Assessing Murine Resistance Artery Function Using Pressure Myography

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.

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.

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 myography, where muscle contraction is assessed by direct mechanical coupling to a force transducer ¹, include (i) that micro resistance arteries, which define the overall resistance developed in vascular system, can be studied, whereas wire myograph is limited to larger conduit arteries, (ii) that the risk to damage the endothelium is reduced since no wires need to be passed through the vessel lumen, (iii) that the natural shape of the vessel is better maintained, and (iv) that vessel dimension can be studied at a wide range of pressures or shear stress ².

Studying micro vessels can be more informative than studying larger conduit arteries to help understand the pathophysiology and molecular mechanisms that contribute to altered vascular tone in cardiovascular diseases such as hypertension. For example, impairment of endothelial function associated with feeding mice a high fat diet for 8 weeks could be demonstrated in 2^nd-order mesenteric arteries ³ but not in aortic rings (Figure 3). Another advantage of pressure myography is that intrinsic myogenic constriction of the pressurized vessel is present, and that the role and function of the endothelium in this phenomenon can be studied. Here, we describe the use of a pressure myograph to study vascular reactivity of mouse mesenteric 3^rd order resistance arteries in a setting of impaired nitric oxide (NO)-cGMP signaling.

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 well. Store at 4 °C. Use within one week.
3. Prepare fresh HEPES-PSS solution on the day of experiment (For 1 liter).
   1. 6.96 g NaCl, 0.35 g KCl, 0.288 g MgSO₄•7H₂O, 0.1605 g KH₂PO₄, 2.38 g HEPES. Dissolve in 100 ml deionized water. Mark as flask A.
   2. 0.312 g CaCl₂•2H₂O. Dissolve in 100 ml deionized water. Mark as flask B.
   3. 1.25 g NaHCO₃, 0.991 g glucose. Dissolve in 98 ml deionized water. Mark as flask C.
4. Add 2 ml of 500X EDTA in flask C. Take 700 ml deionized water in a big flask. Pour contents of flask A, B and C in big flask with continuous vortexing. Adjust the pH to 7.4 with NaOH.

2. Drugs Used

1. Phenylephrine (10^-2 M): Dissolve 20.37 mg in 10 ml deionized water. Aliquot 1 ml and store at -80 °C. Serially dilute to obtain 10^-3, 10^-4, 10^-5, and 10^-6 mol/L on the day of experiment.
2. Acetylcholine (10^-2 M): Dissolve 18.17 mg in 10 ml deionized water. Aliquot 1 ml and store at -80 °C. Serially dilute to obtain 10^-3, 10^-4, 10^-5, and 10^-6 mol/L on the day of experiment.

3. Mice

Male WT and sGCα₁^-/- mice on the S6 and B6 background were studied throughout this study. The sGCα₁^-/- were generated as described previously ^4,5. Housing and procedures involving experimental animals (mice) were approved by the Subcommittee on Research Animal Care of Massachusetts General Hospital, Boston, MA.

4. Measurement of Vascular Reactivity in Mesenteric Artery

1. Euthanize the mice with pentobarbital (200 mg/kg, i.p.). Open the abdominal cavity and dissect out the mesenteric tissue. Isolate and dissect a mesenteric artery of 3^rd order free of connective and adipose tissue, and place the artery in ice-cold HEPES-PSS solution pre-equilibrated with 95% O₂ / 5% CO₂ for 15 min. Cut rings of 2-3 mm length with no branching from the mesenteric artery. Differentiating between arteries and veins at micro vessels level is problematic when there is no blood in the vessels. Therefore, we recommend identifying the vessel while it is still intact in the animal before dissecting it out.
2. Fill in the glass cannulas in the pressure myograph chamber (DMT Model 110P&11P version 1.31, Figure 2) with HEPES-PSS solution with the help of a 10 ml syringe through inlet and outlet valves. Filling of cannula should be done gently and carefully as excessive pressure can damage the fragile transducer connected to cannulas. After filling cannulas close both inlet and outlet valves tightly.
3. Mount one end of the vessel onto right cannula and carefully tie it with one fine strand of nylon suture. With the help of a syringe, flush and fill in the vessel with HEPES solution via inlet valve. Bring in the left cannula closer to the vessel and mount the other end of vessel onto it. Tie it with nylon suture. Fill the chamber with up to 10 ml with HEPES solution. Check for the leak by gently pushing HEPES solution via inlet valve with the help of a syringe.
4. Install the chamber on the myograph and under the video camera. Start the oxygen and heat (37 °C) in the chamber. Connect the inlet valve with tube P1 from the first HEPES solution reservoir, while the valve is closed. Let any bubble and solution pass through the tube until there are no bubbles in the tube. Open the valve.
5. Connect the left valve of the chamber with tube P2 coming from pressure regulator. Again check for any bubbles. There should not be any bubbles or leakage in the entire system.
6. Go to the pressure menu on the myo-interface panel or in the software and turn ON the pump while turning OFF the flow.
7. Open the program and click COLLECT. Vessel should be seen on the screen now (Figure 3). The vessel is visualized with a camera fitted on a microscope, allowing monitoring and analysis of the vessel lumen, vessel diameter, and wall thickness.
8. Gradually raise the interluminal pressure via the P1 valve by selecting P1 pressure in the myo-interface panel or in the program and enter the pressure value as follows; 5 mmHg → 10 → 20 → 40 → 60 mmHg. Vessel sometime tends to twist or convolute while pressure is increasing; adjust the tension or strain accordingly with the help of vertical or longitudinal micropositioner (Figure 2). Equilibrate the vessel at 60 mmHg and 37 °C at least for 45 min. Change the bath solution once with pre-warmed HEPES during equilibration.
9. Apply 10 ml of KCl depolarizing solution, prewarmed at 37 °C, to the bath to fully depolarize smooth muscle cells and achieve maximum constriction. After stable constriction, rinse the bath with HEPES solution 3 times every 10 min.
10. Check the viability of the vessel and integrity of the endothelium by stable and reproducible responses to the addition of phenylephrine (10^-5 M) and acetylcholine (10^-5 M). Rinse 3 times with HEPES solution every 10 min.
11. Add phenylephrine (10^-5 M) to preconstrict the vessel, and when a stable constriction has been obtained, perform a cumulative concentration- vasodilation response curve (CRC) by sequential addition of increasing doses of acetylcholine (10^-9, 3 x 10^-9, 10^-8, 3 x 10^-8, 10^-7, 3 x 10^-7, 10^-6, 3 x 10^-6, and 10^-5 M). After last dose, rinse 3 times with HEPES solution every 10 min before starting new CRC.
12. Determine the passive lumen diameter by applying Ca²⁺-free PSS containing 2 mM EGTA. From the recorded tracings measure lumen diameter for each dose response (Figure 4), which will be used to calculate all parameters.
13. Express all acetylcholine relaxation responses as percentage change in lumen diameter after phenylephrine preconstriction compared with the difference between calcium-free diameter and diameter after phenylephrine constriction, using the following equation:
14. Percentage dilation = 100% x [(D_x - D_i)/(D_Ca-free - D_i)], where D is the measured lumen diameter and x, i, and Ca-free denote arterial diameters at each dose of agonist (x), initial diameter following phenylephrine constriction (i), and in Ca²⁺-free buffer (Ca-free).

5. Statistical Analysis

All continuous measurements are expressed as mean ±SEM. The vascular reactivity in mesenteric arteries is analyzed by repeated-measures two-way ANOVA. In all cases, P < 0.05 is considered statistically significant.

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 pr...

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...

The authors have nothing to disclose.

Authors would like to acknowledge Drs. Paul Huang and Dmitriy Atochin for use of DMT pressure myograph and Drs. Binglan Yu and Chong Lei for providing mice fed with high fat diet or standard diet.

Sources of Funding

This work was supported by Scientist Development Grant 10SDG2610313 from the American Heart Association (to E. S. Buys), and an Eleanor and Miles Shore 50th Anniversary Fellowship program for Scholars in Medicine from Harvard Medical School (to E. S. Buys).