Ageing alters perivascular nerve function of mouse mesenteric arteries in vivo

Abstract

Abstract Mesenteric arteries (MAs) are studied widely in vitro but little is known of their reactivity in vivo. Transgenic animals have enabled Ca(2+) 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.

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).

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.

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.

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.

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).