Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

Summary

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.

Abstract

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.

Introduction

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 µm. Isolated small vessels (about 2 mm long) were inserted by two 40-µm 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 colleagues^2,3,4,5,6. It is now a mature tec....

Protocol

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

1. Prepare HEPES-Tyrode solution (H-T) using 137.0 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂∙6H₂O, 5.6 mM D-glucose, and 10 mM HEPES, pH 7.3-7.4.
2. Prepare HEPES-Tyrode solution without calcium (Ca²⁺-free H-T) using 140.6 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂∙6H₂O, 5.6 mM D-glucose, and 10 mM HEPES, pH 7.3-7.4.
3. Prepare HEPES-Tyrode solution using 124 mM KCl (High K<....

Results

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.......

Discussion

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's Law, the relatively small arteries generate a large proportion of circulatory resistance and serve as the dominant prod.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Multi wire myograph system	DMT	610-M
Stainless steel wire	DMT	400447
Geuder dissection scissor	DMT	400431
Dumont forceps	DMT	300413
PowerLab/8SP	ADInstruments	ML785
Software	ADInstruments	LabChart 5
NaCl	SigmaAldrich	S5886
KCl	SigmaAldrich	P5405
CaCl2	SigmaAldrich	C4901
MgCl2·6H2O	SigmaAldrich	M2393
D-Glucose	SigmaAldrich	G6152
HEPES	Sangon Biotech	A100511-0250
NaOH	SigmaAldrich	S8045
DMSO	SigmaAldrich	D2650