Depressed perivascular sensory innervation of mouse mesenteric arteries with advanced age

Abstract

Key points: The dilatory role for sensory innervation of mesenteric arteries (MAs) is impaired in Old (∼24 months) versus Young (∼4 months) mice. We investigated the nature of this impairment in isolated pressurized MAs. With perivascular sensory nerve stimulation, dilatation and inhibition of sympathetic vasoconstriction observed in Young MAs were lost in Old MAs along with impaired dilatation to calcitonin gene-related peptide (CGRP). Inhibiting NO and prostaglandin synthesis increased CGRP EC50 in Young and Old MAs. Endothelial denudation attenuated dilatation to CGRP in Old MAs yet enhanced dilatation to CGRP in Young MAs while abolishing all dilatations to ACh. In Old MAs, sensory nerve density was reduced and RAMP1 (CGRP receptor component) associated with nuclear regions of endothelial cells in a manner not seen in Young MAs or in smooth muscle cells of either age. With advanced age, loss of dilatory signalling mediated through perivascular sensory nerves may compromise perfusion of visceral organs.

Abstract: Vascular dysfunction and sympathetic nerve activity increase with advancing age. In the gut, blood flow is governed by perivascular sensory and sympathetic nerves but little is known of how their functional role is affected by advanced age. We tested the hypothesis that functional sensory innervation of mesenteric arteries (MAs) is impaired for Old (24 months) versus Young (4 months) C57BL/6 male mice. In cannulated pressurized MAs preconstricted 50% with noradrenaline and treated with guanethidine (to inhibit sympathetic neurotransmission), perivascular nerve stimulation (PNS) evoked dilatation in Young but not Old MAs while dilatations to ACh were not different between age groups. In Young MAs, capsaicin (to inhibit sensory neurotransmission) blocked dilatation and increased constriction during PNS. With no difference in efficacy, the EC50 of CGRP as a vasodilator was ∼6-fold greater in Old versus Young MAs. Inhibiting nitric oxide (l-NAME) and prostaglandin (indomethacin) synthesis increased CGRP EC50 in both age groups. Endothelial denudation reduced the efficacy of dilatation to CGRP by ∼30% in Old MAs yet increased this efficacy ∼15% in Young MAs while all dilatations to ACh were abolished. Immunolabelling revealed reduced density of sensory (CGRP) but not sympathetic (tyrosine hydroxylase) innervation for Old versus Young MAs. Whereas the distribution of CGRP receptor proteins was similar in SMCs, RAMP1 associated with nuclear regions of endothelial cells of Old but not Young MAs. With advanced age, the loss of sensory nerve function and diminished effectiveness of CGRP as a vasodilator is multifaceted and may adversely affect splanchnic perfusion.

Figure 1. Impaired dilatation of MAs to sensory nerve stimulation with advanced age

A, from resting control IDs (Young, 208 ± 12 μm; Old, 172 ± 20 μm), constriction during PNS (8 Hz @ 30 V, 2 ms) was not different between MAs from Young versus Old mice (IDs decreased to 110 ± 11 μm and 89 ± 13 μm, respectively). B, for MAs preconstricted with phenylephrine (1 μm), IDs were 96 ± 4 μm (Young) and 87 ± 10 μm (Old). Following treatment with guanethidine (10 μm), PNS dilated MAs of Young (to 156 ± 20 μm) but not Old mice (86 ± 11 μm). C, in MAs preconstricted as in B, treatment with capsaicin (10 μm) in addition to guanethidine prevented dilatation to PNS (Young, 103 ± 11 μm; Old, 78 ± 10 μm). n = 4–5 MAs per age group. % Constriction or % Max Dilatation calculated as described in Methods. *P < 0.05 compared to Young.

Figure 2. Sensory nerves limit constriction to perivascular nerve stimulation for Young but not Old MAs

A, under control conditions, PNS caused similar constrictions in Young MAs (ID rest, 199 ± 6 μm; ID response, 120 ± 10 μm) and Old MAs (ID rest, 183 ± 14 μm; ID response, 113 ± 13 μm). Repeating PNS during capsaicin treatment (10 μm) increased constriction in Young (ID rest, 200 ± 7 μm; ID response, 97 ± 10 μm) but not Old MAs (ID rest, 182 ± 17 μm; ID response, 110 ± 12 μm). B, tetrodotoxin (TTX, 1 μm) blocked constriction to PNS in Young (ID rest, 201 ± 6; ID response, 193 ± 8 μm) and Old MAs (ID rest, 188 ± 17; ID response, 180 ± 15 μm). % Constriction calculated as described in Methods. n = 4 MAs per age group in A and n = 3 MAs per age group in B. *P < 0.05 compared to Control in same age group.

Figure 3. Increased EC₅₀ of constriction to NA but not dilatation to ACh

A, concentration‐dependent constriction to NA is shifted to the right in Old MAs compared to Young MAs. Initial resting IDs were 199 ± 12 for Young MAs and 187 ± 10 μm for Old MAs; respective IDs were 76 ± 7 and 80 ± 6 μm during peak constriction to NA and were 217 ± 12 and 206 ± 11 μm during maximal dilatation. B, concentration‐dependent dilatation to ACh is similar in Old and Young MAs. Initial baseline IDs during preconstriction with NA (1 μm) were not different between age groups (Young, 107 ± 7 μm; Old, 101 ± 9 μm). Peak IDs during vasodilatation to ACh were 174 ± 2 and 166 ± 12 μm, respectively; corresponding maximal IDs were 211 ± 3 μm and 201 ± 12 μm. % Constriction and % Max dilatation calculated as described in Methods. n = 5 MAs per age group. *P < 0.05, Young versus Old MAs at same NA concentration.

Figure 4. Impaired dilatation to CGRP despite greater dependence on endothelium

A, concentration‐dependent dilatation to CGRP is shifted to the right for Old MAs compared to Young MAs. Intact MAs were preconstricted with NA using EC₅₀ values determined for respective age groups (Table 1). From preconstricted baseline IDs of 111 ± 8 μm (Young) and 108 ± 2 μm (Old), CGRP evoked peak IDs of 193 ± 11 μm and 184 ± 9 μm, respectively; n = 11 per age group. *P < 0.05, Young vs. Old MAs at same CGRP concentration. B, inhibiting endothelium‐derived autacoids with l‐NAME (100 μm) + indomethacin (Indo, 10 μm) shifted response curves to the right for intact MAs of both age groups (dashed lines); n = 6 per age group. Control data (continuous lines) are a subset of values shown in A with same statistical differences.⁺ P < 0.05, Control versus l‐NAME/Indo. C, sequence of evaluations following endothelial denudation. IDs were not different between Young versus Old MAs with control PSS, during EC₅₀ constrictions with NA before and after evaluating responses to CGRP (arrow) during exposure to ACh (1 μm added during preconstriction with EC₅₀ NA; note loss of endothelium‐dependent dilatation), or during maximal dilatation with 0 Ca²⁺ + 10 μm SNP (0Ca/SNP). n = 6 per age group; *P < 0.05 versus resting control diameter within same age group. D, the efficacy of dilatation to CGRP is decreased in Old MAs and increased in Young MAs. Control (intact) data re‐plotted from A for reference. n = 6 (denuded) or 11 (intact) per age group. ⁺ P < 0.05, Young denuded versus Young intact. ^# P < 0.05, Old denuded versus Old intact.

Figure 5. Immunofluorescence of perivascular nerves

Images are representative maximum Z‐projections of MAs double‐stained for both sensory nerves (left panels, labelled for CGRP) and sympathetic nerves (centre panels, labelled for tyrosine hydroxylase, TH) from Young (top row) and Old MAs (bottom row). Right panels are overlays of the fluorescence from respective images. Scale bar = 100 μm and applies to all panels. Images are representative of n = 9–16 MAs from 4–5 mice per age group.

Figure 6. Decreased perivascular sensory but not sympathetic nerve density

A, quantifying CGRP fluorescence following immunolabelling indicates a significant reduction in the density of sensory nerves surrounding Old versus Young MAs. *P < 0.05 versus Young. B, quantifying TH fluorescence following immunolabelling indicates no difference in the density of sympathetic nerves surrounding Old versus Young MAs. n = 9–16 MAs from 4–5 mice per age group.

Figure 7. Nuclear association of RAMP1 with endothelial cell nuclei of Old but not Young MAs

Images are maximum Z‐projections double‐stained for RAMP1 and CRLR. Panels at right are overlays; yellow indicates correspondence in localisation of respective CGRP receptor proteins and is most consistent in endothelium of Young MAs. Staining for RAMP1 is associated with endothelial cell nuclei of Old MAs that is not present in Young MAs (arrows). Scale bar = 20 μm and applies to all panels. Representative of n = 9–12 MAs from 3 mice per age group.

Figure 8. Similar expression of RAMP1 and CRLR proteins in smooth muscle cells of Young and Old MAs

Images are maximum Z‐projections double‐stained for RAMP1 and CRLR. Panels at right are overlays; yellow indicates correspondence in localisation of respective CGRP receptor proteins. Scale bar = 20 μm and applies to all panels. Representative of n = 8–10 MAs from 4 mice per age group.