Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

The overall goal of this procedure is to measure smooth muscle tone. This is accomplished by first dissecting and preparing tissues. The second step is to mount the tissue in an isolated tissue bath.

Then the viability of the tissue is tested and isometric contraction measurements are taken. Ultimately, the isolated tissue bath preparation is used to test receptor function, receptor signaling, and smooth muscle reactivity. In general, The main advantages of this technique are flexibility of the system, the rapidity of achieving results, and keeping the whole tissue intact.

This method can help answer key questions In any field which uses drugs to interfere with receptors signaling or smooth muscle soul, There are a few preparations that must be stressed. It's vital to preheat the tissue bath system to 37 degrees Celsius by turning on the recirculating heated water bath. The water must flow into each component at the lowest barbed connection and flow out at the highest barbed connection.

Each component must be connected. The force transducers should be calibrated and must also be powered up at least 15 minutes prior to the experiment to bring their temperature to an equilibrium. Next, when connecting the gas check for leaks, then pressurize the system.

After filling the system with solution, be sure to prime it and remove any air bubbles. Testing the tissue bath aerators is also warranted. Good aeration will distribute drugs effectively, but not destabilize the tissue immediately prior to the experiment.

Dissect the tissue. After anesthetizing the rat, create a pneumothorax using scissors. Cuss along the diaphragm at the bottom of the rib cage.

Then cut from the bottom of the rib cage to the sternum and bisect the tissue. Clear out the organs over the spinal column to locate the aorta, which is directly along the spinal column. Now sever all the connections to the aorta and cut the aorta perpendicular to the spine at the level of the diaphragm.

Next, gently hold the aorta and dissect the aorta from the spine. Start in the lower diaphragm area adjacent to the spine and work towards the heart. Just be careful not to pull or tug the aortic tissue because that can be damaging to it immediately.

Place the aorta in a dissection dish containing PSS. Now take a wire and make a small 90 degree angle. At one end, anchor the angled end of the wire into the foundation of the dissection dish.

Then carefully thread the guide wire into the lumen of the aorta and slide the aorta onto the wire. When the free end of the wire becomes visible, gently bend it and push it into the foundation, thus stabilizing the tissue. Now using small Venus scissors and forceps, remove the perivascular adipose tissue and other extraneous tissues until the thoracic aorta appears white and somewhat fibrous.

Once cleared of excess tissues, carefully remove the clean aorta from the wire. Finish this section by cutting the aorta into three to five millimeter wide rings. A reach aortic ring.

Have prepared two tissue hooks with sutures to secure it in the bath chamber. One hook should have a suture with a small knotted loop for attachment to a rod. The other hook needs a 10 to 14 centimeter long piece of suture.

To tie a knot, carefully attach a pair of these tissue hooks to each aortic ring. Be careful to avoid tangling the hooks. Next, transfer each tissue piece to a dish of room temperature PSS with a tissue holder secured to a ring stand.

Next, slip the knotted loop onto the peg of the rod and use the long piece of suture to hold the tissue up. Then put the rod into the chamber and make sure the rod is secure. Now, tie the suture from the remaining hook to the force transducer.

Leave enough slack in the suture, so tension between the force transducer and tissue can be adjusted by the micrometer. Repeat this process for each ring in each chamber. Now, set the passive tension.

Use the micrometer to increase the tension to two grams and wait for the tension on the tissue to stop increasing. Then increase the tension by two more grams, and again, wait for the change in the tension. To finish.

The tension should now be around 3.2 grams. Repeat this process on each ring in each chamber. After setting all the passive tensions, allow the tissues to equilibrate for an hour.

During this time, replace the PSS every 15 minutes and make other preparations for the experiment, such as preparation of the drugs at the end of the equilibration period. Wash the tissue one final time, save the data and rero all the inputs. Then following details in the text protocol, make the initial challenge.

Be sure to document these actions in the software. After the initial challenge, the tension change will plateau. Then wash out the challenge drug so the tone of the tissue hits a baseline.

Let the tissue rest at this baseline tone for about 10 minutes now to one bath at the vehicle in this case, water to the other. Add sufficient prazocin for a five nanomolar concentration in the bath. Let the tissue baths incubate for an hour with no washing.

Meanwhile, repair the phenol rine at a wide range of concentrations along a logarithmic curve. After an hour add phenyl rine agonist in increasing concentrations to the bath to generate a cumulative concentration response. After each addition, wait for the tissue response to plateau before adding the next concentration.

Keep making additions until they no longer change the tissue's tone or until the whole response curve has plateaued. Rather, thoracic aorta was incubated in the alpha one adrenergic receptor antagonist POZ sim for one hour prior to adding the alpha one adrenergic agonist phenyl rine to the tissue bath and generating cumulative contraction response curves the maximum and half maximum contractions of phenyl rine. On the vehicle alone control tissue are marked in green.

The half maximum identifies the effective concentration for the drug. The same measurements for the razin treated tissue are seen in black. After watching this video, you should have a good understanding of how to measure smooth muscle function in an isolated tissue bath.