Ageing alters perivascular nerve function of mouse mesenteric arteries in vivo

Abstract

Mesenteric arteries (MAs) are studied widely in vitro but little is known of their reactivity in vivo. Transgenic animals have enabled Ca²⁺ signalling to be studied in isolated MAs but the reactivity of these vessels in vivo is undefined. We tested the hypothesis that ageing alters MA reactivity to perivascular nerve stimulation (PNS) and adrenoreceptor (AR) activation during blood flow control. First- (1A), second- (2A) and third-order (3A) MAs of pentobarbital-anaesthetized Young (3–6 months) and Old (24–26 months) male and female Cx40^BAC-GCaMP2 transgenic mice (C57BL/6 background; positive or negative for the GCaMP2 transgene) were studied with intravital microscopy. A segment of jejunum was exteriorized and an MA network was superfused with physiological salt solution (pH 7.4, 37°C). Resting tone was ≤ 10% in MAs of Young and Old mice; diameters were ∼5% (1A), 20% (2A) and 40% (3A) smaller (P≤ 0.05) in Old mice. Throughout MA networks, vasoconstriction increased with PNS frequency (1–16 Hz) but was ∼20% less in Young vs. Old mice (P≤ 0.05) and was inhibited by tetrodotoxin (1 μm). Capsaicin (10 μm; to inhibit sensory nerves) enhanced MA constriction to PNS (P≤ 0.05) by ∼20% in Young but not Old mice. Phenylephrine (an α₁AR agonist) potency was greater in Young mice (P≤ 0.05) with similar efficacy (∼60% constriction) across ages and MA branches. Constrictions to UK14304 (an α₂AR agonist) were less (∼20%; P≤ 0.05) and were unaffected by ageing. Irrespective of sex or transgene expression, ageing consistently reduced the sensitivity of MAs to α₁AR vasoconstriction while blunting the attenuation of sympathetic vasoconstriction by sensory nerves. These findings imply substantive alterations in splanchnic blood flow control with ageing.

Key points

• Neural control of the circulation is integral to the regulation of tissue blood flow and systemic blood pressure. Vascular dysfunction occurs with ageing but little is known of corresponding changes in the role(s) of perivascular nerves.

• We developed a preparation to study intact mesenteric arteries (MAs) in anaesthetized mice to investigate age-related changes in the function of perivascular sympathetic and sensory nerves in vivo.

• Ageing decreased the diameter of MAs, reduced their sensitivity to α₁-adrenoreceptor stimulation and impaired the ability of sensory nerves to attenuate sympathetic vasoconstriction.

• These changes were manifest in males and females and were unaffected by the expression of the GCaMP2 transgene in endothelial cells, confirming the utility of this model.

• Our results imply that ageing imposes structural and functional limitations to the splanchnic circulation that impair the ability to mobilize blood from the gut in times of physical stress.

Introduction

Ageing is associated with physiological changes throughout the body, with vascular age serving as a better predictor of cardiovascular disease than chronological age (Barodka et al. 2011). With ageing, large conduit arteries stiffen and lose endothelial function (Lakatta & Levy, 2003; Seals et al. 2011), resulting in increased frailty (Newman et al. 2001) with diminished capacity for physical exercise (Heckman & McKelvie, 2008). Preserving exercise capacity is an important goal for the ageing population as physical activity can ameliorate vascular ageing through reducing arterial stiffness and improving endothelium-dependent dilatation (Koch et al. 2005; Heckman & McKelvie, 2008; Seals et al. 2011). In contrast to such documented effects in systemic arteries, the effect of ageing on the smaller resistance arteries and arterioles is less clear. Nevertheless, evidence suggests that resistance vessels also undergo stiffening with impaired function (Laurant et al. 2004; Dumont et al. 2008) along with remodelling of vascular networks (Bearden, 2006; Behnke et al. 2006) that may adversely impact tissue perfusion throughout the body.

Perivascular nerves are integral to vasomotor control in resistance arteries and arterioles. In mesenteric arteries (MAs), these efferent axons include sympathetic (adrenergic) and sensory (peptidergic) fibres (Furness & Marshall, 1974; Kreulen, 2003; Franchini & Cowley, 2004; Haddock & Hill, 2011). Activation of perivascular sympathetic nerves leads to noradrenaline (NA) release, which causes vasoconstriction through activation of adrenoreceptors (ARs) on smooth muscle cells (Furness & Marshall, 1974; Fleming et al. 1987). In contrast, sensory nerves, which release calcitonin gene-related peptide (CGRP), cause vasodilation through activation of CGRP receptors on both smooth muscle and endothelial cells (Kawasaki, 2002; Brain & Grant, 2004). Moreover, sympathetic and sensory nerves can reciprocally regulate each other during neural control of vasomotor function (Kawasaki, 2002).

Little is known of age-related changes in the regulation of splanchnic vasomotor control by perivascular nerves. With nearly 25% of cardiac output directed to the gut, the ability to mobilize splanchnic blood to other areas of the body is integral to maintaining exercise capacity and cardiovascular homeostasis (Rowell, 1974; Flamm et al. 1990). Thus, a key goal of this study was to define the role of perivascular nerves in regulating MA function in vivo. Because sympathetic nerve activity increases with ageing throughout the body (Ng et al. 1993; Dinenno et al. 2000; Seals & Dinenno, 2004) and the mesenteric vasculature is richly innervated (Furness & Marshall, 1974; Long & Segal, 2009; Haddock & Hill, 2011), the mesenteric circulation is a likely target for age-related functional defects in vasomotor control. To investigate this relationship, a second goal of this study was to test the hypotheses that ageing alters the function of perivascular nerves during vasomotor control in vivo.

Experiments were performed using Cx40^BAC-GCaMP2 transgenic mice. In these animals, expression of GCaMP2 (a green fluorescent protein-based Ca²⁺ indicator) is under the control of the connexin40 (Cx40) promoter (Tallini et al. 2007). Because Cx40 expression in the vasculature is restricted to endothelial cells of arteries and arterioles, GCaMP2 is expressed accordingly (Tallini et al. 2007). These mice were first used to study Ca²⁺ signalling during arteriolar reactivity in vivo (Tallini et al. 2007; Bagher et al. 2011b) and have gained acceptance for studying Ca²⁺ signalling in isolated MA preparations (Ledoux et al. 2008; Nausch et al. 2012; Sonkusare et al. 2012) Because previous studies of these mice have been performed exclusively using males, it has not been determined whether sex differences or the presence of GCaMP2 may impact the vascular physiology of animals expressing this transgene. Therefore, a third goal of this study was to evaluate males and females as well as mice that were either positive or negative for the GCaMP2 transgene to further validate this important transgenic model. Our findings show that, irrespective of sex or transgene expression, ageing is associated with reduced inhibition of sympathetic vasoconstriction by sensory nerves concomitantly with desensitization of vascular α₁ARs and inward remodeling. These effects of ageing are manifest throughout MA networks controlling blood flow to the small intestine.

Methods

Animal care and use

All procedures were approved by the Institutional Animal Care and Use Committee of the University of Missouri and performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 8th edn, 2011). Experiments were performed on Young (3–6 months, 26–32 g; n= 16) and Old (24–26 months, 32–41 g; n= 12), Tg (RP24-25504-GCaMP2)1Mik mice (Tallini et al. 2007; Bagher et al. 2011a) bred on a C57BL/6J background in the University of Missouri animal facility. Although not used for Ca²⁺ imaging in the present study, transgenic mice positive for GCaMP2 (and littermates lacking this transgene) were obtained from our breeding colony to characterize the in vivo reactivity of MAs in this strain and thereby substantiate the physiological relevance of data they provide. Animals were genotyped (tail snip) at weaning. Males and females that were positive and negative for the GCaMP2 transgene were studied under identical conditions, with order randomized across transgene expression, age and sex. One MA arcade was studied per mouse.

Surgery and selection of arterial arcades

A mouse was anaesthetized with pentobarbital sodium (60 mg kg⁻¹, i.p. injection) and given supplemental administrations (15 mg kg⁻¹, i.p. injection) as needed throughout each experiment to maintain a stable plane of anaesthesia as confirmed by lack of withdrawal to tail or toe pinch. Hair was removed from the abdomen by shaving and the anaesthetized mouse was placed on a heated aluminium plate to maintain body temperature at 37°C. A midline laparotomy was performed to exteriorize a loop of jejunum with associated mesenteric vasculature. Exposed tissue was superfused continuously (∼5 ml min⁻¹) with bicarbonate-buffered physiological salt solution (PSS, in mm: 131.9 NaCl, 4.7 KCl, 2 CaCl₂, 1.17 MgSO₄, 18 NaHCO₃) equilibrated with 5% CO₂/95% N₂ (pH 7.4, 36°C). Arterial arcades chosen for study were standardized across experiments and typically contained first- (1A), second- (2A) and third- (3A) order MAs with each branch at least 500 μm long. The loop of intestine and vascular arcade were spread over a transparent pedestal (Sylgard 184; Dow Corning, Midland, MI, USA) and secured with pins placed through the edges of mesentery as far as possible from the arcade (Fig. 1). Great care was taken to avoid trauma to the intestine or the vascular supply. The intestine was covered with plastic wrap to prevent evaporation. To clearly visualize vessel edges for diameter measurements (and provide electrode access for perivascular nerve stimulation; described below), peri-arterial fat was carefully dissected away from the superficial aspect of a region (0.5–1 mm) along each branch of the MA arcade while viewing through a stereomicroscope (SMZ 645; Nikon, Melville, NY, USA). Consistent with greater body mass, the MAs of Old mice were surrounded by a greater amount of perivascular adipose than MAs of Young mice.

Figure 1. Illustration of intravital preparation of mesenteric arterial arcade.

A mouse was anaesthetized and a loop of jejunum was exteriorized over a transparent Sylgard pedestal then secured with pins near the edge of the intestine. The mesentery containing an arcade of first- (1A), second- (2A) and third-order (3A) mesenteric arteries was superfused continuously with PSS (pH 7.4, 36°C) with the effluent aspirated. Electrodes for perivascular nerve stimulation were positioned at the proximal end of each branch (as shown for 1A).

Intravital microscopy

The completed preparation was moved to the stage of an intravital microscope based upon an Olympus MVX10 Stereo Zoom platform (Center Valley, PA, USA) and allowed to equilibrate for 30 min prior to starting experiments. Superfusion at 36°C was maintained from a temperature-controlled (SW-60; Warner Instruments; Hamden, CT, USA) 50 ml reservoir (for drug delivery) fed continuously by a supply of control PSS. Images were acquired through an Olympus MV PLAPO 1XC objective using a CCD camera (Hamamatsu, Tokyo, Japan) and visualized on a digital video monitor (Sony Corp., Tokyo, Japan) at a total magnification of 220×. Vessel inner diameters were measured as the width of the red blood cell column using a video calliper (Microcirculation Research Institute, College Station, TX, USA) calibrated to a stage micrometer, with spatial resolution of ∼2 μm. Output from the calliper was recorded at 40 Hz using a PowerLab/400 system (AD Instruments, Colorado Springs, CO, USA).

In vivo pharmacology

Concentration–response experiments were cumulative with appropriate volumes of concentrated drug solutions added to the 50 ml reservoir of superfusion solution. Drug concentrations are given as those to which vessels were exposed. For selective activation of α₁ARs with phenylephrine (PE) or of α₂ARs with UK14304, each concentration was superfused for at least 5 min until stable diameters were recorded for each MA branch and then the next highest concentration was added. After the final concentration, the preparation was superfused with control PSS until baseline diameters recovered (∼20–30 min). The other agonist was then studied in the same fashion, with the order of agonists varied across experiments.

To confirm specificity of α₁AR vs.α₂AR activation, control experiments were performed in Young mice using the sympathetic neurotransmitter NA (10⁻⁹–10⁻⁵ m) in the presence of the βAR antagonist propranolol (10⁻⁷ m) during selective inhibition of α₁ARs or of α₂ARs (Moore et al. 2010). Thus, after responses to NA were evaluated either prazosin (10⁻⁸ m, an α₁AR antagonist) or rauwolscine (10⁻⁷ m, an α₂AR antagonist) was superfused for at least 15 min and was maintained while responses to NA were re-evaluated. The preparation was then superfused with control PSS for ∼20–30 min to wash out the first antagonist, the second antagonist was equilibrated for at least 15 min and cumulative responses to NA were re-evaluated a final time. The order of respective antagonists varied across experiments. At the end of each experiment, sodium nitroprusside (SNP, 10⁻⁵ m) was added to obtain maximal MA diameters.

Perivascular nerve stimulation (PNS)

Perivascular nerves were stimulated in a localized electrical field between two fine wire electrodes (90% platinum/10% iridium; diameter, 250 μm) connected to a stimulation isolation unit (SIU5; Grass, Quincy, MA, USA) driven by a square wave stimulator (S48; Grass). The tip of an electrode was positioned on either side of the MA of interest near its proximal end and diameters were measured at least 500 μm distal to the stimulus site. Stimulus pulses (15 V, 2 ms) were delivered at 1, 2, 4, 8 and 16 Hz until a stable response diameter was achieved (10–15 s). Vessels were allowed to return to their baseline diameters prior the next stimulus. The order of stimulation frequencies was randomized across experiments, as was the order in which the respective MA branches were studied. To evaluate the contribution of sensory nerves, PNS was repeated during superfusion with their inhibitor, capsaicin (10 μm). Separate control experiments were performed in Young mice to evaluate non-specific effects of electrical field stimulation by inhibiting action potentials with tetrodotoxin (1 μm) added to the superfusion solution.

At the end of each experiment the anaesthetized mouse was killed via an overdose of pentobarbital (intracardiac injection) followed by cervical dislocation.

Chemicals and reagents

All drugs were obtained from Sigma-Aldrich (St Louis, MO, USA) and prepared fresh for each day's experiment. Water-insoluble drugs were first dissolved in dimethyl sulphoxide then diluted to their final concentrations in PSS with ≤0.1% dimethyl sulphoxide.

Data presentation and statistical analysis

Data for agonist concentration–response and PNS frequency–response curves are presented as percentage constriction from baseline diameter (with 100% corresponding to lumen closure) calculated as follows: % constriction =[(D_base–D_resp)/D_base]× 100), where D_base= baseline diameter and D_resp= response diameter in the presence of a given agonist concentration or PNS frequency. Measured (internal) diameters of all vessels studied are given for respective age groups in Table 1 (pooled across sex and transgene expression; Table S1 contains values for each sex and genotype). Spontaneous vasomotor tone at rest was calculated as the percentage difference between baseline diameter during superfusion with control PSS and maximal diameter (D_max) during superfusion with SNP (10⁻⁵ m); thus, % tone =[(D_max–D_base)/D_max]× 100. Data were analysed using Student's t tests or analysis of variance, with repeated measures when appropriate (Prism 5, GraphPad Software Inc., La Jolla, CA, USA). When significant F-ratios were obtained with analysis of variance, post hoc comparisons were made using Bonferroni tests. Summary data are expressed as mean values ± SE. Differences were considered statistically significant at P≤ 0.05.

Table 1.

Diameters of mouse mesenteric arteries in vivo

	1A		2A		3A
	Young	Old	Young	Old	Young	Old
Baseline diameter (μm)	190 ± 9	183 ± 9	149 ± 12	121 ± 6*	111 ± 12	69 ± 4*
Maximum diameter (μm)	204 ± 9	193 ± 10*	166 ± 13	131 ± 8*	123 ± 12†	76 ± 4*
Vasomotor tone (%)	7 ± 1	5 ± 1	10 ± 1	7 ± 1	11 ± 1	8 ± 1

Summary data are means ± SE for first- (1A), second- (2A) and third-order (3A) mesenteric arteries. Data are compiled from all mice included in this study (n= 12–16 per age group). Baseline diameters (D_base) were measured during superfusion with control PSS. Maximum diameters (D_max) were measured during superfusion of PSS containing sodium nitroprusside (SNP, 10 μm). Calculated vasomotor tone (%) =[(D_max–D_base)/D_max]× 100. Diameters of respective branch orders were significantly different from each other within each age group (P < 0.05). *P≤ 0.05 versus Young; †P < 0.05, D_max different from D_base.

Results

Diameters and resting tone of MAs in Young and Old mice

Resting baseline and maximum diameters are listed in Table 1. In Young and Old mice, diameters decreased as branch order increased (P < 0.05). Spontaneous resting tone was typically ≤10% with no significant difference between branch orders or age group. Across sex and GCaMP2 expression, resting and maximal diameters of MA in Old mice were ∼5% (1A), 20% (2A) and 40% (3A) smaller vs. Young mice (P≤ 0.05). Thus, the magnitude of diameter difference between age groups increased with vessel branch order (Table 1).

Effect of ageing on vasomotor responses to PNS

To determine their role in vasomotor control of MA networks in vivo, perivascular nerves were stimulated using local electrical field stimulation. In Young mice, PNS constricted 1A, 2A and 3A MAs in a frequency-dependent manner, with respective maximum responses of 34 ± 4, 47 ± 5 and 52 ± 4% in Young (Fig. 2A–C) and 59 ± 2, 54 ± 3 and 58 ± 3% in Old mice (Fig. 2D–F). Constriction was significantly greater in 1A from Old vs. Young mice (P≤ 0.05), but not in 2A or 3A branches (n= 4–6). Repeating PNS in the presence of capsaicin (10 μm) significantly increased constrictions of 1A, 2A and 3A MAs in Young mice with maximum responses of 55 ± 2, 68 ± 5 and 69 ± 4%, respectively (Fig. 2A-C, n= 4, P≤ 0.05 vs. control). In contrast, capsaicin did not significantly affect MA constrictions to PNS in Old animals (Fig. 2D–F, n= 6, P≤ 0.05), nor did it affect MA diameters at rest in either age group. In separate control experiments performed in Young mice, exposure to the voltage-gated sodium channel blocker tetrodotoxin (1 μm) abolished vasoconstrictions to PNS (Fig. S1), thereby excluding non-specific effects of electrical field stimulation (e.g. direct activation of smooth muscle cells).

Figure 2. Sensory nerves attenuate sympathetic vasoconstriction in Young but not Old mice.

A–F, summary data are mean values ± SE for percentage constrictions of first- (A and D), second- (B and E) and third-order (C and F) mesenteric arteries of Young and Old mice in response to electrical field stimulations (see Fig. 1) of 1, 2, 4, 8 and 16 Hz before (filled circles) and following (open circles) treatment with the sensory nerve inhibitor capsaicin (10 μm). *P < 0.05 vs. capsaicin; n= 4–6 mice per age group pooled across sex and GCaMP2 expression.

Relative contributions of α₁ARs and α₂ARs to vasoconstriction in Young and Old mice

To study smooth muscle relaxation and vasodilation, MAs studied in vitro are often preconstricted with PE (an α₁AR agonist) (Arenas et al. 2006; Liu et al. 2006; Zhang et al. 2007). However, the sympathetic neurotransmitter NA can evoke vasoconstriction through α₁ARs and α₂ARs and the role of respective αAR subtypes in mediating MA constriction in vivo is unknown. To define these relationships and determine if they were affected by ageing, we evaluated the actions of selective adrenergic agonists. In Young mice, cumulative superfusion of PE (10⁻⁹–10⁻⁵ m) caused maximum constrictions of 51 ± 5, 55 ± 4 and 47 ± 4% in 1A, 2A and 3A with respective pEC₅₀ values of 6.69 ± 0.08, 6.75 ± 0.13 and 6.80 ± 0.09 (Fig. 3A–C). In Old mice, maximum constrictions to PE in 1A, 2A and 3A were similar to Young mice (51 ± 3, 47 ± 3 and 54 ± 2%, respectively) although response curves were shifted significantly (P < 0.05) to the right with pEC₅₀ values of 5.87 ± 0.20, 5.95 ± 0.11 and 5.92 ± 0.19 in 1A, 2A and 3A, respectively (Fig. 3A–C). Responses to UK14304 (10⁻⁹–10⁻⁵ m) revealed a minor role for α₂ARs with constrictions in 1A, 2A and 3A MA of Young (14 ± 4, 14 ± 5 and 17 ± 3%) and Old mice (14 ± 2, 15 ± 2 and 16 ± 2%) that were <30% of those obtained with PE (P≤ 0.05 across branch orders) with no differences in pEC₅₀ values between age groups (Fig. 3D–F).

Figure 3. α₁ARs dominate adrenergic vasoconstriction with diminished sensitivity in Old mice.

A–F, summary data are mean values ± SE for percentage constrictions of first- (A and D), second- (B and E) and third-order (C and F) MAs of Young (filled circles) and Old (open circles) mice in response to cumulative concentrations of the α₁AR agonist phenylephrine (PE, 10⁻⁹–10⁻⁵ m) or to the α₂AR agonist UK14304 (UK, 10⁻⁹–10⁻⁵ m). *P < 0.05 compared to Old mice; n= 4–6 mice per age group pooled across sex and GCaMP2 expression.

When evaluating specificity of α₁AR vs.α₂AR activation, the βAR antagonist propranolol had no significant effect on MA diameters. NA caused similar concentration-dependent constrictions in all MAs, with maximum responses of 52 ± 4, 53 ± 2 and 52 ± 5% and pEC₅₀ values of 6.54 ± 0.09, 6.65 ± 0.07 and 6.57 ± 0.14 in 1A, 2A and 3A, respectively (Fig. 4A-C). Inhibiting α₁ARs with prazosin reduced respective maximum NA constrictions to 13 ± 3, 11 ± 1 and 9 ± 3% (Fig. 4A–C, P≤ 0.05). In contrast, inhibiting α₂ARs with rauwolscine produced a relatively modest inhibition, decreasing maximum constrictions to 38 ± 2% in 1A and 2A (Fig. 4A and B, P≤ 0.05) and 39 ± 6% in 3A (Fig. 4C P≥ 0.05). Thus, noradrenergic constriction of MAs in vivo is mediated primarily by α₁ARs.

Figure 4. Mesenteric artery constrictions to NA are affected predominantly by α₁ARs.

A–C, summary data are mean values ± SE for percentage constrictions of first- (A), second- (B) and third-order (C) MAs of Young mice in response to cumulative increases in noradrenaline concentration (NA, 10⁻⁹–10⁻⁵ m; upper curves) in the presence and absence of the α₁AR antagonist prazosin (10⁻⁸ m; lower curves) or the α₂AR antagonist rauwolscine (10⁻⁷ m; intermediate curves). All experiments were performed in the presence of the βAR antagonist propranolol (10⁻⁷ m). *P < 0.05 vs. NA alone (Control); ^ΦP < 0.05 vs. NA + rauwolscine; n= 4 pooled across sex and GCaMP2 expression.

Lack of effect of sex or GCaMP2 expression on MAs in vivo

The use of Cx40^BAC-GCaMP2 transgenic mice to study endothelial cell Ca²⁺ signalling in MAs (Ledoux et al. 2008; Nausch et al. 2012; Sonkusare et al. 2012) and arterioles (Tallini et al. 2007; Bagher et al. 2011b) has proceeded using male animals without determining whether expression of the transgene or sex influence vascular reactivity. Thus, an underlying goal of this study was to evaluate whether sex or the expression of GCaMP2 impact vasomotor responses in vivo. Across experiments, there were no trends for differences in resting or maximal diameters (Table S1) or their responsiveness to treatments between male and female mice or between GCaMP2-positive and GCaMP2-negative mice. For example, in light of the predominant role of α₁ARs in mediating MA constriction, the consistency across sex and GCaMP2 expression within age groups for concentration–response relationships to PE for individual Young and Old mice are illustrated in Figs 5 and 6, respectively. Thus, all summary data presented for respective age groups in this study include both male and female mice that were positive or negative for GCaMP2 expression.

Figure 5. Vasoconstriction to PE is independent of sex or GCaMP2 expression in Young mice.

A–F, data are diameter responses (% constriction) to respective concentrations of PE (10⁻⁹–10⁻⁵ m). Scatterplots depict vessels from individual mice, segregated into males vs. females (A–C) and GCaMP2-positive vs. GCaMP2-negative (D–F) in first- (A and D), second- (B and D) and third-order (C and F) MAs. Representative error bars depict ± SE for each group within a panel; n= 4–6 per group. Summary data for these individual observations are presented in Fig. 3A–C (filled circles).

Figure 6. Vasoconstriction to PE is independent of sex or GCaMP2 expression in Old mice.

A–F, data are diameter responses (% constriction) to respective concentrations of PE (10⁻⁹–10⁻⁵ m). Scatterplots depict vessels from individual mice used for data collection, segregated into males vs. females (A–C) and GCaMP2-positive vs. GCaMP2-negative (D–F) in first- (A and D), second- (B and D) and third-order (C and F) MAs. Representative error bars depict ± SE for each group within a panel; n= 4–6 per group. Summary data for these individual observations are presented in Fig. 3A–C (open circles).

Discussion

We have investigated the functional roles of perivascular sympathetic and sensory nerves in MA arcades of anaesthetized mice in vivo. The present data illustrate significant changes that occur with ageing irrespective of sex or the expression of the GCaMP2 transgene. Key physiological findings are that perivascular sensory nerves effectively limit sympathetic vasoconstriction in Young mice and that this ability is lost with ageing, resulting in greater vasoconstriction in response to PNS in MAs of Old mice. The efficacy of α₁ARs predominated over that of α₂ARs in mediating adrenergic vasoconstriction in each vessel branch order, although the sensitivity of α₁AR-mediated responses decreased significantly with ageing in all MA branch orders. Nevertheless, MAs of Old mice were ∼5% (1A) to 40% (3A) smaller in diameter than those of Young mice, thereby imposing a structural limitation to splanchnic blood flow with ageing.

Diameters and resting tone in MAs of Young and Old mice

Across branch orders, MAs of Old mice had smaller resting and maximal diameters than Young mice with no change in myogenic tone (Table 1). These reductions in MA diameters with ageing imply diminished blood flow to the splanchnic circulation. Consistent with reduced tissue perfusion, arterial and arteriolar rarefaction with ageing has been reported in skeletal muscle (Behnke et al. 2006; Faber et al. 2011), brain (Sonntag et al. 1997; Faber et al. 2011), kidneys (Urbieta-Caceres et al. 2012) and the retina (Azemin et al. 2012). Such loss of supply vessels increases blood pressure along with flow and shear stress within prevailing vessels and can thereby stimulate outward remodelling with an increase in diameter (Behnke et al. 2006; Izzo & Mitchell, 2007). Thus, while changes in arterial diameter with ageing are common, they have more often been associated with increased vessel size (Muller-Delp et al. 2002; Behnke et al. 2006; Dumont et al. 2008; Hausman et al. 2012). Remarkably, despite MAs being a widespread model of resistance vessels, the effect of ageing on MA diameter had not been defined. Our present findings indicate that inward rather than outward remodelling with ageing occurs in MAs and imply that age-related changes in MA structure and function can differ from other vascular beds.

The MAs studied here differ from resistance arteries of skeletal muscle (Welsh & Segal, 1996), heart (Chilian et al. 1986) or brain (Heistad et al. 1978) studied in vivo in that they lacked the substantive (i.e. >20%) spontaneous myogenic tone observed in other resistance arteries. While spontaneous tone observed here in vivo was typically <10%, whether MAs isolated for in vitro studies develop tone spontaneously is controversial. When isolated and pressurized, MAs of young rats characteristically did not develop tone (Osol et al. 1991; Thorsgaard et al. 2003; Bergaya et al. 2004; Jackson-Weaver et al. 2011; Sweazea & Walker, 2012). Such lack of smooth muscle activation at rest explains why investigators routinely induce vasoconstriction pharmacologically in MAs used to study mechanisms of vasodilatation. The present findings illustrate that α₁AR activation with PE is ideally suited for this purpose (Fig. 3). However, others have reported ≥20% myogenic tone in MA branches (Dubroca et al. 2007; Koltsova et al. 2009; Zhang et al. 2010; Haddock et al. 2011; Khurana et al. 2012). While the basis of such differences remains unclear, constricting MAs through activation of sympathetic nerves provides a mechanism for effectively redistributing cardiac output away from the gut to augment blood flow to other vascular beds, for example to active skeletal muscle during exercise.

Diminished influence of sensory nerves on sympathetic vasoconstriction in Old vs. Young mice

Our finding that capsaicin increased vasoconstriction during PNS in Young mice (Fig. 2A–C) is consistent with inhibiting the vasodilator actions of perivascular sensory nerves (Bevan & Brayden, 1987; Kawasaki et al. 1988; Kawasaki, 2002). Independent of the endothelium, the release of CGRP from sensory nerve terminals can evoke vasodilatation through activation of CGRP receptors on vascular smooth muscle cells, thereby stimulating protein kinase A to reduce [Ca²⁺]_i and promote relaxation (Drake et al. 2000; Brain & Grant, 2004). The vasodilator actions of CGRP may be enhanced through it acting presynaptically on sympathetic nerve varicosities to inhibit NA release (Kawasaki et al. 1990b; Takenaga & Kawasaki, 1999). In a reciprocal manner, release of CGRP can be inhibited by NA acting on presynaptic α₂ARs of sensory nerves (Kawasaki et al. 1990a). The integrated effect entails reciprocal interaction between perivascular sympathetic and sensory nerves in governing vasomotor activity of MAs (Kawasaki et al. 1990b; Kawasaki, 2002).

A key finding in the present study is the age-related differences in the neural regulation of vasomotor responses in MAs. In Old mice, inhibiting sensory nerves with capsaicin had no significant effect on PNS-induced constrictions (Fig. 2D–F), suggesting that this mechanism of attenuating sympathetic vasoconstriction becomes dysfunctional during ageing. From a clinical perspective, restoring sensory nerve function that is lost with ageing may be of functional significance, as CGRP preconditions vascular endothelial cells against ischaemic injury (Li et al. 2000). Furthermore, a decline in function of CGRP-containing nerves is linked to several peripheral vascular complications including slow wound healing in diabetics and individuals with Raynaud's syndrome (Brain & Grant, 2004). Loss of CGRP release is also associated with headache and increased vasospasm in cerebral arteries (Edvinsson, 2002). Thus, loss of CGRP actions with ageing may well contribute to the aetiology of generalized vascular dysfunction.

The lack of effect of capsaicin in MAs of Old mice may result from an age-related decrease in CGRP release. In the perfused mesentery of spontaneously hypertensive (but not in wild-type) rats, a decrease in vasodilatation was found in MAs of 30- vs. 8-week-old animals in conjunction with diminished CGRP release during nerve stimulation (Kawasaki et al. 1990c; Kawasaki & Takasaki, 1992). While these observations provide insight into factors contributing to hypertension, other mechanisms probably contribute to vascular dysfunction during ageing in normotensive individuals. Furthermore, increases in sympathetic nerve activity with ageing (Seals & Dinenno, 2004; Dinenno & Joyner, 2006) may reduce the influence of sensory nerves. Alternatively, enhanced sympathetic nerve activity may compensate for reduced sensitivity of vasomotor responsiveness to α₁AR activation (Fig. 3).

Age-related increases in sympathetic nerve activity have been most clearly documented in skeletal muscle (Seals & Dinenno, 2004; Dinenno & Joyner, 2006). However, changes in the responsiveness of the vasculature to α-adrenergic stimulation are controversial and probably vary with the vascular bed, vessel diameter and branch order (Dinenno & Joyner, 2006; Muller-Delp, 2006). Remarkably, how ageing may influence adrenergic regulation of MAs has not been determined. Therefore, we developed the intravital MA preparation presented here to characterize the role of α₁ARs and α₂ARs in MAs of Old versus Young mice. We found that activation of α₁ARs with PE evoked the major portion of sympathetic vasoconstriction in 1A–3A MAs (Fig. 3). As confirmed in Young mice, inhibition of α₁ARs with prazosin nearly abolished constriction to NA (Fig. 4). The activation of α₁ARs in Old mice was able to produce maximum constrictions similar to those in Young mice but with significantly decreased sensitivity (Fig. 3). In contrast, α₂ARs do not appear to play a major role in vasomotor control in MAs of either Young or Old mice (Figs 3 and 4).

The expression of α₁AR subtype mRNA declined with aging in the aorta and renal arteries of 3-, 12- and 24-month-old rats, but such changes did not occur in MAs of the same animals (Xu et al. 1997). Thus, changes in receptor expression with ageing can vary between vascular beds. While changes in transcript expression need not correlate with changes in protein expression, post-translational modification of receptors or changes in downstream signalling intermediates may alter the apparent sensitivity of receptors irrespective of their expression level. It is also possible that loss of receptors to sensory neurotransmitters could explain the loss of attenuation for sympathetic vasoconstriction in Old mice (Fig. 2), as could a diminished release of CGRP. Thus, enhanced NA release in Old mice (with loss of sensory modulation) may lead to the desensitization of α₁ARs apparent in Old when compared to Young mice (Fig. 3). In turn, the present data suggest that age-related increases in sympathetic nerve activity (Seals & Dinenno, 2004; Dinenno & Joyner, 2006) may compensate for diminished α₁AR responsiveness in MAs. At the same time, the substantial reduction of MA diameters in Old vs. Young mice (Table 1) results in a physical limitation to splanchnic blood flow irrespective of vasoactive stimuli. When coupled with enhanced sympathetic nerve activity, a structural increase in vascular resistance may well contribute to the elevation in blood pressure shown to accompany ageing in C57BL/6 mice (Gros et al. 2002).

Changes in the density of innervation with ageing could account for some of the functional differences reported here, as sympathetic nerve density within an age group has been correlated with the magnitude of sympathetic vasoconstriction (Furness & Marshall, 1974; Marshall, 1982). Nevertheless, previous studies suggest this is probably not the case with ageing vasculature. For example, no differences were found in the density of perivascular sympathetic nerves of mesenteric, femoral, gracilis and carotid arteries between mice 3 and 20 months of age (Long & Segal, 2009). In basal cerebral arteries of humans, although perivascular nerve density did not change with ageing (62–85 years of age), relative staining for sympathetic versus sensory nerves was not evaluated (Bleys et al. 1996). In MAs and carotid arteries of guinea pigs, adrenergic and peptidergic (CGRP) nerve densities were maintained from 4 weeks to 2 years of age (Dhall et al. 1986). These findings are consistent with maintaining the density of CGRP-containing nerves with ageing in rat MAs (Hobara et al. 2010). However, in renal and femoral arteries of guinea pigs, while noradrenergic and peptidergic nerve densities decreased in Old animals, MAs from all ages had the highest density of both noradrenergic and peptidergic nerves (Dhall et al. 1986). Collectively, previous findings indicate that the densities of perivascular sympathetic and sensory nerve fibres of MAs are preserved with ageing.

Sex, neuroeffector signalling and transgene expression

There was no discernible effect of sex on the diameter, myogenic tone or vasomotor function in 1A–3A MAs (Table S1; Figs 5 and 6). Our findings are thus consistent with studies of rat cerebral arteries (Aukes et al. 2008), which found no differences between males and females in vasomotor responses to CGRP or to NA. Posterior cerebral arteries from female rats had a significantly higher density of perivascular nerves containing CGRP than male rats with no difference between sexes in nerves containing tyrosine hydroxylase (a marker of sympathetic nerves) (Aukes et al. 2008). Vasomotor responses to PNS were not different between sexes, suggesting that nerve density and vasomotor function may not be directly linked in cerebral arteries. In isolated preparations of rat mesentery, sex-based differences were apparent in constriction of MAs to adrenergic nerve stimulation but not to exogenous NA (Li & Duckles, 1994). Nor were there sex-related differences in vasodilatation during CGRP-ergic nerve stimulation (Li & Duckles, 1994). In light of the limited data in the literature, our studies of mouse MAs indicate that sex is unlikely to impact the density of either sympathetic or sensory nerves or the vasomotor responses that they elicit upon stimulation. This conclusion does not exclude the likelihood of sex-based differences in vasomotor control in other vascular beds (e.g. of reproductive organs) or in other species.

The transgenic (Cx40^BAC-GCaMP2) mice studied here serve as a valuable model for studying calcium signalling in the endothelium of resistance vessels (Tallini et al. 2007; Ledoux et al. 2008; Bagher et al. 2011a; Nausch et al. 2012; Sonkusare et al. 2012). Thus, it is essential to determine whether data generated in these mice are physiologically homologous across sex and genotype. All studies of these mice published thus far have utilized only Young males and there have been no comprehensive analyses of whether the expression of GCaMP2 may influence vasomotor control. To provide such insight, we studied both males and females that were bred and raised under identical conditions to determine whether there were trends in our data associated with differences in sex or GCaMP2 expression. Consistent with the lack of sex differences, the present data indicate that neither the function of MAs nor the effects of ageing are influenced by the expression of GCaMP2 in endothelial cells (Figs 5 & 6). Thus, GCaMP2 may be considered an effective tracer molecule that functions as a calcium sensor without altering vascular reactivity or vasomotor control. These findings are also relevant economically and with respect to the responsible use of animals for research: if only male GCaMP2-positive mice were used, approximately 75% of the resources expended in maintaining a breeding colony would be lost to killing of the GCaMP2-negative and female animals. Thus, the ability to study both sexes (irrespective of GCaMP2 expression) optimizes total costs and numbers of animals bred and used.

Summary and conclusion

The present findings support the hypothesis that ageing alters the function of perivascular sympathetic and sensory nerves of MAs in vivo irrespective of sex or the expression of a Ca²⁺ sensitive transgene in the endothelium. While sensory nerves effectively limit sympathetic vasoconstriction of MAs during PNS in Young mice, this modulation is lost in Old mice. Sympathetic vasoconstriction is mediated predominantly by α₁ARs in MAs of both Young and Old mice. However, α₁ARs are desensitized in Old mice across MA branch orders, suggesting that ageing affected adrenergic reactivity of all MAs similarly. The smaller diameters throughout MA arcades of Old mice impose a structural limitation to splanchnic blood flow. The diminished sensitivity of MAs to α₁AR stimulation in Old mice would impair the ability to constrict and mobilize blood from the splanchnic circulation, for example to support the energetic demands of contracting skeletal muscle during physical activity. At the same time, this limitation in α₁AR-mediated vasoconstriction is offset through loss of the ability of sensory nerves to attenuate sympathetic vasoconstriction. With advancing age, such changes to the splanchnic circulation may compromise the quality of life and contribute to vascular disease.

Glossary

1A, 2A, 3A

first, second and third order

ARs

adrenoreceptors

CGRP

calcitonin gene-related peptide

Cx40

connexin40

MAs

mesenteric arteries

NA

noradrenaline

pEC50

negative logarithm of agonist concentration evoking half-maximal response

PNS

perivascular nerve stimulation

PE

phenylephrine

PSS

physiological salt solution

SNP

sodium nitroprusside

Author contributions

Conception and design of the experiments: E.B.W. and S.S.S.; data collection: E.B.W; analysis and interpretation of data: E.B.W. and S.S.S.; drafting and revising the article for content: E.B.W. and S.S.S.

Supplementary material

Supplementry Material