Ageing alters perivascular nerve function of mouse mesenteric arteries in vivo

J Physiol 591.5 (2013) pp 1251–1263 1251

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Ageing alters perivascular nerve function of mousemesenteric arteries in vivo

Erika B. Westcott1 and Steven S. Segal1,2

1Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO 65212, USA2Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO 65211, USA

Key points

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

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

• Ageing decreased the diameter of MAs, reduced their sensitivity to α1-adrenoreceptorstimulation and impaired the ability of sensory nerves to attenuate sympatheticvasoconstriction.

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

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

Abstract Mesenteric arteries (MAs) are studied widely in vitro but little is known of theirreactivity in vivo. Transgenic animals have enabled Ca2+ signalling to be studied in isolatedMAs but the reactivity of these vessels in vivo is undefined. We tested the hypothesis thatageing alters MA reactivity to perivascular nerve stimulation (PNS) and adrenoreceptor (AR)activation during blood flow control. First- (1A), second- (2A) and third-order (3A) MAsof pentobarbital-anaesthetized Young (3–6 months) and Old (24–26 months) male and femaleCx40BAC-GCaMP2 transgenic mice (C57BL/6 background; positive or negative for the GCaMP2transgene) were studied with intravital microscopy. A segment of jejunum was exteriorized andan MA network was superfused with physiological salt solution (pH 7.4, 37◦C). Resting tonewas ≤ 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 PNSfrequency (1–16 Hz) but was ∼20% less in Young vs. Old mice (P ≤ 0.05) and was inhibited bytetrodotoxin (1 μM). Capsaicin (10 μM; to inhibit sensory nerves) enhanced MA constriction toPNS (P ≤ 0.05) by ∼20% in Young but not Old mice. Phenylephrine (an α1AR agonist) potencywas greater in Young mice (P ≤ 0.05) with similar efficacy (∼60% constriction) across ages andMA branches. Constrictions to UK14304 (an α2AR agonist) were less (∼20%; P ≤ 0.05) and wereunaffected by ageing. Irrespective of sex or transgene expression, ageing consistently reducedthe sensitivity of MAs to α1AR vasoconstriction while blunting the attenuation of sympatheticvasoconstriction by sensory nerves. These findings imply substantive alterations in splanchnicblood flow control with ageing.

C© 2013 The Authors. The Journal of Physiology C© 2013 The Physiological Society DOI: 10.1113/jphysiol.2012.244483

1252 E. B. Westcott and S. S. Segal J Physiol 591.5

(Received 4 September 2012; accepted after revision 10 December 2012; first published online 17 December 2012)Corresponding author S. S. Segal: Department of Medical Pharmacology and Physiology, MA415 Medical SciencesBuilding – 1 Hospital Drive, University of Missouri, Columbia, MO 65212. Email: [email protected]

Abbreviations 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 concentrationevoking half-maximal response; PNS, perivascular nerve stimulation; PE, phenylephrine; PSS, physiological saltsolution; SNP, sodium nitroprusside.

Introduction

Ageing is associated with physiological changesthroughout the body, with vascular age serving as a betterpredictor of cardiovascular disease than chronological age(Barodka et al. 2011). With ageing, large conduit arteriesstiffen and lose endothelial function (Lakatta & Levy, 2003;Seals et al. 2011), resulting in increased frailty (Newmanet al. 2001) with diminished capacity for physical exercise(Heckman & McKelvie, 2008). Preserving exercise capacityis an important goal for the ageing population as physicalactivity can ameliorate vascular ageing through reducingarterial stiffness and improving endothelium-dependentdilatation (Koch et al. 2005; Heckman & McKelvie, 2008;Seals et al. 2011). In contrast to such documented effectsin systemic arteries, the effect of ageing on the smallerresistance arteries and arterioles is less clear. Nevertheless,evidence suggests that resistance vessels also undergostiffening with impaired function (Laurant et al. 2004;Dumont et al. 2008) along with remodelling of vascularnetworks (Bearden, 2006; Behnke et al. 2006) that mayadversely impact tissue perfusion throughout the body.

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

Little is known of age-related changes in the regulationof 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 ofthe body is integral to maintaining exercise capacity andcardiovascular 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 ageingthroughout the body (Ng et al. 1993; Dinenno et al. 2000;Seals & Dinenno, 2004) and the mesenteric vasculature isrichly innervated (Furness & Marshall, 1974; Long & Segal,2009; Haddock & Hill, 2011), the mesenteric circulationis a likely target for age-related functional defects in vaso-motor control. To investigate this relationship, a secondgoal of this study was to test the hypotheses that ageingalters the function of perivascular nerves during vaso-motor control in vivo.

Experiments were performed using Cx40BAC-GCaMP2transgenic mice. In these animals, expression of GCaMP2(a green fluorescent protein-based Ca2+ indicator) isunder the control of the connexin40 (Cx40) promoter(Tallini et al. 2007). Because Cx40 expression in thevasculature is restricted to endothelial cells of arteriesand arterioles, GCaMP2 is expressed accordingly (Talliniet 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 acceptancefor studying Ca2+ signalling in isolated MA preparations(Ledoux et al. 2008; Nausch et al. 2012; Sonkusareet al. 2012) Because previous studies of these mice havebeen performed exclusively using males, it has not beendetermined whether sex differences or the presence ofGCaMP2 may impact the vascular physiology of animalsexpressing this transgene. Therefore, a third goal of thisstudy was to evaluate males and females as well as micethat were either positive or negative for the GCaMP2transgene to further validate this important transgenicmodel. Our findings show that, irrespective of sex ortransgene expression, ageing is associated with reducedinhibition of sympathetic vasoconstriction by sensorynerves concomitantly with desensitization of vascularα1ARs and inward remodeling. These effects of ageingare manifest throughout MA networks controlling bloodflow to the small intestine.

Methods

Animal care and use

All procedures were approved by the Institutional AnimalCare and Use Committee of the University of Missouri andperformed in accordance with the Guide for the Care and

C© 2013 The Authors. The Journal of Physiology C© 2013 The Physiological Society

J Physiol 591.5 Ageing and perivascular nerves in vivo 1253

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/6Jbackground in the University of Missouri animal facility.Although not used for Ca2+ imaging in the present study,transgenic mice positive for GCaMP2 (and littermateslacking this transgene) were obtained from our breedingcolony to characterize the in vivo reactivity of MAs in thisstrain and thereby substantiate the physiological relevanceof data they provide. Animals were genotyped (tail snip)at weaning. Males and females that were positive andnegative for the GCaMP2 transgene were studied underidentical conditions, with order randomized across trans-gene expression, age and sex. One MA arcade was studiedper mouse.

Surgery and selection of arterial arcades

A mouse was anaesthetized with pentobarbital sodium(60 mg kg−1, I.P. injection) and given supplementaladministrations (15 mg kg−1, I.P. injection) as neededthroughout each experiment to maintain a stable plane ofanaesthesia as confirmed by lack of withdrawal to tail ortoe pinch. Hair was removed from the abdomen by shavingand the anaesthetized mouse was placed on a heatedaluminium plate to maintain body temperature at 37◦C. Amidline laparotomy was performed to exteriorize a loop ofjejunum with associated mesenteric vasculature. Exposedtissue was superfused continuously (∼5 ml min−1) withbicarbonate-buffered physiological salt solution (PSS, inmM: 131.9 NaCl, 4.7 KCl, 2 CaCl2, 1.17 MgSO4, 18NaHCO3) equilibrated with 5% CO2/95% N2 (pH 7.4,36◦C). Arterial arcades chosen for study were standardizedacross experiments and typically contained first- (1A),second- (2A) and third- (3A) order MAs with each branchat least 500 μm long. The loop of intestine and vasculararcade were spread over a transparent pedestal (Sylgard184; Dow Corning, Midland, MI, USA) and secured withpins placed through the edges of mesentery as far aspossible from the arcade (Fig. 1). Great care was takento avoid trauma to the intestine or the vascular supply.The intestine was covered with plastic wrap to preventevaporation. To clearly visualize vessel edges for diametermeasurements (and provide electrode access for peri-vascular nerve stimulation; described below), peri-arterialfat was carefully dissected away from the superficial aspectof a region (0.5–1 mm) along each branch of the MAarcade while viewing through a stereomicroscope (SMZ645; Nikon, Melville, NY, USA). Consistent with greaterbody mass, the MAs of Old mice were surrounded by agreater amount of perivascular adipose than MAs of Youngmice.

Intravital microscopy

The completed preparation was moved to the stageof an intravital microscope based upon an OlympusMVX10 Stereo Zoom platform (Center Valley, PA, USA)and allowed to equilibrate for 30 min prior to startingexperiments. Superfusion at 36◦C was maintained froma temperature-controlled (SW-60; Warner Instruments;Hamden, CT, USA) 50 ml reservoir (for drug delivery)fed continuously by a supply of control PSS. Imageswere acquired through an Olympus MV PLAPO 1XCobjective using a CCD camera (Hamamatsu, Tokyo, Japan)and visualized on a digital video monitor (Sony Corp.,Tokyo, Japan) at a total magnification of 220×. Vesselinner diameters were measured as the width of thered blood cell column using a video calliper (Micro-circulation 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 Hzusing a PowerLab/400 system (AD Instruments, ColoradoSprings, CO, USA).

In vivo pharmacology

Concentration–response experiments were cumulativewith appropriate volumes of concentrated drug solutionsadded to the 50 ml reservoir of superfusion solution.Drug concentrations are given as those to which vesselswere exposed. For selective activation of α1ARs withphenylephrine (PE) or of α2ARs with UK14304, eachconcentration was superfused for at least 5 min untilstable diameters were recorded for each MA branchand then the next highest concentration was added.After the final concentration, the preparation was super-fused with control PSS until baseline diameters recovered(∼20–30 min). The other agonist was then studied in thesame fashion, with the order of agonists varied acrossexperiments.

To confirm specificity of α1AR vs. α2AR activation,control experiments were performed in Young mice usingthe 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 wereevaluated either prazosin (10−8 M, an α1AR antagonist) orrauwolscine (10−7 M, an α2AR antagonist) was superfusedfor at least 15 min and was maintained while responses toNA were re-evaluated. The preparation was then super-fused with control PSS for ∼20–30 min to wash out thefirst antagonist, the second antagonist was equilibratedfor at least 15 min and cumulative responses to NAwere re-evaluated a final time. The order of respectiveantagonists varied across experiments. At the end ofeach experiment, sodium nitroprusside (SNP, 10−5 M) wasadded to obtain maximal MA diameters.



Perivascular nerve stimulation (PNS)

Perivascular nerves were stimulated in a localized electricalfield between two fine wire electrodes (90% platinum/10%iridium; diameter, 250 μm) connected to a stimulationisolation unit (SIU5; Grass, Quincy, MA, USA) drivenby a square wave stimulator (S48; Grass). The tip of anelectrode was positioned on either side of the MA of inter-est near its proximal end and diameters were measured atleast 500 μm distal to the stimulus site. Stimulus pulses(15 V, 2 ms) were delivered at 1, 2, 4, 8 and 16 Hz until astable response diameter was achieved (10–15 s). Vesselswere allowed to return to their baseline diameters priorthe next stimulus. The order of stimulation frequencieswas randomized across experiments, as was the orderin which the respective MA branches were studied. Toevaluate the contribution of sensory nerves, PNS wasrepeated during superfusion with their inhibitor, capsaicin(10 μM). Separate control experiments were performedin Young mice to evaluate non-specific effects ofelectrical field stimulation by inhibiting action potentialswith tetrodotoxin (1 μM) added to the superfusionsolution.

At the end of each experiment the anaesthetized mousewas killed via an overdose of pentobarbital (intracardiacinjection) 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 dimethylsulphoxide then diluted to their final concentrations inPSS with ≤0.1% dimethyl sulphoxide.

Data presentation and statistical analysis

Data for agonist concentration–response and PNSfrequency–response curves are presented as percentageconstriction from baseline diameter (with 100%corresponding to lumen closure) calculated as follows:% constriction = [(Dbase – Dresp)/Dbase] × 100), whereDbase = baseline diameter and Dresp = response diameterin the presence of a given agonist concentration or PNSfrequency. Measured (internal) diameters of all vesselsstudied are given for respective age groups in Table 1(pooled across sex and transgene expression; Table S1contains values for each sex and genotype). Spontaneousvasomotor tone at rest was calculated as the percentagedifference between baseline diameter during superfusionwith control PSS and maximal diameter (Dmax) duringsuperfusion with SNP (10−5 M); thus, % tone = [(Dmax –Dbase)/Dmax] × 100. Data were analysed using Student’st tests or analysis of variance, with repeated measureswhen appropriate (Prism 5, GraphPad Software Inc., La

Jolla, CA, USA). When significant F-ratios were obtainedwith analysis of variance, post hoc comparisons weremade using Bonferroni tests. Summary data are expressedas mean values ± SE. Differences were consideredstatistically significant at P ≤ 0.05.

Results

Diameters and resting tone of MAs in Young and Oldmice

Resting baseline and maximum diameters are listed inTable 1. In Young and Old mice, diameters decreased asbranch order increased (P < 0.05). Spontaneous restingtone was typically ≤10% with no significant differencebetween branch orders or age group. Across sex andGCaMP2 expression, resting and maximal diameters ofMA in Old mice were ∼5% (1A), 20% (2A) and 40% (3A)smaller vs. Young mice (P ≤ 0.05). Thus, the magnitudeof diameter difference between age groups increased withvessel branch order (Table 1).

Effect of ageing on vasomotor responses to PNS

To determine their role in vasomotor control of MAnetworks in vivo, perivascular nerves were stimulatedusing local electrical field stimulation. In Young mice, PNSconstricted 1A, 2A and 3A MAs in a frequency-dependentmanner, 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). Constrictionwas significantly greater in 1A from Old vs. Young mice(P ≤ 0.05), but not in 2A or 3A branches (n = 4–6).

Figure 1. Illustration of intravital preparation of mesentericarterial arcadeA mouse was anaesthetized and a loop of jejunum was exteriorizedover a transparent Sylgard pedestal then secured with pins near theedge of the intestine. The mesentery containing an arcade of first-(1A), second- (2A) and third-order (3A) mesenteric arteries wassuperfused continuously with PSS (pH 7.4, 36◦C) with the effluentaspirated. Electrodes for perivascular nerve stimulation werepositioned at the proximal end of each branch (as shown for 1A).



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 allmice included in this study (n = 12–16 per age group). Baseline diameters (Dbase) were measured during superfusion with controlPSS. Maximum diameters (Dmax) were measured during superfusion of PSS containing sodium nitroprusside (SNP, 10 μM). Calculatedvasomotor tone (%) = [(Dmax – Dbase)/Dmax] × 100. Diameters of respective branch orders were significantly different from each otherwithin each age group (P < 0.05). ∗P ≤ 0.05 versus Young; †P < 0.05, Dmax different from Dbase.

Repeating PNS in the presence of capsaicin (10 μM)significantly increased constrictions of 1A, 2A and 3A MAsin Young mice with maximum responses of 55 ± 2, 68 ± 5and 69 ± 4%, respectively (Fig. 2A-C, n = 4, P ≤ 0.05 vs.control). In contrast, capsaicin did not significantly affectMA constrictions to PNS in Old animals (Fig. 2D–F , n = 6,P ≤ 0.05), nor did it affect MA diameters at rest in eitherage group. In separate control experiments performed inYoung mice, exposure to the voltage-gated sodium channelblocker tetrodotoxin (1 μM) abolished vasoconstrictions

to PNS (Fig. S1), thereby excluding non-specific effects ofelectrical field stimulation (e.g. direct activation of smoothmuscle cells).

Relative contributions of α1ARs and α2ARs tovasoconstriction 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;

Figure 2. Sensory nerves attenuate sympatheticvasoconstriction in Young but not Old miceA–F, summary data are mean values ± SE forpercentage constrictions of first- (A and D), second- (Band E) and third-order (C and F) mesenteric arteries ofYoung and Old mice in response to electrical fieldstimulations (see Fig. 1) of 1, 2, 4, 8 and 16 Hz before(filled circles) and following (open circles) treatmentwith the sensory nerve inhibitor capsaicin (10 μM).∗P < 0.05 vs. capsaicin; n = 4–6 mice per age grouppooled across sex and GCaMP2 expression.



Zhang et al. 2007). However, the sympathetic neuro-transmitter NA can evoke vasoconstriction through α1ARsand α2ARs and the role of respective αAR subtypes inmediating MA constriction in vivo is unknown. To definethese relationships and determine if they were affected byageing, we evaluated the actions of selective adrenergicagonists. 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 respectivepEC50 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 in1A, 2A and 3A were similar to Young mice (51 ± 3, 47 ± 3and 54 ± 2%, respectively) although response curves wereshifted significantly (P < 0.05) to the right with pEC50

values of 5.87 ± 0.20, 5.95 ± 0.11 and 5.92 ± 0.19 in1A, 2A and 3A, respectively (Fig. 3A–C). Responses toUK14304 (10−9–10−5 M) revealed a minor role for α2ARswith constrictions in 1A, 2A and 3A MA of Young (14 ± 4,14 ± 5 and 17 ± 3%) and Old mice (14 ± 2, 15 ± 2 and16 ± 2%) that were <30% of those obtained with PE(P ≤ 0.05 across branch orders) with no differences inpEC50 values between age groups (Fig. 3D–F).

When evaluating specificity of α1AR vs. α2ARactivation, the βAR antagonist propranolol had no

significant effect on MA diameters. NA caused similarconcentration-dependent constrictions in all MAs, withmaximum responses of 52 ± 4, 53 ± 2 and 52 ± 5% andpEC50 values of 6.54 ± 0.09, 6.65 ± 0.07 and 6.57 ± 0.14in 1A, 2A and 3A, respectively (Fig. 4A-C). Inhibitingα1ARs with prazosin reduced respective maximum NAconstrictions to 13 ± 3, 11 ± 1 and 9 ± 3% (Fig. 4A–C,P ≤ 0.05). In contrast, inhibiting α2ARs with rauwolscineproduced a relatively modest inhibition, decreasingmaximum constrictions to 38 ± 2% in 1A and 2A (Fig. 4Aand B, P ≤ 0.05) and 39 ± 6% in 3A (Fig. 4C P ≥ 0.05).Thus, noradrenergic constriction of MAs in vivo ismediated primarily by α1ARs.

Lack of effect of sex or GCaMP2 expression on MAsin vivo

The use of Cx40BAC-GCaMP2 transgenic mice to studyendothelial 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 usingmale animals without determining whether expression ofthe transgene or sex influence vascular reactivity. Thus,an underlying goal of this study was to evaluate whether

Figure 3. α1ARs dominate adrenergicvasoconstriction with diminished sensitivity in OldmiceA–F, summary data are mean values ± SE forpercentage constrictions of first- (A and D), second- (Band E) and third-order (C and F) MAs of Young (filledcircles) and Old (open circles) mice in response tocumulative concentrations of the α1AR agonistphenylephrine (PE, 10−9–10−5 M) or to the α2ARagonist UK14304 (UK, 10−9–10−5 M). ∗P < 0.05compared to Old mice; n = 4–6 mice per age grouppooled across sex and GCaMP2 expression.



sex or the expression of GCaMP2 impact vasomotorresponses in vivo. Across experiments, there were notrends for differences in resting or maximal diameters(Table S1) or their responsiveness to treatments betweenmale and female mice or between GCaMP2-positive andGCaMP2-negative mice. For example, in light of the pre-dominant role of α1ARs in mediating MA constriction,the consistency across sex and GCaMP2 expression withinage groups for concentration–response relationships to PEfor individual Young and Old mice are illustrated in Figs 5and 6, respectively. Thus, all summary data presented forrespective age groups in this study include both male andfemale mice that were positive or negative for GCaMP2expression.

Figure 4. Mesenteric artery constrictions to NA are affectedpredominantly by α1ARsA–C, summary data are mean values ± SE for percentageconstrictions of first- (A), second- (B) and third-order (C) MAs ofYoung mice in response to cumulative increases in noradrenalineconcentration (NA, 10−9–10−5 M; upper curves) in the presence andabsence of the α1AR antagonist prazosin (10−8 M; lower curves) orthe α2AR antagonist rauwolscine (10−7 M; intermediate curves). Allexperiments were performed in the presence of the βAR antagonistpropranolol (10−7 M). ∗P < 0.05 vs. NA alone (Control); �P < 0.05vs. NA + rauwolscine; n = 4 pooled across sex and GCaMP2expression.

Discussion

We have investigated the functional roles of perivascularsympathetic and sensory nerves in MA arcades ofanaesthetized mice in vivo. The present data illustratesignificant changes that occur with ageing irrespectiveof sex or the expression of the GCaMP2 transgene. Keyphysiological findings are that perivascular sensory nerveseffectively limit sympathetic vasoconstriction in Youngmice and that this ability is lost with ageing, resultingin greater vasoconstriction in response to PNS in MAsof Old mice. The efficacy of α1ARs predominated overthat of α2ARs in mediating adrenergic vasoconstrictionin each vessel branch order, although the sensitivity ofα1AR-mediated responses decreased significantly withageing in all MA branch orders. Nevertheless, MAs of Oldmice were ∼5% (1A) to 40% (3A) smaller in diameterthan those of Young mice, thereby imposing a structurallimitation to splanchnic blood flow with ageing.

Diameters and resting tone in MAs of Young andOld mice

Across branch orders, MAs of Old mice had smallerresting and maximal diameters than Young mice with nochange in myogenic tone (Table 1). These reductions inMA diameters with ageing imply diminished blood flowto the splanchnic circulation. Consistent with reducedtissue perfusion, arterial and arteriolar rarefaction withageing 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 supplyvessels increases blood pressure along with flow and shearstress within prevailing vessels and can thereby stimulateoutward remodelling with an increase in diameter (Behnkeet al. 2006; Izzo & Mitchell, 2007). Thus, while changesin arterial diameter with ageing are common, they havemore often been associated with increased vessel size(Muller-Delp et al. 2002; Behnke et al. 2006; Dumontet al. 2008; Hausman et al. 2012). Remarkably, despiteMAs being a widespread model of resistance vessels, theeffect of ageing on MA diameter had not been defined. Ourpresent findings indicate that inward rather than outwardremodelling with ageing occurs in MAs and imply thatage-related changes in MA structure and function candiffer from other vascular beds.

The MAs studied here differ from resistance arteriesof skeletal muscle (Welsh & Segal, 1996), heart (Chilianet al. 1986) or brain (Heistad et al. 1978) studied invivo in that they lacked the substantive (i.e. >20%)spontaneous myogenic tone observed in other resistancearteries. While spontaneous tone observed here in vivo wastypically <10%, whether MAs isolated for in vitro studiesdevelop tone spontaneously is controversial. When iso-lated 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 muscleactivation at rest explains why investigators routinelyinduce vasoconstriction pharmacologically in MAs usedto study mechanisms of vasodilatation. The presentfindings illustrate that α1AR activation with PE is ideallysuited for this purpose (Fig. 3). However, others havereported ≥20% myogenic tone in MA branches (Dubrocaet al. 2007; Koltsova et al. 2009; Zhang et al. 2010;Haddock et al. 2011; Khurana et al. 2012). While thebasis of such differences remains unclear, constrictingMAs through activation of sympathetic nerves providesa mechanism for effectively redistributing cardiac outputaway from the gut to augment blood flow to othervascular beds, for example to active skeletal muscle duringexercise.

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

Our finding that capsaicin increased vasoconstrictionduring PNS in Young mice (Fig. 2A–C) is consistent withinhibiting the vasodilator actions of perivascular sensory

nerves (Bevan & Brayden, 1987; Kawasaki et al. 1988;Kawasaki, 2002). Independent of the endothelium, therelease of CGRP from sensory nerve terminals can evokevasodilatation through activation of CGRP receptors onvascular smooth muscle cells, thereby stimulating proteinkinase A to reduce [Ca2+]i and promote relaxation (Drakeet al. 2000; Brain & Grant, 2004). The vasodilator actions ofCGRP may be enhanced through it acting presynapticallyon sympathetic nerve varicosities to inhibit NA release(Kawasaki et al. 1990b; Takenaga & Kawasaki, 1999). Ina reciprocal manner, release of CGRP can be inhibitedby NA acting on presynaptic α2ARs of sensory nerves(Kawasaki et al. 1990a). The integrated effect entailsreciprocal interaction between perivascular sympatheticand sensory nerves in governing vasomotor activity ofMAs (Kawasaki et al. 1990b; Kawasaki, 2002).

A key finding in the present study is the age-relateddifferences in the neural regulation of vasomotorresponses in MAs. In Old mice, inhibiting sensory nerveswith capsaicin had no significant effect on PNS-inducedconstrictions (Fig. 2D–F), suggesting that this mechanismof attenuating sympathetic vasoconstriction becomesdysfunctional during ageing. From a clinical perspective,restoring sensory nerve function that is lost with ageingmay be of functional significance, as CGRP preconditions

Figure 5. Vasoconstriction to PE is independent ofsex or GCaMP2 expression in Young miceA–F, data are diameter responses (% constriction) torespective concentrations of PE (10−9–10−5 M).Scatterplots depict vessels from individual mice,segregated into males vs. females (A–C) andGCaMP2-positive vs. GCaMP2-negative (D–F) in first- (Aand D), second- (B and D) and third-order (C and F)MAs. Representative error bars depict ± SE for eachgroup within a panel; n = 4–6 per group. Summarydata for these individual observations are presented inFig. 3A–C (filled circles).



vascular endothelial cells against ischaemic injury (Liet al. 2000). Furthermore, a decline in function ofCGRP-containing nerves is linked to several peripheralvascular complications including slow wound healingin diabetics and individuals with Raynaud’s syndrome(Brain & Grant, 2004). Loss of CGRP release is alsoassociated with headache and increased vasospasm incerebral arteries (Edvinsson, 2002). Thus, loss of CGRPactions with ageing may well contribute to the aetiologyof generalized vascular dysfunction.

The lack of effect of capsaicin in MAs of Old micemay 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 vasodilatationwas found in MAs of 30- vs. 8-week-old animals inconjunction with diminished CGRP release during nervestimulation (Kawasaki et al. 1990c; Kawasaki & Takasaki,1992). While these observations provide insight intofactors contributing to hypertension, other mechanismsprobably contribute to vascular dysfunction during ageingin normotensive individuals. Furthermore, increases insympathetic nerve activity with ageing (Seals & Dinenno,2004; Dinenno & Joyner, 2006) may reduce the influence ofsensory nerves. Alternatively, enhanced sympathetic nerve

activity may compensate for reduced sensitivity of vaso-motor responsiveness to α1AR activation (Fig. 3).

Age-related increases in sympathetic nerve activity havebeen most clearly documented in skeletal muscle (Seals& Dinenno, 2004; Dinenno & Joyner, 2006). However,changes in the responsiveness of the vasculature toα-adrenergic stimulation are controversial and probablyvary with the vascular bed, vessel diameter and branchorder (Dinenno & Joyner, 2006; Muller-Delp, 2006).Remarkably, how ageing may influence adrenergicregulation of MAs has not been determined. Therefore,we developed the intravital MA preparation presentedhere to characterize the role of α1ARs and α2ARs in MAsof Old versus Young mice. We found that activation ofα1ARs with PE evoked the major portion of sympatheticvasoconstriction in 1A–3A MAs (Fig. 3). As confirmedin Young mice, inhibition of α1ARs with prazosin nearlyabolished constriction to NA (Fig. 4). The activationof α1ARs in Old mice was able to produce maximumconstrictions similar to those in Young mice but withsignificantly decreased sensitivity (Fig. 3). In contrast,α2ARs do not appear to play a major role in vaso-motor control in MAs of either Young or Old mice(Figs 3 and 4).

Figure 6. Vasoconstriction to PE is independent ofsex or GCaMP2 expression in Old miceA–F, data are diameter responses (% constriction) torespective concentrations of PE (10−9–10−5 M).Scatterplots depict vessels from individual mice used fordata collection, segregated into males vs. females (A–C)and GCaMP2-positive vs. GCaMP2-negative (D–F) infirst- (A and D), second- (B and D) and third-order (Cand F) MAs. Representative error bars depict ± SE foreach group within a panel; n = 4–6 per group.Summary data for these individual observations arepresented in Fig. 3A–C (open circles).



The expression of α1AR subtype mRNA declined withaging in the aorta and renal arteries of 3-, 12- and24-month-old rats, but such changes did not occur inMAs of the same animals (Xu et al. 1997). Thus, changesin receptor expression with ageing can vary betweenvascular beds. While changes in transcript expressionneed not correlate with changes in protein expression,post-translational modification of receptors or changesin downstream signalling intermediates may alter theapparent sensitivity of receptors irrespective of theirexpression level. It is also possible that loss of receptorsto sensory neurotransmitters could explain the lossof attenuation for sympathetic vasoconstriction in Oldmice (Fig. 2), as could a diminished release of CGRP.Thus, enhanced NA release in Old mice (with loss ofsensory modulation) may lead to the desensitization ofα1ARs apparent in Old when compared to Young mice(Fig. 3). In turn, the present data suggest that age-relatedincreases in sympathetic nerve activity (Seals & Dinenno,2004; Dinenno & Joyner, 2006) may compensate fordiminished α1AR responsiveness in MAs. At the sametime, the substantial reduction of MA diameters in Oldvs. Young mice (Table 1) results in a physical limitation tosplanchnic blood flow irrespective of vasoactive stimuli.When coupled with enhanced sympathetic nerve activity,a structural increase in vascular resistance may wellcontribute to the elevation in blood pressure shown toaccompany ageing in C57BL/6 mice (Gros et al. 2002).

Changes in the density of innervation with ageingcould account for some of the functional differencesreported here, as sympathetic nerve density within anage group has been correlated with the magnitude ofsympathetic vasoconstriction (Furness & Marshall, 1974;Marshall, 1982). Nevertheless, previous studies suggestthis is probably not the case with ageing vasculature.For example, no differences were found in the densityof perivascular sympathetic nerves of mesenteric, femoral,gracilis and carotid arteries between mice 3 and 20 monthsof age (Long & Segal, 2009). In basal cerebral arteriesof humans, although perivascular nerve density did notchange with ageing (62–85 years of age), relative stainingfor sympathetic versus sensory nerves was not evaluated(Bleys et al. 1996). In MAs and carotid arteries of guineapigs, adrenergic and peptidergic (CGRP) nerve densitieswere maintained from 4 weeks to 2 years of age (Dhallet al. 1986). These findings are consistent with maintainingthe density of CGRP-containing nerves with ageing inrat MAs (Hobara et al. 2010). However, in renal andfemoral arteries of guinea pigs, while noradrenergic andpeptidergic nerve densities decreased in Old animals, MAsfrom all ages had the highest density of both noradrenergicand peptidergic nerves (Dhall et al. 1986). Collectively,previous findings indicate that the densities of perivascularsympathetic and sensory nerve fibres of MAs are preservedwith ageing.

Sex, neuroeffector signalling and transgeneexpression

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 consistentwith studies of rat cerebral arteries (Aukes et al. 2008),which found no differences between males and females invasomotor responses to CGRP or to NA. Posterior cerebralarteries from female rats had a significantly higher densityof perivascular nerves containing CGRP than male ratswith no difference between sexes in nerves containingtyrosine hydroxylase (a marker of sympathetic nerves)(Aukes et al. 2008). Vasomotor responses to PNS werenot different between sexes, suggesting that nerve densityand vasomotor function may not be directly linked incerebral arteries. In isolated preparations of rat mesentery,sex-based differences were apparent in constriction of MAsto adrenergic nerve stimulation but not to exogenousNA (Li & Duckles, 1994). Nor were there sex-relateddifferences in vasodilatation during CGRP-ergic nervestimulation (Li & Duckles, 1994). In light of the limiteddata in the literature, our studies of mouse MAs indicatethat sex is unlikely to impact the density of eithersympathetic or sensory nerves or the vasomotor responsesthat they elicit upon stimulation. This conclusion does notexclude the likelihood of sex-based differences in vaso-motor control in other vascular beds (e.g. of reproductiveorgans) or in other species.

The transgenic (Cx40BAC-GCaMP2) mice studied hereserve as a valuable model for studying calcium signallingin the endothelium of resistance vessels (Tallini et al.2007; Ledoux et al. 2008; Bagher et al. 2011a; Nauschet al. 2012; Sonkusare et al. 2012). Thus, it is essentialto determine whether data generated in these mice arephysiologically homologous across sex and genotype. Allstudies of these mice published thus far have utilizedonly Young males and there have been no comprehensiveanalyses of whether the expression of GCaMP2 mayinfluence vasomotor control. To provide such insight,we studied both males and females that were bred andraised under identical conditions to determine whetherthere were trends in our data associated with differencesin sex or GCaMP2 expression. Consistent with the lack ofsex differences, the present data indicate that neither thefunction of MAs nor the effects of ageing are influencedby the expression of GCaMP2 in endothelial cells (Figs5 & 6). Thus, GCaMP2 may be considered an effectivetracer molecule that functions as a calcium sensor withoutaltering vascular reactivity or vasomotor control. Thesefindings are also relevant economically and with respectto the responsible use of animals for research: if onlymale GCaMP2-positive mice were used, approximately75% of the resources expended in maintaining a breedingcolony 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 costsand numbers of animals bred and used.

Summary and conclusion

The present findings support the hypothesis that ageingalters the function of perivascular sympathetic and sensorynerves of MAs in vivo irrespective of sex or the expressionof a Ca2+ sensitive transgene in the endothelium.While sensory nerves effectively limit sympathetic vaso-constriction of MAs during PNS in Young mice, thismodulation is lost in Old mice. Sympathetic vaso-constriction is mediated predominantly by α1ARs inMAs of both Young and Old mice. However, α1ARsare desensitized in Old mice across MA branch orders,suggesting that ageing affected adrenergic reactivity ofall MAs similarly. The smaller diameters throughout MAarcades of Old mice impose a structural limitation tosplanchnic blood flow. The diminished sensitivity of MAsto α1AR stimulation in Old mice would impair the abilityto constrict and mobilize blood from the splanchniccirculation, for example to support the energetic demandsof contracting skeletal muscle during physical activity. Atthe same time, this limitation in α1AR-mediated vaso-constriction is offset through loss of the ability of sensorynerves to attenuate sympathetic vasoconstriction. Withadvancing age, such changes to the splanchnic circulationmay compromise the quality of life and contribute tovascular disease.

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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.

Acknowledgements

This research was supported by grants R01-HL086483 andR37-HL41026 from the National Institutes of Health, UnitedStates Public Health Service. Conflicts of interest: none.


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Figure S1. Vasoconstrictions induced by local electric field stimulation are mediated by

perivascular nerves. Data are mean percent constrictions ± SE of (A) first-, (B) second- and

(C) third-order MAs of Young mice in response to local electric field stimulations of 1, 2, 4, 8,

and 16 Hz in the presence and absence of the voltage-gated sodium channel blocker, tetrodotoxin

(TTX, 1 µM). * P < 0.05 vs. TTX (n = 4).

Documents

Ageing alters perivascular nerve function of mouse mesenteric arteries in vivo