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

The overall goal of this procedure is to evaluate the physiological and pharmacological properties of bladder smooth muscle tissue. This is accomplished by dissecting the rat bladder, opening it into a flat sheet and cutting strips containing the urothelium smooth muscle and nerves. In the second step, the tissue is attached to a force transducer connected to amplifiers and stimulators for measuring the muscle contractility.

In the next step, baseline tone and tissue viability are established, and then the strips are exposed to pharmacological and or electrical stimulation. Ultimately, the effects of the stimuli on the contractility of the bladder smooth muscle tissue can be measured. This method is used to characterize the effects of pathological and pharmacological compounds on the whole bladder, as well as specific bladder tissue components such as the smooth muscle nerves in urothelium, in humans and in animals.

This technique has been extensively used during the development of therapies for various bladder dysfunctions because it represents a fast, easy, and reproducible screening method for pharmacological agents in a well-defined and controlled environment. After confirming anesthesia by loss of rear limb, withdraw reflex, shave the animal's abdomen, and then expose the pelvic organs via a midline abdominal incision. Next, identify the bladder and urethra, and then cut at the bladder neck close to the proximal urethra.

To remove the bladder, place the tissue immediately in a sill guard coated dish filled with aerated Creb solution. Then after sacrificing the animal, insert tissue dissecting pins through the bladder dome neck and ureters to stabilize the tissue for further dissection without stretching the tissue. Trim the bladder of extraneous tissue and then open the tissue from the base to the dome to create a flat sheet cirrhosis side down and luminal side up.

Place dissecting pins on each corner of the tissue and then remove the bladder, neck and dome tissue. Next, cut the tissue lengthwise from the base to the dome into about two by eight millimeter strips, attaching a tissue clip to both ends of each strip. Transfer the strips to the experimental chambers attaching one end of each strip to a force transducer and the other to a fixed rod.

Now, gently stretch the tissues until baseline tension reaches one gram. Initially, the tissue tends to relax, which is recorded as a decrease in baseline tension. Wash the strips approximately every 15 minutes with warm aerated crebs, and adjust the baseline tension to one gram.

After each wash, allow the tissues to equilibrate for about one to two hours until they no longer relax. Once the baseline tension is stable, add potassium chloride directly to the bath for five minutes or until a plateau response is reached to test the tissue viability. Then wash the tissues three to five times with warm aerated crebs to allow the tissue to return to their pre-treatment conditions.

For pharmacological smooth muscle stimulation, add drugs from concentrated solutions directly to the bath. For example, for carbahol stimulation, add 10 microliters of each of the concentrations of the stimulus to each 10 milliliter tissue bath. As soon as the response from the previous concentration reaches a plateau.

In parallel strips, add equal amounts of the vehicle for neural stimulation of the smooth muscle or electrical field stimulation. Set the train duration to three to 10 seconds and the intertrain interval to at least one minute apart. After establishing the frequency of the train stimuli, run a frequency response curve ranging from 0.5 to 50 hertz to establish the intensity of the stimulus.

Systematically increase the voltage until the amplitude of the contractions reaches a plateau, keeping the frequency constant. Once the stimulation parameters have been established, allow about 20 to 30 minutes for the electrical field stimulation, evoked contractions to stabilize prior to drug testing. Then to test the effects of alpha beta methylene a TP and atropine on the electrical field stimulation, for example, perform two control frequency response curves, and then add 10 microliters of alpha beta methylene a TP to the test strip, and 10 microliters of vehicle to the control strips.

Alpha beta methylene A TP directly stimulates the puric receptors in the smooth muscle and desensitizes these receptors causing an initial baseline contraction. After the response returns to baseline, repeat the frequency response curves in all strips. Add 10 microliters of atropine to the test strip, and 10 microliters of vehicle to the control strips, and repeat the frequency response curves at the end of the electric field stimulation.

Verify the selectivity of the electric field stimulation by blocking the neural transmission with the sodium channel blocker tetra to toxin. Finally, unclip the strips. Block them gently on a piece of tissue paper to eliminate any extra fluid and measure the weight of each strip on a balance to determine the effects on smooth muscle tone.

Use the appropriate data analysis software to select a window of at least 10 to 30 seconds before and at the peak of the drug induced response, and measure the amplitude of the contraction to determine the effects of the experimental treatments on neurally evoked contractions. Measure the amplitude, duration, and area under the curve of at least three contractions before and at the peak of the drug induced response. Finally, normalize the data to compare the results across the strips and pharmacological treatments.

Spontaneous myogenic activity is a smooth muscle characteristic that changes during postnatal development and pathology. In this representative experiment strips from neonatal rats exhibit large amplitude, low frequency rhythmic contractions while strips from adult rats exhibit small amplitude high frequency activity after spinal cord injury. The neonatal pattern reemerges otin, A-K-C-N-Q channel opener decreases the amplitude of spontaneous activity in strips from adult rat smooth muscle tone and contractility properties, which can be influenced by the urothelium and mucosa are important factors for proper bladder function during urine storage and voiding.

For example, in this graph, the concentration dependent increases in smooth muscle tone. By caric call activation of muscarinic receptors in pig bladder strips is illustrated. A reduction in muscle contractility in response to carbahol treatment is observed in the presence of the mucosa.

As shown in this graph, five HT four receptors are expressed pre ally in parasympathetic nerves, and their activation increases acetylcholine levels. These graphs illustrate the different efficacy and potency of the excitatory effect of the five HT four receptor agonist cisapride on human bladder versus ileum strips. Species differences that exist in a number of receptors can be assessed using this bladder strip method.

For example, these data show that bombesin receptor antagonists have excitatory effects on the rat bladder while having no effects on mouse or pig bladder strips. For pharmacological stimulation, it is important to pay attention to the concentration, timing, and duration of the drug application. In order to avoid unspecific effects on other receptors or desensitization of the targeted receptors for neural Stimulation is important to choose the correct parameters for electrical field stimulation according to the aim of the experiment, and to ensure that the stimulus does not directly activate the smooth muscle.