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Objective: Reproducibility of the reflex cutaneous vasoconstriction response is currently unknown. Our aim was to determine the test-retest reproducibility of laser speckle contrast imaging (LSCI) and varying sampling depths of laser Doppler flowmetry (LDF) in response to whole-body cooling., Methods: Over two studies, nine and fourteen healthy, young adults underwent a 40-min cooling bout over two separate experiments. Participants were cooled from 34.0 °C to 30.5 °C and held at a 30.5 °C plateau for 10-min prior to rewarming. Throughout the cooling bout, changes in blood flow were measured as LSCI flux and LDF flux for Study 1 and LDF flux by three different LDF sampling depths in Study 2. Test-retest reproducibility and reliability were evaluated by the coefficient of variation (CV) and intraclass correlation coefficients (ICC), respectively. Vasoconstriction was presented as cutaneous vascular conductance (CVC = flux / mean arterial pressure) and expressed as a percent change from baseline (%ΔCVC BASELINE )., Results: For Study 1, test-retest reproducibility displayed good reproducibility for LSCI (CV: <9.0%) and good-to-moderate for LDF (CV: <17.0%) throughout the cooling bout and at plateau (LSCI CV: 1.0%; LDF CV: 1.9%). For Study 2, all Doppler depths displayed good reproducibility during the cooling bout (CV: <9.0%) and at plateau (CV: 0.9-2.0%). Only LSCI demonstrated reliability across both studies (ICC: 0.58-0.88). A reduced vasoconstriction response was measured with the shallowest penetration in the skin (LSCI: 26 ± 0.9%ΔCVC BASELINE ) compared to the Doppler with the deepest penetration (35 ± 0.6%ΔCVC BASELINE , p < 0.001)., Conclusions: Although Dopplers better discriminate the reflex cutaneous vasoconstriction response, LSCI exhibits greater test-retest reproducibility and reliability, and thus may be more suitable for longitudinal assessments., (Copyright © 2022 Elsevier Inc. All rights reserved.)

New Findings: What is the central question of this study? Ageing is associated with altered sympathetic responses to stress, which are explained in part by reduced noradrenergic function. The impact of supplementation with oral l-tyrosine, the amino acid precursor for catecholamine synthesis, on the effector responses to cold and exercise stress has yet to be examined. What is the main finding and its importance? Oral l-tyrosine ingestion augmented the sympathetically mediated vasoconstriction response to cold exposure in aged skin. This suggests that l-tyrosine supplementation might improve thermoregulatory function in older adults. l-Tyrosine is the primary substrate for noradrenaline biosynthesis within sympathetic axon terminals. In stressful conditions requiring increased catecholamine production, the axonal l-tyrosine concentration may limit the full expression of the sympathetic effector response and this may be particularly evident in older adults. We hypothesize that oral l-tyrosine supplementation will increase the sympathetic response to whole-body cooling and muscle metaboreflex activation. In a randomized, double-blind design, 11 young (Y = 24 ± 1 years) and 11 older participants (O = 68 ± 4 years) ingested either 150 mg kg -1 of l-tyrosine or placebo before commencing 30 min of whole-body cooling to induce a gradual decline in skin temperature from 34 to 30.5°C. Laser Doppler flux (LDF) was measured at the ventral forearm, and cutaneous vascular conductance (CVC) was calculated as CVC = LDF/mean arterial pressure and expressed as a percentage change from baseline (%ΔCVC). Two minutes of static hand-grip exercise (35% maximal voluntary contraction) followed by 3 min of postexercise ischaemia were implemented before and toward the end of the cooling bout. l-Tyrosine supplementation did not affect blood pressure or heart rate responses to exercise or postexercise ischaemia. However, the blunted vasoconstriction response to whole-body cooling in older adults (placebo: Y = 39 ± 5%ΔCVC and O = 16 ± 2 %ΔCVC; P < 0.05) was augmented after l-tyrosine supplementation (l-tyrosine: Y = 40 ± 4%ΔCVC and O = 32 ± 5 %ΔCVC; P < 0.05). These results suggest that l-tyrosine bioavailability might limit thermoregulatory function in an older population., (© 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.)

New Findings: What is the central question of this study? In young adults, about half of the cold-related reduction in skin blood flow during cold exposure is mediated by noradrenaline, while the remainder is attributable to other substances co-released with noradrenaline that have yet to be identified. What is the main finding and its importance? Purinergic receptor blockade blunted the vasoconstriction response to whole-body cooling and to intradermal administration of tyramine. These results indicate that ATP is necessary to vasoconstrict blood vessels in the skin adequately and prevent heat loss in a cold environment. Noradrenaline is responsible for eliciting ∼60% of the reflex cutaneous vasoconstriction (VC) response in young adults, while the remainder is attributable to one or more unidentified co-released sympathetic adrenergic neurotransmitter(s). Inconsistent evidence has placed neuropeptide Y in this role; however, other putative cotransmitters have yet to be tested. We hypothesize that ATP contributes to the reflex cutaneous VC response. Two protocols were conducted in young adults (n = 10); both involved the placement of three microdialysis probes in forearm skin and whole-body cooling (skin temperature = 30.5°C). In protocol 1, the following solutions were infused: (i) lactated Ringer solution (control); (ii) 10 mm l-NAME; and (iii) purinergic receptor blockade with 1 mm suramin plus l-NAME. In protocol 2, the following solutions were infused: (i) lactated Ringer solution; (ii) suramin plus l-NAME; and (iii) suramin plus l-NAME plus adrenoreceptor blockade with 5 mm yohimbine plus 1 mm propranolol. Laser Doppler flux (LDF) was measured over each microdialysis site, and cutaneous vascular conductance (CVC) was calculated (CVC = LDF/MAP) and expressed as percentage changes from baseline (%ΔCVC BASELINE ). l-NAME was used to block the vasodilatory influence of ATP and unmask the P 2 X-mediated VC response to exogenous ATP infusion (-21 ± 6%ΔCVC BASELINE ). During cooling, the VC response (control, -39 ± 8%ΔCVC BASELINE ) was attenuated at the suramin site (-21 ± 4%ΔCVC BASELINE ) and further blunted with combined adrenoreceptor blockade (-9 ± 3%ΔCVC BASELINE ; P < 0.05). Compared with the control site (-22 ± 5%ΔCVC BASELINE ), suramin inhibited pharmacologically induced VC to tyramine (-12 ± 6%ΔCVC BASELINE ; P < 0.05), which displaces adrenergic neurotransmitters from axon terminals. These data indicate that ATP contributes to the cutaneous VC response in humans., (© 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.)

Stimulation of angiotensin II type I receptors (AT1R) elicits vasoconstriction (VC) that may be occurring through the activation of a pathogenic vascular pathway such as Rho kinase (ROCK). We hypothesize that reflex cutaneous VC to whole body cooling (mean skin temperature = 30.5°C) in older humans relies in part on AT1R activation, which may explain greater ROCK activity attendant with aging. Two microdialysis (MD) fibers were placed in the forearm skin of 10 young (Y; 24 ± 1 yr) and 10 older (O; 70 ± 2 yr) individuals for infusion of 1) lactated Ringer's solution (switched to fasudil, a ROCK antagonist, after cooling); and 2) AT1R blockade with losartan. Laser Doppler flux (LDF) was measured over each MD site and cutaneous vascular conductance (CVC) was calculated (CVC = LDF/mean arterial pressure) and expressed as percent change from baseline (%ΔCVCBASELINE). In older individuals the VC response to whole body cooling was blunted (Y = -34 ± 2, O = -17 ± 3%ΔCVC) and was further attenuated at the losartan site (Y = -34 ± 3, O = -9 ± 3%ΔCVC; P < 0.05). The VC response to an exogenous 10-μM dose of angiotensin II (Y = -27 ± 3, O = -42 ± 5%ΔCVC) was completely blocked in sites pretreated with losartan or with fasudil. These data suggest that AT1R activation contributes to the reflex VC response in aged but not young skin. Furthermore, the angiotensin II component of the VC response appears to occur primarily through a ROCK-mediated mechanism., (Copyright © 2015 the American Physiological Society.)

We have recently demonstrated that tetrahydrobiopterin (BH(4)) augments reflex vasoconstriction (VC) in aged skin. Although this appears to occur through its role in norepinephrine (NE) biosynthesis, the extent with which vascular mechanisms are affected are unknown. We hypothesized that localized BH(4) supplementation would not affect the VC response to exogenous NE when sympathetic nerves were blocked. Two microdialysis fibers were placed in bretylium tosylate pretreated (presynaptically blocks neurotransmitter release from sympathetic adrenergic nerve terminals; iontophoresis, 200 μA for 20 min) 3-cm(2) forearm skin of 10 young (Y) and 10 older (O) subjects for perfusion of 1) Ringer (control) and 2) 5 mM BH(4). While local skin temperature was clamped at 34°C, six concentrations of NE (10(-12), 10(-10), 10(-8), 10(-6), 10(-4), 10(-2) M) were infused at each drug-treated site. Cutaneous vascular conductance (CVC) was calculated (CVC = laser Doppler flux/mean arterial pressure) and normalized to baseline (%ΔCVC(base)). Despite prejunctional adrenergic blockade, NE-mediated VC was blunted in aged skin at each NE dose (10(-12): -12 ± 2 vs. -21 ± 2; 10(-10): -15 ± 2 vs. -27 ± 1; 10(-8): -22 ± 2 vs. -32 ± 2; 10(-6): -27 ± 2 vs. -38 ± 1; 10(-4): -52 ± 3 vs. -66 ± 5; 10(-2): -62 ± 3 vs. -75 ± 4%ΔCVC(base); P < 0.01), and this response was not affected by pretreatment with BH(4) (P > 0.05). Localized BH(4) did not affect end-organ responsiveness to exogenous NE, suggesting that the effects of BH(4) on cutaneous VC are primarily isolated to the NE biosynthetic pathway.

The attenuated reflex vasoconstriction in aged skin may be partly mediated by oxidant-induced reduction in functional substrate and cofactor availability for noradrenaline biosynthesis. We hypothesized that localized supplementation of tyrosine and tetrahydrobiopterin (BH(4)) in aged human skin could augment reflex- (whole-body cooling) and pharmacologically (tyramine, which displaces noradrenaline from axon terminals) induced vasoconstriction. Four microdialysis fibres were placed in the forearm skin of 10 young and 10 older subjects for infusion of (1) Ringer solution (control), (2) 0.5 mm L-tyrosine, (3) 5 mm BH(4), and (4) BH(4) + L-tyrosine. Cutaneous vascular conductance (CVC) was calculated (laser Doppler flux/mean arterial pressure) and normalized to baseline (% Delta CVC(base)). Vasoconstriction was attenuated at the control site in the older subjects during both whole-body cooling (young: 39 +/- 3, older: 17 +/- 3% Delta CVC(base); P < 0.01) and tyramine infusion (young: 41 +/- 3, older: 21 +/- 4% Delta CVC(base); P < 0.01). BH(4) (cold, young: 37 +/- 3, older: 36 +/- 3; tyramine, young: 41 +/- 2, older: 36 +/- 3% Delta CVC(base)) and tyrosine (cold, young: 37 +/- 4, older: 34 +/- 4; tyramine, young: 40 +/- 4, older: 45 +/- 4% Delta CVC(base)) both resolved the age-related decrease in cutaneous vasoconstriction, but BH(4) + tyrosine did not further augment vasoconstriction (cold, young: 38 +/- 4, older: 31 +/- 3; tyramine, young: 36 +/- 3, older: 36 +/- 5 Delta %CVC(base)). These data are consistent with the concept that reduced bioavailability of BH(4) and/or tyrosine may impair noradrenaline synthesis and contribute to the attenuated vasoconstrictor response in aged skin.

Primary human aging may be associated with augmented Rho kinase (ROCK)-mediated contraction of vascular smooth muscle and ROCK-mediated inhibition of nitric oxide synthase (NOS). We hypothesized that the contribution of ROCK to reflex vasoconstriction (VC) is greater in aged skin. Cutaneous VC was elicited by 1) whole body cooling [mean skin temperature (T(sk)) = 30.5 degrees C] and 2) local norepinephrine (NE) infusion (1 x 10(-6) M). Four microdialysis fibers were placed in the forearm skin of eight young (Y) and eight older (O) subjects for infusion of 1) Ringer solution (control), 2) 3 mM fasudil (ROCK inhibition), 3) 20 mM N(G)-nitro-l-arginine methyl ester (NOS inhibition), and 4) both ROCK + NOS inhibitors. Red cell flux was measured by laser-Doppler flowmetry over each site. Cutaneous vascular conductance (CVC) was calculated as flux/mean arterial pressure and normalized to baseline CVC (%DeltaCVC(baseline)). VC was reduced at the control site in O during cooling (Y, -34 + or - 3; and O, -18 + or - 3%DeltaCVC(baseline); P < 0.001) and NE infusion (Y, -53 + or - 4, and O, -41 + or - 9%DeltaCVC(baseline); P = 0.006). Fasudil attenuated VC in both age groups during mild cooling; however, this reduction remained only in O but not in Y skin during moderate cooling (Y, -30 + or - 5; and O, -7 + or - 1%DeltaCVC(baseline); P = 0.016) and was not altered by NOS inhibition. Fasudil blunted NE-mediated VC in both age groups (Y, -23 + or - 4; and O, -7 + or - 3%DeltaCVC(baseline); P < 0.01). Cumulatively, these data indicate that reflex VC is more reliant on ROCK in aged skin such that approximately half of the total VC response to whole body cooling is ROCK dependent.

In young healthy humans full expression of reflex cutaneous vasodilation is dependent on cyclooxygenase (COX)- and nitric oxide synthase (NOS)-dependent mechanisms. Chronic low-dose aspirin therapy attenuates reflex cutaneous vasodilation potentially through both platelet and vascular COX-dependent mechanisms. We hypothesized the contribution of COX-dependent vasodilators to reflex cutaneous vasodilation during localized acute COX inhibition would be attenuated in healthy middle-aged humans due to a shift toward COX-dependent vasoconstrictors. Four microdialysis fibers were placed in forearm skin of 13 middle-aged (53 +/- 2 yr) normotensive healthy humans, serving as control (Ringer), COX-inhibited (10 mM ketorolac), NOS-inhibited (10 mM N(G)-nitro-l-arginine methyl ester), and combined NOS- and COX-inhibited sites. Red blood cell flux was measured over each site by laser-Doppler flowmetry as reflex vasodilation was induced by increasing oral temperature (T(or)) 1.0 degrees C using a water-perfused suit. Cutaneous vascular conductance was calculated (CVC = flux/mean arterial pressure) and normalized to maximal CVC (CVC(max); 28 mM sodium nitroprusside). CVC(max) was not affected by localized microdialysis drug treatment (P > 0.05). Localized COX inhibition increased baseline (18 +/- 3%CVC(max); P < 0.001) compared with control (9 +/- 1%CVC(max)), NOS-inhibited (7 +/- 1%CVC(max)), and combined sites (10 +/- 1%CVC(max)). %CVC(max) in the COX-inhibited site remained greater than the control site with DeltaT(or) < or = 0.3 degrees C; however, there was no difference between these sites with DeltaT(or) > or = 0.4 degrees C. NOS inhibition and combined COX and NOS inhibition attenuated reflex vasodilation compared with control (P < 0.001), but there was no difference between these sites. Localized COX inhibition with ketorolac significantly augments baseline CVC but does not alter the subsequent skin blood flow response to hyperthermia, suggesting a limited role for COX-derived vasodilator prostanoids in reflex cutaneous vasodilation and a shift toward COX-derived vasoconstrictors in middle-aged human skin.

Reflex vasoconstriction (VC) is attenuated in aged skin resulting in greater skin blood flow and heat loss during cold exposure. We hypothesized that adrenergic function is compromised due to depletion of tetrahydrobiopterin (BH(4)), an essential cofactor required for catecholamine synthesis, and therefore local BH(4) supplementation would functionally augment reflex and pharmacologically induced VC elicited by gradual whole-body cooling (skin temperature (T(sk)) = 30.5 degrees C) and tyramine infusion, respectively. Four microdialysis (MD) fibres were placed in the forearm skin of 11 young (Y) and 11 older (O) human subjects for infusion of (1) Ringer solution (control), (2) 5 mM BH(4), (3) 5 mM BH(4) + 10 mM ascorbate, and (4) 5 mM BH(4) + adrenoreceptor blockade (5 mM yohimbine + 1 mM propranolol). Laser Doppler flux (LDF) was measured over each MD site and cutaneous vascular conductance was calculated as CVC = LDF/MAP and expressed as per cent change from baseline (% DeltaCVC(base)). The VC response was lower at the control site in O during cooling (Y: -34 +/- 2% DeltaCVC(base), O: -17 +/- 2% DeltaCVC(base); P < 0.001) and tyramine infusion (Y: - 33 +/- 4% DeltaCVC(base), O: -15 +/- 3% DeltaCVC(base); P < 0.001). BH(4) infusion normalized O to Y values during both cooling (Y: -34 +/- 4% DeltaCVC(base), O: -34 +/- 2% DeltaCVC(base); P < 0.001) and tyramine (Y: -38 +/- 4% DeltaCVC(base), O: -35 +/- 3% DeltaCVC(base); P < 0.001), however, adding adrenoreceptor blockade abolished VC in aged skin indicating that BH(4) acts through adrenergic, not cotransmitter, mechanisms. Local BH(4) supplementation augments reflex and tyramine-induced VC in aged skin, suggesting that reduced BH(4) bioavailability may contribute to attenuated VC during whole-body cooling.