ワイヤー図法を使用してマウスの腸間膜動脈の等尺性収縮力測定

Our lab has been using the wire myograph method to measure vascular smooth muscle contractility for many years. We use this technique to study the mechanism of smooth muscle contraction. We use this method to explore novel drugs for hypertension therapy.

The main advantage of this technique is that the vessel segment survives at least four hours after mounting and keeps the contractility induced by high potassium for many times. The multi-channel system gives the opportunity for high therapy screening of new drugs. Pin the body of the euthanized mouse with its abdomen facing up and moisten the abdomen with 70%ethanol.

Then, after using scissors to cut the skin along the ventral midline from the groin, make incisions down each leg from the start of the first incision. Pull the skin back on both sides, then make similar incisions to open the peritoneum. Next, use scissors to cut the esophagus, colon and other connective tissues to completely isolate the gastrointestinal tract with feeding vasculature from the body.

After rinsing the blood from the GI tract with HEPES-Tyrode, or H-T solution, transfer the isolated segment into a coated Petri dish for mesenteric artery dissection at room temperature. Smooth out the stomach, jejunum, ileum and cecum in a clockwise direction and pin the stomach and cecum on the left-and right-hand side respectively. Turn on the transmission light source of a stereoscopic microscope and fix the intestine with pins to expose the dissected mesenteric arteries.

Then clamp the adipose tissues around the arteries with forceps and carefully isolate the arteries by cutting off all the connective tissues with dissection scissors. Grasp an excess artery with forceps and transfer and immerse the mesenteric artery tree into cold calcium ion-free H-T solution. Cut off a 1.4 millimeter portion of the artery proximal to the intestinal wall of a mesenteric arcade and use two forceps to open both sides of this artery segment carefully.

Prepare two segments of stainless steel wire of 2.5 centimeters in length and place them into the same dish. Use forceps to gently clamp one end of the artery. Then use another forceps to carefully insert two wires into the lumen of the artery one by one.

Insure that the wires are kept straight and do not touch the endothelium. Using two forceps, clamp both wires outside of the threaded vessel simultaneously and carefully transfer the vessel from the Petri dish to a myograph chamber filled with H-T solution. Screw the jaws of the chamber apart to make space for mounting, then grasp both sides of one of the two inserted wires using two forceps and place the vessel in the jaw gap.

Wrap both sides of the clamped wire around the screws of the jaw connected to the micrometer. Fix the left-side screw by twisting clockwise. Then use forceps to straighten the wire.

Next, fix the right-side screw by twisting clockwise. Make sure that the vessel is always inside the jaw gap but do not touch the jaw to avoid damage. Close the two jaws using the micrometer.

Make sure the two jaws are close enough but that they do not touch each other, and that the unfixed wire is on top of the fixed wire. Using the right-hand forceps, carefully fold the unscrewed wire at the corner of the jaw connected to force transducer and wrap it clockwise around the right-side screw. Then, fix the screw.

Repeat the unfolding and wrapping of the left side of the wire and fix the left-side screw. Next, move the jaws slightly apart without stretching the vessel by carefully rotating the micrometer so that the gap between the two jaws can just accommodate the two wires. Then use forceps to move the wire at the micrometer side to the horizontal plane of the wire at the transducer side.

After mounting arteries in the other chambers, connect all the chambers to the equipment and cover the chambers. Attach the 100%oxygen supply and a temperature probe and start heating to 37 degrees Celsius. Open the charting software.

Press the Start'button to start recording. Select the channel of interest from the DMT menu to open the DMT normalization window for the corresponding channel. Enter the following constant values into the window:Tissue end points a1, 0.1;Tissue end points a2, 4;Wire diameter, 40.

The window then displays the calculated vessel length as 1.40 millimeters. Read the micrometer of the appropriate tissue chamber. Enter the value into the micrometer reading box and click Add Point'button.

This value is the initial value of X.After a 60 second delay, the window displays the force and the effective pressure corresponding to this micrometer value. Simultaneously, the micrometer reading box becomes active. Stretch the vessel being normalized by turning the micrometer in a counter-clockwise direction.

Enter the micrometer value into the micrometer reading box and click the Add Point'button. Wait for a delay time of 60 seconds again. Continue to stretch the vessel and add micrometer values until the window displays the value of micrometer X-one, which is the calculated micrometer setting required to stretch the vessel to its IC-one.

Set the micrometer to the X-one value. After normalization, equilibrate the vessel in the chamber for 15 to 20 minutes. Once equilibrated, challenge the vessel with high potassium solution by replacing the H-T solution with 5 milliliters of high potassium solution to induce contraction for 10 minutes.

After repeating the 10 minute incubation in fresh high-potassium solution, wash with five milliliters of H-T solution three to four times. Again, challenge the vessel with five milliliters of high potassium solution to induce contraction. After five minutes, add 0.5 microliters of the Neoliensinine stock solution into the chamber to relax the vessel at a final concentration of one micromolar Neoliensinine.

When the force is stable after several minutes, add another 0.5 microliters of Neoliensinine stock solution to increase the concentration to two micromolar. Add one microliter of the stock solution each time to increase the concentration to four, six, eight and 10 micromolar to generate the dose response curve. During the normalization procedure, the vessel was stretched several times until reaching the value of IC-100 and each stretched cycle included a robust contraction, rapid relaxation and a force maintenance in 60 seconds.

The contraction of the vascular smooth muscle induced by high potassium solution usually showed two phases, a robust phase and a sustained phase. We described a method to measure isometric contractility of mouse mesenteric artery using a multi-wire myograph system. This method can be used to assess the functions of vascular smooth muscle and to screen relaxants of smooth muscle.

The main difference between this method and others is in the mounting steps. This step used here is easy to perform and does not damage the tissue or the device. Following this procedure, other stimulators such as EP and Angiotensin II can also be used to stimulate smooth muscle contraction in order to find some novel drugs that are sensitive to this agonist.