Guidelines for the measurement of vascular function and structure in isolated arteries and veins

Abstract

The measurement of vascular function in isolated vessels has revealed important insights into the structural, functional, and biomechanical features of the normal and diseased cardiovascular system and has provided a molecular understanding of the cells that constitutes arteries and veins and their interaction. Further, this approach has allowed the discovery of vital pharmacological treatments for cardiovascular diseases. However, the expansion of the vascular physiology field has also brought new concerns over scientific rigor and reproducibility. Therefore, it is appropriate to set guidelines for the best practices of evaluating vascular function in isolated vessels. These guidelines are a comprehensive document detailing the best practices and pitfalls for the assessment of function in large and small arteries and veins. Herein, we bring together experts in the field of vascular physiology with the purpose of developing guidelines for evaluating ex vivo vascular function. By using this document, vascular physiologists will have consistency among methodological approaches, producing more reliable and reproducible results.

Keywords: arteries; contraction; methods; relaxation; veins.

Figure 1.

In a pressure myograph system, blood vessel segments are cannulated with glass pipettes (A) to regulate the intraluminal pressure. In isometric force myograph systems, wires (B) or pins (C) are passed through the lumen of a blood vessel and is stretched to level that approximates physiological conditions.

Figure 2.

Blood vessel segments can be cannulated at both ends for flow-through applications (A), or tied off and secured to the cannula at one end to create a “blind sack” (B).

Figure 3.

Binocular (A) and a trinocular (B) dissecting microscope connected to a weighted base via a boom arm.

Figure 4.

Essential tools for dissecting macro- and microvessels for ex vivo experiments. Straight forceps with fine tips (A), curved forceps with fine tips (B), Vannas spring scissors with straight cutting edge (C) (curved scissors are also available, not shown), shallow and deep glass dissecting dishes with black silicone elastometer on the bottom (D, E), and insect pins (F). Created with Biorender.com.

Figure 5.

Diagram showing method to evaluate myogenic tone in an isolated, pressurized artery. VSMC, vascular smooth muscle cell.

Figure 6.

Isolation of small mesenteric arteries for vascular studies. Isolated rat mesenteric bed that is pinned out in a petri dish containing colored silicon elastometer and filled with physiological salt solution (A and B). C is magnified to illustrate the 5th order artery selected for cannulation after removal of the adipose, veins, lymph vessels, and connective tissue. Figure provided by Perenkita Mendiola at the Dept. of Cell Biology and Physiology, Univ. of New Mexico, Albuquerque, NM.

Figure 7.

Image shows a cardiac septum with right ventricle (RV) removed (A), and an amplified image for a septal coronary resistance microvessel (B).

Figure 8.

Image shows a muscle artery predissection. Thoracodorsal artery (TDA; ∼200 µm in lumen diameter) from mouse lays on top of the spinotrapezius muscle. Lateral view of the right shoulder of a mouse (A). Enlargement of the red box shown in A describing the anatomy of the superficial muscles of the right shoulder of a mouse: 1: spinotrapezius muscle; 2: latissimus dorsi muscle; 3: triceps brachii muscle (forelimb); 4: position of the scapula (B). Lateral view of the right shoulder of a mouse with a forceps pulling the latissimus dorsi muscle, revealing the TDA (C). Enlargement of the red box shown in C (D). Enlargement of the blue box in D (E). The purple box on the upper right shows a zoom in view of the TDA (Ar) surrounded by two veins (V) located on the caudal boarder of the scapula (designated by the arrow). Used with permission from Billaud et al. (32).

Figure 9.

Schematic of uterine artery bed from mice (A) (created with Biorender.com). Image shows uterine artery bed predissection (B). Lines indicate uterus, cervix, ovary, pups, and different arterial segments branched from ovarian arteries (A and B). Figure provided by Dr. Ramon A. Lorca at the Univ. of Colorado-Anschutz Medical Campus.

Figure 10.

Image shows left lung and numbers indicate pulmonary artery (PA) divided into five branches (A). Please note that the lumen diameter of the fifth artery branch is ∼ 50 µm before cannulation and pressurization (A). Amplified image for a fifth-order PA cannulated at both ends (B).

Figure 11.

Anatomy of surface pial arteries (A and B). Created with Biorender.com.

Figure 12.

A section of the brain cut around the middle cerebral artery and parenchymal arterioles are carefully dissected from this section (A). Image of a parenchymal arteriole (arrow) that is embedded within the parenchyma (B).

Figure 13.

Representative illustrations from a brain slice cannulation technique. A coronal brain slice with three parenchymal arteriole examples corresponding to a highly vascularized downstream network (high resistance), a short parenchymal arteriole (low resistance), and an averaged length and branched parenchymal arteriole (A). Coronal brain slice showing a pial arteriole with a branching downstream parenchymal arteriole not suitable for cannulation (B). The expanded imaged of an ideal cannulated parenchymal arteriole (C). The perfusion system which includes the syringe pump, pressure transducer, and cannula (D). Complete system which includes the perfusion system plus the vascular network (E). Example of the pressure-flow relationship corresponding to the experimental steps used to determine the resistance of the cannula (R_C) as well as the resistance of the entire system (F). Equations used to determine the resistance of the vascular network (R_A) as well as the equation used to determine the flow rate (Q) needed to bring the perfused system to a desired pressure (P) (G).

Figure 14.

Representative image from a cortical brain slice showing the presence of dead neurons (A), and arteriole, venule, and a capillary (B). Calibration bar, 10 μm. Image shows cannulated arteriole labeled with FITC (postexperiment) to define the downstream vascular network and the factors that contribute to the total resistance of the system (C).

Figure 15.

A: three colors stain (Trichrome) and vascular smooth muscle cells (VSMC) alpha actin staining in the rat thoracic aorta vs. vena cava (above the liver). L, lumen. B: veins have valves to promote the one-way movement of blood from tissues back to the heart. Used with permission from Hartmannsgruber et al. (51).

Figure 16.

Experimental protocols assessing the vasoactive effects of perivascular adipose tissue (PVAT). Two segments of the same vessel are tested, with one segment being PVAT-denuded and the other PVAT-intact (A). A vascular segment denuded from PVAT is mounted into a wire myograph and is tested in the absence of PVAT (left). Subsequently, the same vascular segment is tested in the presence of its neighboring PVAT (right), which had been initially removed and stored at 4°C physiological salt solution until experimentation. In this protocol, PVAT from comparing groups and conditions can be used to determine whether the observed effect is specific to (patho)physiological state (B). A vascular segment is cleaned from its PVAT, which is incubated in physiological salt solution at 37°C to create PVAT-conditioned media. The PVAT solution is filtered and added into the wire myograph chamber. Concentration-response curves to selected stimuli are performed in the absence and presence of PVAT-conditioned media (C). Created with Biorender.com.

Figure 17.

Step-by-step description of mounting small resistance arteries and veins on the wire myograph.

Figure 18.

Example traces demonstrating loss of lumen diameter in a pressurized vessel with a leak. Representative traces showing (A) lumen diameter is stable in vessel without a leak and (B) is reduced in a preparation with a leak (either due to a branch/hole in the vessel or it was not tied to the cannulas correctly) in preparations pressurized to 100 mmHg and closed to external pressure. Lumen diameter is restored in the leaky vessel once pressure is reintroduced.

Figure 19.

A cumulative concentration response curve (A) and noncumulative curve (B) using increasing concentrations of serotonin (5-HT, 5-hydroxytryptamine).

Figure 20.

Cumulative concentration response curve to the agonist serotonin (5-HT, 5-hydroxytryptamine) in the isolated abdominal vena cava, reported as a percentage of an initial challenge to the adrenergic agonist norepinephrine (NE). CRC, concentration response curve.

Figure 21.

GraphPad Prism software allows a number of different methods for analyzing sigmoidal pharmacological curve (A). Concentration-response curve to phenylephrine (PE) in the presence or absence of Prazosin (alpha-1 antagonist, 5 nM) in rat thoracic aorta (B). GraphPad Prism software presents the option of Log (agonist) vs. response—variable slope (four parameters) (C), to calculate data that are derived from the antagonist experiment in B.

Figure 22.

Logistic function (logit) is used for fitting sigmoidal concentration-response curves.