Assessing Murine Resistance Artery Function Using Pressure Myography

Summary

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3^rd order mesenteric arteries in c57 and sGCα₁^-/- mice using pressure myography.

Abstract

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3^rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.

To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the α₁ subunit of soluble guanylate cyclase (sGCα₁^-/-) are hypertensive when on a 129S6 (S6) background (sGCα₁^-/-S6) but not when on a C57BL/6 (B6) background (sGCα₁^-/-B6). Using pressure myography, we demonstrate that sGCα₁-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGCα₁^-/-S6 than in sGCα₁^-/-B6 mice, likely contributing to the higher blood pressure in sGCα₁^-/-S6 than in sGCα₁^-/-B6 mice.

Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

Introduction

Pressure myograph systems are used to measure the physiological function and properties of small arteries, veins and other vessels. An intact small segment of an artery or vein is mounted onto two small glass cannulas and pressurized to a suitable luminal pressure, allowing the vessel to maintain most of its in vivo characteristics (Figures 1 and 2). The near physiological condition in a pressure myograph reflects the in vivo behavior of the vascular bed, allowing the investigation of intrinsic properties (e.g. myogenic tone) of isolated vessels. Some of the advantages of pressure myography over wire myograp....

Protocol

1. Preparation of Solution

1. 500X EDTA stock: weigh 500 mg EDTA-Na₂•2H₂O and dissolve in 50 ml deionized water. Store at room temperature.
2. KCl depolarizing solution.
   1. Prepare K10X stock solution: 3.69 g NaCl, 18.64 g KCl, 0.36 g MgSO₄ anhydrous, 0.41 g KH₂PO₄, 0.46 g CaCl₂•2H₂O. Dissolve in 250 ml deionized water. Store at room temperature.
   2. For 100 ml final KCl depolarizing solution 1X: Dissolve 0.21 NaHCO₃, 0.18 g glucose in 90 ml deionized water. Add 10 ml of K10X stock solution. Add 95 μl of 500X EDTA solution. Mix wel....

Results

NO is centrally involved in maintenance of blood pressure homeostasis both in humans ⁶ and in animal models ⁷. The ability of NO to control vascular smooth muscle relaxation is mediated by soluble guanylate cyclase (sGC), a heme-containing heterodimeric enzyme that generates cGMP ⁸. Recently, a blood pressure-modifying genetic variant was identified in a locus that contains the sGCα₁ and sGCβ₁ genes, illustrating the relevance of sGC in regulating blood pressure in h.......

Discussion

Mice are the experimental model of choice for many investigators, in part because of the possibility to introduce genetic modifications, thereby generating mouse models for human pathophysiology. The vasoactive status of small resistance but not of larger conduit vessels largely defines the regulation of blood flow throughout the vascular system ¹¹. The size of resistance arteries in small animals, such as mice, prevents the use of a wire myograph to study microvascular function. Pressure myographs not only ov.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NaCl	Fisher Scientific	BP358
CaCl2 (2H2O)	Fisher Scientific	C79-500
KCl	Sigma	P9333
MgSO4	Fisher Scientific	M65-500
KH2PO4	Sigma	P3786
NaHCO3	Fisher Scientific	BP328
NaOH	Fisher Scientific	S318
D-Glucose	Sigma	G8270
EDTA	Fisher Scientific	BP121
HEPES	Sigma	H3375
Phenylephrine	Acros Organics	AC20724
Acetylcholine	Sigma	A6625
Pressure Myograph System	DMT