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. Author manuscript; available in PMC: 2021 Jun 15.
Published in final edited form as: J Vasc Res. 2020 Jun 15;57(5):291–301. doi: 10.1159/000508129

Vasoreactivity of the murine external jugular vein and carotid artery

Jae Min Cho 1,2, Yan-Ting Shiu 3,4, J David Symons 1,2,*, Timmy Lee 5,6,*
PMCID: PMC7486270  NIHMSID: NIHMS1593594  PMID: 32541137

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 three 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 artery versus vein. Nitric oxide synthase (NOS) 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: Vascular reactivity, Endothelial Dysfunction, Endothelial Cells, Nitric Oxide, Smooth Muscle Cell Relaxation

INTRODUCTION

Arteriovenous fistula (AVF) maturation failure is a significant clinical problem for hemodialysis patients and its underlying pathogenic mechanisms are poorly understood [1, 2]. One of the working hypotheses in the field of AVF research is that endothelium-dependent and / or - independent dysfunction of the venous segment might precipitate and / or amplify adverse remodeling and contribute to AVF failure. Before testing this hypothesis, procedures must be established to evaluate endothelium-dependent and -independent vasomotor responses in vascular segments that comprise the commonly used pre-clinical model of AVF [1, 3, 4], i.e., the carotid artery and the external jugular vein in mice. We and others have documented procedures used to evaluate vascular responses of the carotid artery in rodents [5, 6]. However, in our review of the literature, we found few reports that measured external jugular vein reactivity using isometric procedures [7-10], none that examined external jugular vein reactivity in mice, and none that provided sufficiently detailed methods for other investigators to replicate results. As such, the purpose of the present study was to establish and document isometric procedures to comprehensively characterize endothelium-dependent and vascular smooth muscle function of the murine carotid artery and external jugular vein.

MATERIALS AND METHODS

Animals

Five-month old male C57BL/6 mice (n=9) were housed under controlled temperature (22°C) and light conditions (12-h light/12-h dark cycle) and were provided with food and water ad libitum. All experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Utah.

Tissue collection

Mice were anesthetized using 2-5% isoflurane, the chest was opened, a small incision was made in the right atrium, and 1 ml of physiological saline solution (PSS; pH 7.35-7.40) was slowly perfused through the left ventricle. Next, the heart was excised, and the carotid artery and external jugular vein was dissected free from adherent tissue while bathed in ice-cold PSS.

Vascular function: carotid artery

Once removed from the mouse, the carotid artery was immersed in iced PSS in a sylgard lined dissecting dish that was affixed to an ice pack. One 20-μm thick tungsten wire was placed through the lumen of the carotid artery. The artery was then transferred to a myograph chamber (Figure 1, Panel A) containing 8 ml PSS, where a second 20-μm thick tungsten wire was inserted through the vessel. Next, one wire was attached to a stationary micrometer, while the second wire was fastened to a force transducer. After the vessel mounting procedure was complete, the tissue chamber was heated over 45-min from room temperature to 37°C with vessels at 0 mg tension. Tension on the artery then was increased by 100 mg every 10-min by manually increasing the distance between the two wires, until 400 mg tension was achieved i.e., 0-100 mg (10-min), 100-200 mg (20-min), 200-300 mg (30-min), 300-400 mg (40-min). The vessel bathing medium was exchanged with fresh PSS at the conclusion of each manual tension increase. A schematic of experimental procedures is shown in Figure 1, Panel B.

Figure 1.

Figure 1.

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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 NG-methyl-L-arginine acetate salt; indomethacin, the cyclooxygenase 1 and 2 inhibitor; SNP, sodium nitroprusside. See text for a detailed explanation.

At 40-min (i.e., at 400 mg tension) tension was manually increased to 500 mg but this time the vessel bathing medium was exchanged with 100 mM potassium chloride (KCl). KCl opens voltage-gated Ca2+ channels (i.e., L-type Ca2+ channels) resulting in vasocontraction (i.e., active tension development) of vascular smooth muscle. After tension development to KCl was stable, the vessel bathing medium was exchanged with fresh PSS, and tension returned to pre-KCl baseline values, i.e., ~500 mg. Next, tension was manually increased to ~600 mg, KCl was administered, the vessel bathing medium was exchanged with fresh PSS when tension development was stable, and tension returned to pre-KCl values, i.e., ~600 mg. This procedure was repeated, using ~100 mg manual increments, until active tension development to KCl from a given resting tension was ≤ 10% of the KCl-evoked tension development in response to the previous (i.e., lower) resting tension. Using an example from our study, a carotid artery developed 277 mg, 316 mg, and 322 mg tension when KCl was administered at resting tensions of 760 mg, 873 mg, and 970 mg, respectively. The increase from 277-316 mg represents a 14% increase, which necessitated a further manual increase. However, since the increase from 316-322 represents a 2% increase, further manual increases from 970 mg were not necessary, and 970 mg was deemed Lmax tension for this particular vascular segment.

After determining Lmax for each artery, the vessel bathing medium was exchanged at 10 and 20-min with fresh PSS. At 30-min, non-receptor mediated vasocontractile responses to KCl (20-100 mM) were completed, followed 30-min later by receptor-mediated vasocontractile responses to phenylephrine (PE, 10−8-10−5 M) and (30-min later) U-46619 (10−8-10−5 M). During each 30-min interval the vessel bathing medium was exchanged twice immediately following the dose-response curve, once at 10 min, and once at 20-min. We and others have shown that this time interval avoids tachyphylaxis.

To evaluate arterial endothelial function, U-46619 was used to contract the arteries above the resting baseline tension by ~65% of their maximal response to U-46619. When tension development was stable, responses to acetylcholine (ACh, 10−8-10−6 M) were assessed to determine stimulated endothelium-dependent vasorelaxation. Upon completion of this concentration response curve, the vessel bathing medium was exchanged twice immediately with PSS, and again at 10-min with PSS. In arteries from 5 of 9 mice, a second ACh dose-response curve was completed in the presence of nitric oxide synthase (NOS) isoform 3 inhibition using NG-methyl-L-arginine acetate salt (L-NMMA). In this regard, at 20-min, 10−3 M L-NMMA was administered to, and remained in, the vessel bathing medium for 30-min. After 30-min, and in the presence of L-NMMA, U-46619 was used to contract the arteries above resting baseline tension by ~65% of the maximal response to U-46619. When tension development was stable, responses to ACh again were assessed to determine ACh-induced, NO-independent vasorelaxation. In arteries from 4 of 9 mice, a second ACh dose-response curve was completed in the presence of cyclooxygenase 1 and cyclooxygenase 2 inhibition using 10−3 M indomethacin. Procedures were identical to those described for NOS3 inhibition using L-NMMA.

Upon completion of the ACh dose-response curve in the presence of L-NMMA or indomethacin, the vessel bathing medium was exchanged twice with PSS, once 10-min later, and once 20-min later, with PSS. At 30-min, U-46619-induced vasocontraction again was performed as described, and a sodium nitroprusside (SNP, 10−9-10−4 M) concentration-response curve was completed to assess vascular smooth muscle relaxation i.e., endothelium-independent vasorelaxation in arteries from all 9 mice.

Vascular function: external jugular vein

After veins from 9 mice were mounted on the myograph unit (Figure 1, Panel A), and the temperature of the chamber was elevated to 37°C, the distance between the two wires at 0 mg tension was calculated e.g., 200 μm internal diameter (i.d.). This value was multiplied by 4 e.g., 800 μm. Next, at 10-min intervals, the distance between the wires was increased by 25% until the 4 x distance was achieved i.e., 200-350 μm, 350-500 μm, 500-650 μm, 650-800 μm. 100 mM KCl was administered to the venous bathing medium at the 75% (e.g., 650 μm) and 100% (e.g., 800 μm) distance. In all cases, KCl-evoked tension development at the 4 x resting internal diameter (i.e., 100%) was ≤ 10% when compared to tension development evoked by the 75% resting internal diameter. This procedure was used to determine Lmax of the venous segment, and evolved from 5 pilot experiments wherein different approaches were evaluated.

After a 30-min interval during which the vessel bathing medium was exchanged with PSS at 10 and 20-min, non-receptor mediated vasocontractile responses to KCl, and receptor-mediated vasocontractile responses to PE and U-46619 were completed. A 30-min interval separated each dose-response curve, during which time the vessel bathing medium was exchanged twice with PSS at the conclusion of the intervention, once at 10-min, and once at 20-min with PSS.

Next, veins were contracted using U-46619 above baseline to 65% of their maximal response to U-46619. When tension development to this precontraction was stable, responses to ACh (10−8-10−6 M) were assessed to determine stimulated endothelium-dependent vasorelaxation. Upon completion of the curve, the vessel bathing medium was exchanged twice immediately with PSS, once at 10-min, and once at 20-min. In veins from 5 of 9 mice, a second ACh dose-response curve was completed in the presence of L-NMMA. Specifically, at 20-min, 10−3 M L-NMMA was added to, and remained in, the vessel chamber. After 30-min incubation with L-NMMA, veins again were precontracted using U-46619 above baseline tension to 65% of maximal U-46619-induced tension development. When tension development was stable, responses to ACh were assessed in the presence of L-NMMA to determine ACh-induced, NO-independent vasorelaxation. In veins from 4 of 9 mice, a second ACh dose-response curve was completed in the presence of 10−3 M indomethacin. Procedures were identical to those described for NOS3 inhibition using L-NMMA.

Upon completion of the ACh dose-response curve in the presence of L-NMMA or indomethacin, the vessel bathing medium was exchanged with PSS twice immediately, once at 10-min, and once at 20-min. At 30-min, U-46619-induced precontraction again was performed as described, and a sodium nitroprusside (SNP, 10−9-10−4 M) concentration-response curve was completed to assess function of the vascular smooth muscle. A schematic of our experimental protocol is shown in Figure 1B.

Immunohistochemistry.

To assess expression of α-1A adrenergic receptor, 1 carotid artery and 1 external jugular vein was obtained from each of 3 C57BL/6 mice. Vessels were formalin-fixed, paraffin-embedded, and cut into 5-μm thin sections [1]. After deparaffinization and rehydration, epitope retrieval was performed by immersing the tissue sections in citrate buffer [pH 6.0, at 1X dilution (Abcam, Cambridge, MA)] at 95°C for 10 min. Subsequently, endogenous peroxidases were quenched by treating with 1% hydrogen peroxide, and non-specific binding was minimized by incubating with 2% blocking solution (Invitrogen, Camarillo, CA). Sections then were incubated with : (0 primary antibody to α-1A adrenergic receptor (1:200, polyclonal rabbit IgG) (Invitrogen, Camarillo, CA) at room temperature overnight; followed by (ii) incubation with a biotinylated secondary antibody (1:200, biotinylated goat anti-rabbit IgG) (Vector Laboratories, Burlingame, CA) at room temperature for 30 min. Color was developed using the ultra-sensitive avidin-biotin complex (ABC) peroxidase staining kit (ThermoFisher, Camarillo, CA), metal-enhanced 3, 3′diaminobenzidine tetrahydrochloride (DAB) substrate (1X) (ThermoFisher, Camarillo, CA), followed by counterstaining with hematoxylin. Images of the entire cross-section were acquired using the Olympus FLUOVIEW FV1000 under bright-field with the same settings. The intimal and medial layer of each vessel were delineated on Fiji, and the intensity of the brown staining (α-1A adrenergic receptor) was measured after separation using the color deconvolution plugin and selection using the threshold function. Two sections per vessel from each of 3 mice were evaluated. One section that did not receive primary antibody was used to determine background intensity. This intensity was subtracted from values obtained from the section that did receive primary antibody. As such, three staining intensities per vessel type were obtained and averaged.

Data acquisition

For studies involving both vessel types, all tension data were recorded continuously using an analog-to-digital interface card (Biopac Systems Inc., Santa Barbara, CA) that allowed for subsequent off-line quantitative analyses. We have used these procedures to evaluate reactivity in arteries obtained from humans [11-15], rodents [16-22], pigs [23], and rabbits [24].

Statistical analyses

Values are expressed as mean ± standard error of the mean (SEM). Comparison of one endpoint (e.g., maximal tension development to U-46619) between the carotid artery and external jugular vein was performed using an unpaired t-test. The comparison of multiple data points (e.g., 10−10-10−6 M ACh) between carotid artery and jugular vein was made using a repeated measures two-way analysis of variance. Tukey post-hoc tests were performed when significant interactive effects were identified. Significance was accepted when p<0.05.

RESULTS

Vasocontraction responses in the carotid artery and external jugular vein.

The resting internal diameter and vessel length were similar between the artery and vein when vessel types were assessed at 0 mg tension (Figure 1, Panel A). KCl depolarizes the vascular smooth muscle cell membrane potential to open voltage-gated Ca2+ channels (i.e., L-type Ca2+ channels), resulting in vasocontraction of vascular smooth muscle. Relative to baseline tension, KCl-evoked tension development occurred (p<0.05) in both vessel types, but responses to this non-receptor mediated agonist were more robust (p<0.05) in carotid artery vs. external jugular vein from 40-100 mM (Figure 2, Panel A).

Figure 2.

Figure 2.

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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.

PE binds to α1-adrenergic receptors that are coupled to Gq proteins. PE-mediated α1-activation stimulates inositol triphosphate (IP3) mediated release of Ca2+, which triggers Rho-kinase, inhibits myosin light-chain phosphatase, and evokes vascular smooth muscle contraction. External jugular veins were refractory to PE throughout the range of doses administered. PE-evoked tension development was greater (p<0.05) from 10−6 M to 10−5 M in carotid artery, vs. external jugular vein (Figure 2, Panel B).

U-46619 is a stable synthetic analog of the endoperoxide prostaglandin PGH2, and acts as a thromboxane A2 receptor (TP) agonist. U-46619-mediated TP activation couples with and mobilizes one or more G proteins to activate phospholipase C, IP3, and intracellular Ca2+ in vascular smooth muscle cells. Relative to baseline tension, U-46619-evoked tension development occurred (p<0.05) in both vessel types, but responses to this non-receptor mediated agonist were more robust (p<0.05) in carotid artery at doses from 3 x 10−7 M to 10−5 M (Figure 2, Panel C). For KCl, PE, and U-46619, maximal tension development was greater (p<0.05) in carotid artery vs. external jugular vein (Figure 2, Panel D).

α1-adrenergic receptor staining intensity in the carotid artery and external jugular vein.

Results shown in Figure 2 Panel C indicate the external jugular vein was not responsive to PE, whereas vasocontraction to the same dose of PE was robust in the carotid artery. This could result from a relative paucity of α-1A adrenergic receptor in vein vs. artery. We tested this. Indeed, α-1A adrenergic receptor staining intensity was lower (p<0.05) in the external jugular vein vs. the carotid artery (Figure 3, Panels A, B).

Figure 3.

Figure 3.

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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.

Vasorelaxation responses in the carotid artery and external jugular vein.

ACh binds to M3 muscarinic receptors on the vascular endothelium to increase intracellular Ca2+. Elevated intracellular Ca2+ then activates constitutive type III nitric oxide (NO) synthase (NOS), which facilitates conversion of the amino acid substrate L-arginine to the products L-citrulline and NO. As such, ACh is used to stimulate endothelium-dependent vasorelaxation. In carotid arteries that were stably precontracted using U-46619, ACh evoked vasorelaxation that was maximal at ~68%. In external jugular vein segments that were precontracted similarly, ACh evoked vasorelaxation that was maximal at ~ 58% (Figure 4, Panel A). Maximal ACh-evoked vasorelaxation was not different between carotid artery and external jugular vein (Figure 4, Panel B).

Figure 4.

Figure 4.

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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.

NO that is produced by endothelial cells diffuses abluminally to the vascular smooth muscle where it activates guanylyl cyclase (GC). GC stimulation increases cGMP formation which inhibits Ca2+ entry into the vascular smooth muscle and precipitates vasorelaxation. SNP is a nitrodilator that releases NO spontaneously by activating GC directly. As such, SNP is used to assess endothelium-independent vasorelaxation. In carotid arteries, after precontraction using U-46619, SNP-evoked vasorelaxation occurred in a dose-dependent manner that was maximal at ~ 85%. In external jugular vein segments that were precontracted similarly, SNP evoked vasorelaxation in a concentration-dependent fashion that was maximal at ~ 63% (Figure 4, Panel C). While differences did not attain significance when vasorelaxation at each dose of SNP was compared between carotid artery and external jugular vein using a repeated measures ANOVA, maximal SNP-evoked vasorelaxation was greater (p<0.05) in carotid artery vs. external jugular vein when results were compared using an unpaired t-test (Figure 4, Panel D).

The influence of NOS3 inhibition on ACh-evoked vasorelaxation in carotid artery and external jugular vein.

To assess the contribution from NO to vasorelaxation in the carotid artery and external jugular vein, we completed ACh concentration – response curves after vessels had incubated for 30-min with L-NMMA. L-NMMA competes with the endogenous NOS substrate L-arginine. Because L-NMMA is administered to the vessel bathing medium in excess, it outcompetes L-arginine for NOS binding sites and thereby limits the capability for NO generation. In both vessel types i.e., the carotid artery (Figure 5, Panel A) and external jugular vein (Figure 5, Panel B). ACh-evoked vasorelaxation was attenuated (p<0.05) in the presence (i.e., +L-NMMA) vs. the absence (i.e., −L-NMMA) of NOS inhibition. Next we determined whether NOS inhibition suppressed ACh-evoked vasorelaxation to a greater extent in carotid artery vs. external jugular vein. Maximal ACh-evoked vasorelaxation in the presence of L-NMMA vs. in the absence of L-NMMA was calculated for each of 9 carotid a (e.g., 25 ± 11% vs. 81 ± 9%, respectively) and each of 9 external jugular v (e.g., 48 ± 10% vs. 75 ± 7%, respectively; Figure 5, Panel C). NOS inhibition suppressed maximal ACh-evoked vasorelaxation to a greater (p<0.05) extent in the carotid artery (61 ± 1%) vs. external jugular vein (35 ± 1%)(Figure 5D).

Figure 5.

Figure 5.

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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 NG - 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.

The influence of cyclooxygenase inhibition on ACh-evoked vasorelaxation in carotid artery and external jugular vein.

To assess the contribution from cyclooxygenase products to vasorelaxation in the carotid artery and external jugular vein, we completed ACh concentration – response curves after vessels had incubated for 30-min with indomethacin. Indomethacin inhibits cyclooxygenase 1 and cyclooxygenase 2, enzymes required for the conversion of arachidonic acid to prostaglandins. In both vessel types i.e., the carotid artery (Figure 6, Panel A, C) and external jugular vein (Figure 6, Panel B, C), ACh-evoked vasorelaxation was not different when assessed in the absence vs. the presence of indomethacin.

Figure 6.

Figure 6.

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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.

DISCUSSION

Here we provide sufficient methodological detail for investigators to evaluate endothelium-dependent and vascular smooth muscle function in the external jugular vein of healthy male C57BL/6 mice using isometric procedures. Regarding functional responses of the two vessel types, external jugular veins developed tension to KCl and U-46619, but not to PE, whereas common carotid arteries responded to all three vasocontractile agents in a more robust manner. Second, while maximal responses to the endothelium-dependent vasodilator ACh were similar between the venous and arterial segments, the dose at which maximal responses were observed was lower in the artery vs. the vein. Third, NOS inhibition attenuated but did not abolish ACh-evoked vasorelaxation in both vascular segments, indicating L-NMMA-resistant dilators exist in carotid arteries and external jugular veins. One of the L-NMMA-resistant dilators is not a product of cyclooxygenase metabolism because ACh-induced vasorelaxation was similar in carotid artery and external jugular vein in the absence and presence of indomethacin. Finally, while dose-response curves to the endothelium-independent vasodilator SNP were similar between vascular segments, maximal responses were greater in arteries vs. veins. Collectively, our results demonstrate that endothelium-dependent and vascular smooth muscle function can be reliably assessed in the external jugular vein of healthy male C57BL/6 mice using isometric procedures.

Vasocontraction responses.

It is most common for investigators to assess function in arterial segments from preclinical models of cardiovascular disease [16-22]. Because of the importance in evaluating the venous segment of the AVF, we reviewed the literature in an attempt to design protocols for conducting these experiments. We found few reports that measured external jugular vein reactivity using isometric procedures [7-10], and none that examined external jugular vein reactivity in mice. External jugular veins from rabbits display vasocontraction to KCl, U-46619. and PE [7-9]. In our study the murine external jugular vein was markedly less sensitive to both non-receptor (KCl) and receptor mediated (PE, U-46619) vasocontractile agonists vs. the murine carotid artery (Figure 2). In the case of non-receptor mediated vasocontraction, these results may indicate that veins have fewer L-type Ca2+ channels and / or their sensitivity is less. Regarding receptor-mediated vasocontraction, external jugular veins were refractory to PE even though a large concentration range was used. This (lack of) response could be secondary to relatively few α-1A adrenergic receptors present in the external jugular vein vs. the artery and this was confirmed (Figure 3). It is not unreasonable to speculate that a paucity of α-1A adrenergic receptors exists in the external jugular vein so that vasocontraction of this vascular segment does not occur in situations involving sympathetic stimulation to an extent that compromises venous return from the cerebral circulation to the superior vena cava.

Vasorelaxation responses.

The magnitude of ACh-evoked vasorelaxation we observed in the external jugular vein from mice was similar to that reported by others in the corresponding vascular segment from rabbits [7, 8]. Although maximal vasodilation to ACh was not different between the venous and arterial segments in our investigation, sensitivity to ACh was significantly reduced in the vein as compared to the artery (Figure 4). While both vessel types have a single layer of endothelial cells, vascular smooth muscle cells are more abundant in arteries vs. veins. As such, it is possible that even though endothelial cells from both vessel types release NO to a significant extent, the arteries respond sooner (i.e., at lower doses) as a result of the greater abundance of vascular smooth muscle cells.

Although NO contributes to ACh-evoked vasorelaxation in both vascular segments, its role is greater in carotid a vs. external jugular v. Evidence for this statement is that L-NMMA suppressed ACh-evoked vasorelaxation by ~ 60% in carotid a vs. ~ 35% in external jugular v (Figure 5D). Significant L-NMMA resistant, ACh-evoked vasorelaxation was observed in murine external jugular vein (Figure 5) and this is consistent with a previous report in external jugular vein from rabbits [9]. In the latter study, because the same dose of L-NMMA prevented 5-hydroxytryptamine receptor-stimulated endothelium-dependent vasorelaxation, the authors concluded that analogues of L-arginine (i.e., L-NMMA) demonstrate agonist-dependence in terms of their ability to inhibit receptor-mediated vasorelaxation. Products of cyclooxygenase metabolism did not contribute to ACh-evoked, L-NMMA-resistant vasorelaxation in external jugular vein (or carotid artery) from our mice because ACh produced vasorelaxaton that was similar in the absence and presence of indomethacin (Figure 6). Likewise, cyclooxygenase inhibition using indomethacin was without effect concerning ACh-evoked vasorelaxation in external jugular vein from rabbits [9]. Future studies will explore the role of additional L-NMMA - resistant / indomethacin-resistant, ACh evoked vasorelaxation (e.g., endothelium-derived hyperpolarizing factors) in the external jugular vein.

We assessed endothelium-independent vasorelaxation using SNP to measure vascular smooth muscle function in the two vascular segments (Figure 4C). Results from the carotid artery substantiate our earlier findings using this vessel [5, 6]. Furthermore, data obtained from the murine external jugular vein concerning SNP-evoked vasorelaxation are similar to those reported from the external jugular vein of rabbits [7-9]. While the SNP dose-response curve was not different between the artery and vein when results were assessed using a repeated measures ANOVA (i.e., multiple comparisons), arteries responded to a greater extent when maximal responses were compared via an unpaired t-test (i.e., one comparison). This latter finding is not unexpected because the vascular smooth muscle cell layer is more abundant in the arterial segment, and as such, is more capable of responding to an agonist. On the other hand, since vasorelaxation to SNP was generally similar between arteries and veins, and because vascular smooth muscle cells are less abundant in veins, it is plausible that if responses were normalized per vascular smooth muscle cell, the venous portion might be significantly more responsive.

Experimental considerations.

Several limitations should be considered when our findings are integrated into the current literature. We examined the common carotid artery and external jugular vein which are medium-sized vessels, and the vasomotor responses may be, and likely are, quite different in other vascular beds. However, the carotid artery and external jugular vein comprise the vascular components of our preclinical AVF model [1], and the purpose of this report was to establish isometric procedures to evaluate vasoreactivity of these particular segments. Second, male mice were used and it is well-known that sex-specific vasoreactivity responses exist, at least in the arterial circulation [25]. Third, vascular segments were obtained from healthy mice, and we acknowledge (and hypothesize) that aging and/or the presence of disease known to impact arterial function likely precipitates altered vasoreactivity in veins. Fourth, human veins used for AVF creation in CKD patients have a thick intima [26] that likely results from advanced age and concurrent vascular complications. We measured reactivity of naïve mouse veins that are void of concurrent pathology and possess a relatively thin media. Finally, we chose to assess vasoreactivity using isometric vs. isobaric procedures. In an earlier study [1] we observed that side-branches often exist in the venous segment of the AVF. Isobaric approaches reguire that these side branches be ligated so that constant pressure can be maintained within the vessel. While this can be done, ligation has potential for additional damage and / or mechanical hindrance secondary to suture placement. To circumvent this potential pitfall, we sought to determine an alternative approach (i.e., isometric procedures). After reviewing the literature, we could find no instances wherein this procedure was described comprehensively for the murine external jugular vein.

Conclusions.

We have established and documented isometric procedures to comprehensively characterize endothelium-dependent and vascular smooth muscle function of the murine carotid artery and external jugular vein. These procedures will provide a viable alternative to evaluate vascular function in venous vascular disease (e.g., venous segment of an AVF) particularly in cases when multiple side branches preclude the use of isobaric approaches.

Acknowledgements

The authors thank Yuxia He for animal husbandry and tissue collection and Jason Chieh Sheng Tey for performing immunohistochemistry experiments.

Funding Disclosure

Support was provided for : JMC by University of Utah Graduate Research Fellowship and American Heart Association (AHA) Predoctoral Fellowship 20PRE35110066; YTS by NIDDK (R01DK100505, R01DK121227) and the Department of Veterans Affairs (Merit award I01BX004133); JDS by AHA (16GRNT31050004), NIA (RO3AG052848) and NHLBI (RO1HL141540); and TL by NIDDK (R44DK109789), NHLBI (R01HL139692), and the Department of Veterans Affairs (Merit award I01BX003387).

Footnotes

Data Availability

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

Ethics Statement

The Institutional Animal Care and Use Committee of the University of Utah approved these experiments.

Conflict of Interest Statement

TL is a consultant for Proteon Therapeutics, Merck, and Boston Scientific. The other co-authors declare no competing interests.

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