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

Summary

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.

Abstract

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.

Introduction

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 (β₃AR 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 interstit....

Protocol

All procedures described here are approved by the IACUC committee at University of Pittsburgh.

1. Solutions

1. 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.
2. Aerate Krebs with 95% O₂, 5% CO₂ and place it in a 37 ºC 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.
3. Measure pH (~7.4) and osmolarity (~ 300 mOsm) of aerated Krebs.

Results

Spontaneous Myogenic Activity

Spontaneous myogenic activity is an important smooth muscle characteristic that undergoes changes with postnatal development^6-9 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.......

Discussion

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

Materials

List of materials used in this article

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