Vasoreactivity of the Murine External Jugular Vein and Carotid Artery

Abstract

Introduction: Impaired venous reactivity has potential to contribute to clinically significant pathologies such as arteriovenous fistula (AVF) maturation failure. Vascular segments commonly used in murine preclinical models of AVF include the carotid artery and external jugular vein. Detailed descriptions of isometric procedures to evaluate function of murine external jugular vein ex vivo have not been previously published.

Objective: To establish isometric procedures to measure naive murine external jugular vein reactivity ex vivo.

Methods: Vasomotor responses of external jugular veins and ipsilateral common carotid arteries from C57BL/6 mice were evaluated using isometric tension procedures.

Results: External jugular veins developed tension (p < 0.05) to potassium chloride and U-46619, but not to phenylephrine, whereas common carotid arteries responded to all 3 agents (p < 0.05). While maximal responses to acetylcholine (ACh) were similar between the venous and arterial segments, the dose required to achieve this value was lower (p < 0.05) in the artery versus vein. Nitric oxide synthase inhibition attenuated (p < 0.05) but did not abolish ACh-evoked vasorelaxation in both vascular segments, whereas cyclooxygenase blockade had no effect. Endothelium-independent vasorelaxation to sodium nitroprusside was similar in the artery and vein.

Conclusion: Vasorelaxation and vasocontraction can be reliably assessed in the external jugular vein in C57BL/6 mice using isometric procedures.

Keywords: Endothelial cells; Endothelial dysfunction; Nitric oxide; Smooth muscle cell relaxation; Vascular reactivity.

Figure 1.

A. Carotid artery (a) and external jugular vein (v) mounted in a myograph apparatus. Each vessel is shown at 0 mg tension. Values on the x and y axis represent mean ± SE of the width (y axis, μm) and length (x axis, μm) of the respective vessel types from 16 carotid a and 14 external jugular v. B. Schematic of our experimental protocol. d/r, dose-response curve; KCl, potassium chloride; PE, phenylephrine; ACh, acetylcholine; L-NMMA, the nitric oxide synthase inhibitor N^G-methyl-L-arginine acetate salt; indomethacin, the cyclooxygenase 1 and 2 inhibitor; SNP, sodium nitroprusside. See text for a detailed explanation.

Figure 2.

A. Non-receptor mediated tension development (mg) / vessel length (μm) to potassium chloride (KCl) in the carotid a and external jugular v. Receptor-mediated tension development to phenylephrine (PE; B) and U-46619 (C) in both vessel types. D. Maximal responses to KCl, PE, and U-46619 in carotid a and external jugular v. Values represent mean ± SEM from 16 carotid a and 14 external jugular v obtained from 9 mice. For A-D, *p<0.05 vs. external jugular v. In Panels A, B, and C, the SEM for the v is too small to show.

Figure 3.

A. Representative images of immunohistochemistry staining for α-1A adrenergic receptors in the carotid artery (a) and external jugular vein (v). Scale bar = 50 μm at 20X magnification. B. Staining intensity (mean ± SEM) of α-1A adrenergic receptors from the carotid a and external jugular v of 3 mice per group x 2 sections per mouse. Background intensity Quantified from one section without stain was subtracted from α-1A adrenergic receptor staining intensity Quantified from the second section with primary antibody. *p<0.05 vs. external jugular v.

Figure 4.

A. Endothelium-dependent vasorelaxation to acetylcholine (ACh) in carotid artery (a) and external jugular vein (v). EC₅₀ values were −7.819 ± 0.157 for carotid a and −7.269 ± 0.105 for external jugular v. B. Maximal responses to ACh in a and v. C. Endothelium-independent vasorelaxation to sodium nitroprusside (SNP) in a and v. EC₅₀ values were −7.511 ± 0.113 for carotid a and −7.371 ± 0.237 for external jugular v. D. Maximal responses to SNP in a and v. Values represent mean ± SEM from 16 carotid a and 14 external jugular v from 9 mice. For A-D, *p<0.05 vs. external jugular v.

Figure 5.

A. Endothelium-dependent vasorelaxation to acetylcholine in the absence (− L-NMMA) and presence (+ L-NMMA) of nitric oxide synthase isoform 3 (NOS3) inhibition using 10⁻³ M N^G - methyl-L-arginine acetate salt (L-NMMA) in carotid artery (a) and (B) external jugular vein (v). EC₅₀ values were −7.912 ± 0.220 for −L-NMMA and −7.287 ± 0.310 for +L-NMMA in carotid a: −7.472 ± 0.088 for −L-NMMA and −6.835 ± 0.112 for +L-NMMA in external jugular v. C. Maximal acetylcholine evoked vasorelaxation in the absence and presence of L-NMMA in the a and v. D. The difference between maximal ACh-evoked vasorelaxation in the absence vs. the presence of L-NMMA (i.e., bar 1 vs. bar 2 in Figure 5C for carotid a, and bar 3 vs. bar 4 in Figure 5C for external jugular v). All values represent mean ± SEM from 9 carotid a and 9 external jugular v from 5 mice. For A - D, *p<0.05 + L-NMMA vs. − L-NMMA.

Figure 6.

A. Endothelium-dependent vasorelaxation to acetylcholine in the absence (− indomethacin) and presence (+ indomethacin) of cyclooxygenase 1 and cyclooxygenase 2 inhibition using 10⁻³ M indomethacin in carotid artery (a) and B. external jugular vein (v). EC₅₀ values were −7.428 ± 0118 for − indomethacin and −7.028 ± 0057 for + indomethacin in carotid a; −7.457 ± 0.222 for − indomethacin and −7.022 ± 0.134 for + indomethacin in external jugular v. C. Maximal acetylcholine evoked vasorelaxation in the absence and presence of indomethacin in the a and v. Values represent mean ± SEM from 7 carotid a and 5 external jugular v from 4 mice.