Name: isometric contractility measurement of the mouse mesenteric artery using wire myography
Uploaded: 2018-08-20T12:30:00
Description: Watch this Scientific Journal Video about Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography at JoVE.com

We thank Dr. Wei Qi He (Soochow University, Suzhou, China) and Dr. Yan Ning Qiao (Shaanxi Normal University, Xi&#39;an, China) for the technical assistance. This work was supported by the National Natural Science Foundation of China (Grant 31272311, 81373295 and 81473420) and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (Grant No. ysxk-2016).

Animal manipulations were approved by the Institutional Animal Care and Use Committee (IACUC) of the Model Animal Research Center of Nanjing University.
1. Solution Preparation
<ol>
	<li>Prepare HEPES-Tyrode solution (H-T) using 137.0 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2&#8729;6H2O, 5.6 mM D-glucose, and 10 mM HEPES, pH 7.3-7.4.</li>
	<li>Prepare HEPES-Tyrode solution without calcium (Ca2+-free H-T) using 140.6 mM NaCl, 2.7 mM KCl, 1 mM Mg...

<ol>
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Hypertension is a widespread public health challenge due to its severe complications, including cardiovascular and kidney diseases16. Understanding the pathogenesis of hypertension and exploring more anti-hypertensive drugs has become an urgent task in this field. Blood pressure is generated and maintained by peripheral resistance of the circulation. According to Poiseuille&#39;s Law, the relatively small arteries generate a large proportion of circulatory resistance and serve as the dominant prod...

The small vessel myograph system used here was for measuring the isometric contraction of small resistance vessels with internal diameters ranging from 100 to 400 &#181;m. Isolated small vessels (about 2 mm long) were inserted by two 40-&#181;m diameter wires and were then mounted on the micrometer-side and transducer-side jaws sequentially. This myograph technique was first suggested in 19721 and then developed primarily by Mulvany and his colleagues2,3,4,5,6. It is now a mature technique with stable equipment, easy performance and a standard normalization procedure7,8,9. We utilized this method with some modifications for measurements in the mouse mesenteric artery.
Vascular smooth muscle lines the walls of almost all blood vessels. Their fundamental function is to generate forces through contraction in response to various stimuli. The normal contractility of vascular smooth muscle is essential for blood pressure regulation and nutrition supplement10. Abnormal regulation of blood pressure results in a variety of diseases, including hypertension, heart failure and ischaemia. Several studies have suggested that abnormal blood pressure is always associated with dysfunctional vascular smooth muscle contractility7,11,12,13. The myograph method allows investigation of isometric contractility of mouse vessels induced by various stimuli including vasoconstrictors, inhibitors and drugs. Successful measurements of contraction will help us understand the mechanisms of blood pressure maintenance and the pathogenesis of vascular smooth muscle-associated diseases and to explore novel therapeutic approaches.
Many Chinese herbs have been widely used for clinical treatment of vascular diseases; however, their effective ingredients usually remain unknown. Thus, isolation and identification of the effective components is very important for the development of novel drugs. Multi-wire myograph technology offers a simple approach for screening active components in herbs. We have reported several studies using the small vessel myograph system to investigate mouse mesenteric artery contraction and identified natural compounds with anti-hypertension activity12,13,14. Here, we describe the detailed protocol for the myograph method and assess the relaxant effect of neoliensinine isolated from embryos of lotus seed (Nelumbo nucifera Gaertn.)14.

<table>
	<tbody>
		<tr>
			<td>Multi wire myograph system</td>
			<td>DMT</td>
			<td>610-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel wire</td>
			<td>DMT</td>
			<td>400447</td>
			<td></td>
		</tr>
		<tr>
			<td>Geuder dissection scissor</td>
			<td>DMT</td>
			<td>400431</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont forceps</td>
			<td>DMT</td>
			<td>300413</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLab/8SP</td>
			<td>ADInstruments</td>
			<td>ML785</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td>ADInstruments</td>
			<td>LabChart 5</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>SigmaAldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>SigmaAldrich</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>SigmaAldrich</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>SigmaAldrich</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>SigmaAldrich</td>
			<td>G6152</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sangon Biotech</td>
			<td>A100511-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>SigmaAldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>SigmaAldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
	</tbody>
</table>

isometric contractility measurement of the mouse mesenteric artery using wire myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

We measured the isometric contractility of mouse mesenteric artery using a multi-wire myograph system and assessed the relaxant effect of neoliensinine purified from embryos of lotus seed (Nelumbo nucifera Gaertn.)14. The mouse mesenteric resistance artery was isolated, cleaned of connective tissues and cut into 1.4-mm segments. The artery segment was inserted by two steel wires in Ca2+-free H-T solution in a Petri dish, and then the segment was...

Watch this Scientific Journal Video about Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography at JoVE.com

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

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.

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