As principais aplicações são triagem de drogas e fisiologia do tecido entendimento, farmacologia e patologia. 