Ten days of repeated local forearm heating does not affect cutaneous vascular function

We show for the first time that 10 days of repeated forearm heating is not sufficient to improve cutaneous vascular responsiveness in recreationally active young adults. In addition, this is the first study to investigate cutaneous cholinergic sensitivity and forearm blood flow following repeated local heat exposure. Our data add to the limited studies regarding repeated local heating of the cutaneous vasculature.

Abstract

The aim of the present study was to determine whether 10 days of repeated local heating could induce peripheral adaptations in the cutaneous vasculature and to investigate potential mechanisms of adaptation. We also assessed maximal forearm blood flow to determine whether repeated local heating affects maximal dilator capacity. Before and after 10 days of heat training consisting of 1-h exposures of the forearm to 42°C water or 32°C water (control) in the contralateral arm (randomized and counterbalanced), we assessed hyperemia to rapid local heating of the skin (n = 14 recreationally active young subjects). In addition, sequential doses of acetylcholine (ACh, 1 and 10 mM) were infused in a subset of subjects (n = 7) via microdialysis to study potential nonthermal microvascular adaptations following 10 days of repeated forearm heat training. Skin blood flow was assessed using laser-Doppler flowmetry, and cutaneous vascular conductance (CVC) was calculated as laser-Doppler red blood cell flux divided by mean arterial pressure. Maximal cutaneous vasodilation was achieved by heating the arm in a water-spray device for 45 min and assessed using venous occlusion plethysmography. Forearm vascular conductance (FVC) was calculated as forearm blood flow divided by mean arterial pressure. Repeated forearm heating did not increase plateau percent maximal CVC (CVC_max) responses to local heating (89 ± 3 vs. 89 ± 2% CVC_max, P = 0.19), 1 mM ACh (43 ± 9 vs. 53 ± 7% CVC_max, P = 0.76), or 10 mM ACh (61 ± 9 vs. 85 ± 7% CVC_max, P = 0.37, by 2-way repeated-measures ANOVA). There was a main effect of time at 10 mM ACh (P = 0.03). Maximal FVC remained unchanged (0.12 ± 0.02 vs. 0.14 ± 0.02 FVC, P = 0.30). No differences were observed in the control arm. Ten days of repeated forearm heating in recreationally active young adults did not improve the microvascular responsiveness to ACh or local heating.

NEW & NOTEWORTHY We show for the first time that 10 days of repeated forearm heating is not sufficient to improve cutaneous vascular responsiveness in recreationally active young adults. In addition, this is the first study to investigate cutaneous cholinergic sensitivity and forearm blood flow following repeated local heat exposure. Our data add to the limited studies regarding repeated local heating of the cutaneous vasculature.

previous studies have demonstrated an improved ability to thermoregulate in humans following a 10-day exercise heat acclimation protocol (12, 28, 34, 38, 40). This improvement manifests as an increase in both sweat rate (SR) and skin blood flow (SkBF) at a given core temperature, which enhances heat dissipation (12, 40). For many years, it was thought that improvements in SkBF and SR were centrally mediated, implying a greater neural output and/or a lowered threshold for activation to a given thermal stimulus, resulting in higher SR and SkBF at a given core body temperature (26, 38, 42, 44). Others have suggested that the increased SR and SkBF following heat acclimation are partly due to a potentiated capacity of the sweat glands and cutaneous vasculature themselves (7, 11, 15, 16, 23, 41a). Our laboratory previously demonstrated that 10 days of exercise heat acclimation in trained cyclists could improve both cutaneous vascular responsiveness and sweating to doses of acetylcholine (ACh) infused via microdialysis in the forearm during thermoneutral resting conditions (28). This finding was important, as it demonstrated that exercise heat acclimation results in functional improvements within the sweat glands and cutaneous vasculature over 10 days without central changes in neural outflow. More recently, 14 days of exercise heat acclimation has been shown to induce local improvements in forearm sweat gland output during a whole body heat stress test (35). This was not observed in the upper back or chest, suggesting regional variability in sweat gland adaptation following whole body heat acclimation. Additionally, a number of studies have demonstrated that sweat glands can be locally trained following repeated stimulation using electrical and pharmacological techniques, as well as repeated local heating (10, 11, 15, 22, 23). However, less is known about the cutaneous vasculature and the potential adaptive responses to repeated local heating.

Using lower limb heating to raise core body temperature, Carter et al. (8) demonstrated that 8 wk of modest heat acclimation improved cutaneous vascular responsiveness to local heating of the skin of the ventral forearm. However, when arm temperature was clamped to 30°C in this study, no improvements in cutaneous vascular function were observed. This finding suggests that the improvement in cutaneous vascular responsiveness during exercise or passive heat acclimation may be due, in part, to an increase in local skin temperature. More recently, Brunt et al. (5), using N^ω-nitro-l-arginine [a nonspecific nitric oxide (NO) synthase (NOS) inhibitor] with microdialysis, demonstrated that improved NO-dependent vasodilation underlies the improvement in SkBF following 8 wk of intermittent whole body heat exposure. However, laser-Doppler flowmetry showed no increase in maximal cutaneous vascular conductance (CVC_max) in response to local heating to 43.5°C and infusion of 56 mM sodium nitroprusside (SNP).

Green et al. (16) demonstrated that repeated local heat training via water immersion of the forearm over 8 wk could improve SkBF responsiveness to a gradual local heating protocol. During immersion, shear stress was manipulated in one arm via a forearm cuff inflated to 100 mmHg proximal to the water bath. Interestingly, SkBF responses to local heating were not improved in the cuffed arm. It was concluded that an increase in shear stress is requisite for improving cutaneous microcirculation following local heat training. Maximal SkBF was not measured in this study. This is important, because it is unknown if the SkBF responses following repeated local heat training were improved by an increased ability of the skin vessels to vasodilate (indicative of a structural improvement) or by an improved responsiveness to a given stimulus (indicative of a functional improvement).

We designed the present study to determine whether 10 days of repeated local heat training could induce peripheral adaptations to the cutaneous vasculature, similar to observations in our laboratory following 10 days of whole body heat acclimation. We examined the hypothesis that 10 days of local forearm heat training would augment microvascular function in response to a local heating protocol as well as to local ACh administration. The SkBF response to rapid local heating is bimodal, with the initial peak mediated by local sensory nerves and the plateau phase mediated by NO, with a lesser role for endothelium-derived hyperpolarizing factors (EDHF) (4, 21, 24, 33). ACh is an endothelium-dependent vasodilator that is prostanoid-dependent, with smaller roles for NO and EDHF (3, 20, 25). Thus the use of ACh would allow assessment of other vasodilator systems that may contribute to improvements in SkBF following repeated forearm heating. We further hypothesized that local forearm heat acclimation would not increase CVC_max, demonstrating that a structural change in the microvasculature does not explain the improved vascular responses to local heating and ACh infusion.

METHODS

Subjects

All protocols used in the current study were approved by the Institutional Review Board at the University of Oregon. Prior to participation in the study, subjects gave both written and verbal informed consent in accordance with the Declaration of Helsinki. Seventeen recreationally active (2–8 h of exercise per week) young (18–29 yr old) subjects who had no history of cardiovascular disease and were not taking medications completed the local heat acclimation protocol. Because of several technical difficulties during the earlier phases of the study, microdialysis data from one subject and maximal forearm blood flow data from three subjects were excluded. Additionally, because of difficulty maintaining consistent water temperatures through the water-spray devices in three subjects, such that these subjects did not receive proper local heat acclimation, all data from these subjects were excluded. Because some subjects had a localized reaction to the microdialysis probes early in the study, these experiments were completed in a subset of the most recent subjects with use of fibers from a different manufacturer. As such, data were analyzed from 14 subjects [7 men (25 ± 1 yr old) and 7 women (20 ± 1 yr old)] as follows: SkBF during local heating in 14 subjects, SkBF during administration of ACh via microdialysis in 7 subjects, and maximal forearm blood flow in 11 subjects. To rule out the possibility that local forearm heating was affecting core body temperature, two of the initial subjects were instrumented with a rectal thermistor (series 400, Yellow Springs Instruments, Yellow Springs, OH) during forearm heating. All female participants were taking oral contraceptives and were studied during the active phase to minimize the impact of fluctuating sex steroids on cutaneous vascular function (6, 9).

Study Design

Participants underwent a number of physiological tests to assess SkBF and maximal forearm blood flow in both arms followed by a 10-day local forearm heat-training protocol in one arm and a thermoneutral temperature clamp in the contralateral arm; then these tests were repeated. This allowed for intrasubject and intra-arm comparisons. The local heat-training protocol consisted of 10 consecutive days of 60-min local forearm heat exposures of the experimental arm at 42°C using a cylindrical arm water-spray device (29, 45). During these sessions, the control forearm was fully submerged in a thermoneutral (32°C) water bath to clamp skin temperature. Control and experimental arms were randomly assigned and counterbalanced across subjects. Preacclimation studies were performed within 1–2 days before the beginning of repeated local forearm heat training, and postacclimation studies were completed within 1–2 days of completion of local forearm heat training.

Subject Monitoring

Subjects were instructed to fast for 4 h before reporting to the laboratory on the study days. They were also instructed to abstain from alcohol and caffeine for ≥12 h and not to exercise within 24 h before the study day. In addition, they were asked to avoid all supplements and nonprescription medications other than oral contraceptives for the entirety of the study and to maintain their current exercise regimens. All studies were performed in a temperature-controlled (22–24°C) laboratory, with subjects in the seated position. Blood pressure (Cardiocap, Datex Oheda, Louisville, CO) was assessed via the oscillatory method every 5–10 min throughout the study days.

Skin Blood Flow

SkBF was assessed on days 1 and 12. SkBF was measured as red blood cell flux using noninvasive laser-Doppler flowmetry (MoorLab, Moor Instruments, Devon, UK). Two laser-Doppler probes were used in combination with local skin heating units to investigate SkBF responses to local heating in each arm. In addition, a laser-Doppler probe was used to assess SkBF responses to 1, 10, and 100 mM ACh, as well as 56 mM SNP, administered via microdialysis in each arm. All laser-Doppler site locations were recorded and measured with reference to the antecubital fossa. The same laser-Doppler probes were used for pre- and posttreatment measurements on the experimental and control arms within subject and counterbalanced across subjects.

Specific Protocol (Days 1 and 12)

Local heating.

Once the subject had been seated for ≥20 min, the local heating devices (2 on each arm) with laser-Doppler flow probes were mounted on the ventral side of the arm in areas void of superficial veins. The local heaters were turned on and held at 33°C for 10 min while baseline data were collected. After collection of baseline data, the temperature of the local heaters was increased to 42°C at a rate of 0.5°C every 5 s. The local heaters were held at 42°C until a 10-min stable plateau in SkBF was reached. The temperature of the local heaters was then raised to 44°C to elicit maximal cutaneous vasodilation (31, 33). Because both arms were being studied simultaneously, blood pressure measurements were obtained during noncritical moments to minimize the effect this procedure might have on collection of blood flow data. Additionally, blood flow data were checked before and after each blood pressure measurement to ensure consistency.

Microdialysis.

A microdialysis fiber (MD 2000, 30-kDa cutoff, Bioanalytical Systems, West Lafayette, IN) was placed in the ventral forearm skin in both of the subject’s arms. [Note: because of manufacturing problems at Bioanalytical Systems, microdialysis fibers from a different manufacturer (CMA 31 linear probe, 55-kDa cutoff; CMA Microdialysi, Kista, Sweden) were used for the final 2 subjects. Because pre- and posttreatment comparisons were made and subjects were their own controls, we believed this to be of minimal consequence.] Microdialysis fibers were placed by insertion of a 25-gauge needle with entry and exit points ~2.5 cm apart. The microdialysis fiber was then threaded through the needle, and the needle was withdrawn from the skin with the fiber still in place. A period of 90–120 min was allowed for trauma from the needle placement to subside. Meanwhile, Ringer solution was continuously perfused through the fiber at a rate of 2.0 μl/min to keep the fiber membrane patent. Single-point laser-Doppler probes were placed over the microdialysis sites. The sites on both arms were monitored continuously and independently until a stable 10-min baseline was recorded. A perfusate containing 1, 10, and 100 mM ACh dissolved in Ringer solution was administered; 1 and 10 mM concentrations were chosen on the basis of previous research done in humans, including studies in our own laboratory (3, 28, 32, 43). Administration of 100 mM ACh was used to provide an endothelium-dependent method of achieving maximal dilation. ACh infusions were administered incrementally in order of increasing concentration. Each infusion lasted ≥20 min until a stable plateau in SkBF was achieved. After the 100 mM ACh infusion, 56 mM SNP was administered to confirm maximal dilation of the skin sites to 100 mM ACh. Administration of 100 mM ACh in the current study elicited maximal dilation in all subjects tested.

Maximal forearm blood flow.

Maximal forearm blood flow was assessed on days 2 and 11. Both the experimental and control arms were independently and sequentially locally heated in a cylindrical water-spray device that sprayed heated water (42°C) via nozzles encircling the arm for 45 min (41b). Again, the distance from the antecubital fossa to the point of forearm insertion in the spray device was measured to ensure that the same area of skin was being heated during the training days and the postmeasurement days. A thermistor applied to the ventral side of the forearm was used to ensure that skin temperature was 42°C, and water temperature was adjusted to maintain a constant thermal load (42°C) on the arm. Subjects were instrumented with a mercury-in-Silastic strain gauge (Hokanson, Bellevue, WA) for measurement of forearm blood flow using venous occlusion plethysmography. Forearm blood flow was measured at rest and at 15-min increments into heating.

Specific Protocol (Days 2 and 11)

The subject was seated in a phlebotomy chair and rested for 20 min. During this time, the subject was instrumented with a strain gauge and the arm was inserted into the cylindrical spray device, and resting forearm blood flow was measured using venous occlusion plethysmography. Water was then heated and perfused (Thermostat 2100, Fisher Scientific) through the cylindrical arm water-spray device at 44°C, the temperature necessary to raise forearm skin temperature to 42°C (assessed via thermistor). Forearm blood flow measurements were repeated at 15, 30, and 45 min into heating (41b). After arm heating, the subject rested for 20 min, and the protocol was repeated in the contralateral arm. The order in which experimental and control arms were heated was randomized; however, the order was maintained within subject pre- to posttreatment. The experimental arm was heated for an additional 15 min to ensure that days 2 and 11 could be considered “training” days.

Analysis

Data were digitized and saved using Windaq data acquisition software (Dataq Instruments, Akron, OH) at 40 Hz. CVC was calculated as red blood cell flux divided by mean arterial blood pressure. Local heating data are reported as a percentage of CVC_max. The initial peak during the local heating protocol was analyzed using a 20- to 30-s period of averaged SkBF. For the plateau during local heating and during drug infusions, a stable 5-min period of averaged SkBF was used for analysis. Forearm vascular conductance (FVC) was calculated as limb blood flow divided by mean arterial blood pressure. SkBF at each dose of ACh, each phase of local heating, and maximal FVC were compared using two-way repeated-measures analysis of variance with repeated factors of arm (experimental vs. control) and time into forearm heating (before vs. after 10 days of forearm heating). Tukey’s post hoc test was performed where significance was detected at α = 0.05. To also confirm that there were no differences between experimental and control arms before intervention, Student’s paired t-tests were performed for each measurement across ACh dose, local heating phase, and FVC. Values are means ± SE.

RESULTS

Figure 1 shows the cutaneous vascular responses to local heating (peak, nadir, and plateau) before and after repeated forearm heating of the experimental and control arms. There was no statistically significant interaction between before (Pre) and after (Post) repeated forearm heating and between control and experimental arms in the plateau phase of local heating [89 ± 3 (Pre) vs. 89 ± 2% CVC_max (Post) for experimental arm and 89 ± 2% CVC_max (Pre) vs. 89 ± 1% CVC_max (Post) for control arm (P = 0.19)]. In addition, there was no main effect of time (P = 0.24) or arm (P = 0.36). Despite the trending increase in the control arm during the nadir phase of heating, there was no statistically significant interaction between time and arm [64 ± 3% CVC_max (Pre) vs. 65 ± 2% CVC_max (Post) for experimental arm and 60 ± 3% CVC_max (Pre) vs. 66 ± 3% CVC_max (Post) for control arm (P = 0.36)]. Also, there was no main effect of time (P = 0.18) or arm (P = 0.69). The initial peak phase of heating increased slightly in the experimental and control arms; however, there was no interaction between Pre and Post and between control and experimental arms [73 ± 3% CVC_max (Pre) vs. 76 ± 2% CVC_max (Post) for experimental arm and 68 ± 2% CVC_max (Pre) vs. 75 ± 3% CVC_max (Post) for control arm (P = 0.47)]. There was no main effect of arm (P = 0.30); however, there was a main effect of time (P = 0.03). There were no significant differences between experimental and control arms before intervention.

Fig. 1.

Cutaneous vascular conductance (CVC) during local heating: initial peak, nadir, and plateau responses are presented in experimental and control arms before (light gray) and after (dark gray) training. Values are means ± SE for 14 subjects.

Figure 2 shows the cutaneous vascular responses to 1 and 10 mM ACh before and after repeated forearm heating of the experimental and control arms. There were no statistically significant interactions between Pre and Post and between experimental and control conditions at 1 mM ACh [43 ± 9% CVC_max (Pre) vs. 51 ± 6% CVC_max (Post) for experimental arm and 46 ± 11% CVC_max (Pre) vs. 59 ± 8% CVC_max (Post) for control arm (P = 0.76)] and 10 mM ACh [61 ± 9% CVC_max (Pre) vs. 82 ± 5% CVC_max (Post) for experimental arm and 75 ± 6% CVC_max (Pre) vs. 83 ± 5% CVC_max (Post) for control arm (P = 0.37)]. In addition, there was no main effect of time (P = 0.23) or arm (P = 0.57) for 1 mM ACh. There was no main effect of arm at 10 mM ACh (P = 0.16); however, there was a main effect of time (P = 0.03).

Fig. 2.

CVC during ACh administration (1 and 10 mM) in experimental and control arms before (light gray) and after (dark gray) training. Preheating intervention values to 10 mM ACh were lower on average in the experimental than the control arm. Values are means ± SE for 7 subjects; individual subject data are also plotted.

Figure 3 shows the forearm vascular responses to 45 min of forearm heating to elicit maximal forearm blood flow before and after repeated forearm heating in the experimental and control arms. There was no statistically significant interaction between Pre and Post and between experimental and control conditions [0.12 ± 0.02 FVC (Pre) vs. 0.14 ± 0.02 FVC (Post) for experimental arm and 0.11 ± 0.01 FVC (Pre) vs. 0.12 ± 0.02 FVC (Post) for control arm (P = 0.30)]. In addition, there was no main effect of time (P = 0.10) or arm (P = 0.78). There were no significant differences between the experimental and control arms preintervention.

Fig. 3.

Effect of repeated forearm heating on maximal forearm vascular conductance in control (Con) and experimental (Exp) arms before (Pre, light gray bars) and after (Post, dark gray bars) intervention. Assessment of maximal forearm skin blood flow showed no changes after repeated local forearm heating. Values are means ± SE for 11 subjects. VOP, venous occlusion plethysmography.

No rise in core temperature was observed in the two subjects instrumented with rectal thermistors.

DISCUSSION

We previously demonstrated that 10 days of whole body heat acclimation can confer peripheral adaptations to the cutaneous vasculature (28). This was important, because it demonstrated that exercise heat acclimation confers functional improvements to the cutaneous vasculature independent of centrally mediated changes in neural outflow. The aim of the present study was to determine whether 10 days of repeated local forearm heat training, independent of changes in core temperature, could augment cutaneous microvascular function, similar to previous observations in our laboratory, as well as after 8 wk of chronic local or whole body heat exposure (5, 8, 16, 28). We also assessed maximal SkBF to determine whether 10 days of repeated local heating affects overall maximal dilator capacity.

The major findings of the current study in young healthy individuals and relative to the thermoneutral water-treated (sham) arm are as follows. 1) Short-term local forearm heat acclimation does not improve the cutaneous vascular responses to local thermal hyperemia or to locally administered ACh in the skin microcirculation. 2) Maximal forearm SkBF does not change following 10 days of local forearm heating. 3) Ten days of local forearm heat acclimation is insufficient to elicit local adaptations to the cutaneous vasculature.

The results of the present study are inconsistent with the few previous reports that have assessed peripheral adaptations in the cutaneous vasculature following 10 days of whole body exercise heat acclimation or 8 wk of chronic local heat training or passive whole body heating (5, 8, 16, 28). Lorenzo and Minson (28) demonstrated increased SkBF responsiveness to ACh locally applied under thermoneutral conditions after 10 days of whole body heat acclimation in trained cyclists. However, there was no change in the response to thermal hyperemia with local heating. Furthermore, there was no change in maximal forearm SkBF. It is generally agreed that ACh is an endothelium-dependent vasodilator that works through a prostanoid-dependent pathway, with smaller roles for NO and EDHF (3, 20, 24), whereas vasodilation to gradual or rapid local heating of the skin is mediated primarily by local sensory nerves and local NO production, as well as EDHF (4, 21, 25, 33). To our knowledge, this is the first study to investigate potential improvements in cutaneous vascular responsiveness to an endothelium-dependent, nonthermal stimulus following a 10-day period of repeated forearm heating. The present study indicates that 10 days of local forearm heating was not associated with a significant increase in the cholinergic responsiveness of the cutaneous microcirculation, as previously observed after 10 days of whole body heat acclimation (Fig. 2). Based on our data and the findings of others, we believe that 10 days is on the cusp of the time necessary to induce local microvascular adaptations using repeated local heating. That is, adaptation is beginning in some subjects, but not in others. Evidence by Fox et al. (15) suggests that 15 days of heating the arm in water at 43°C for 2 h/day was sufficient to improve SR in the treated arm during a bout of controlled hyperthermia. This would seem to indicate that peripheral adaptations may occur on a time course similar to our study; however, the following factors must be considered. 1) Fox et al. studied the local training effects of heat and sweating in a small sample size (n = 2). It may be that sweat glands are faster to adapt in the presence of repeated local heat or that these individuals were fast to respond. 2) Fox et al. heated the subjects’ forearms for ~30 h over the course of 15 days, whereas in the present study the subjects’ forearms were heated for ~10 h over 10 days. Despite their small sample size, Fox et al. observed significant changes in forearm SR. The magnitude of the daily heating stimulus and the duration of our study suggest that perhaps we did not adequately heat the forearm to cause local adaptations. It is also possible that the peripheral vascular adaptations from repeated forearm heating are mechanistically different from the peripheral adaptations from whole body heating. This may be due to differences in the mode of heat stimulus. For example, local heating of the forearm to 42°C increases SkBF to ~90% of CVC_max (Fig. 1) and significantly increases local skin temperature. Conversely, exercise whole body heating raises internal temperature and neurally mediates an increase in SkBF to ~50–70% of CVC_max with only a modest increase in local skin temperature.

Brunt et al. (5) recently demonstrated improved cutaneous thermal hyperemia via improved NO bioavailability following 8 wk (4–5 days/wk) of whole body passive heat therapy. Pharmacological blockade via microdialysis allowed for interrogation of the mechanisms of cutaneous microvascular adaptation following heat acclimation. In particular, use of N^ω-nitro-l-arginine (a nonspecific NOS inhibitor) demonstrated that heat therapy improved NO-dependent dilation. These investigators proposed that an increase in SkBF and shear stress and an upregulation of heat shock proteins (Hsps) via elevated core temperature were essential for these adaptations.

Green et al. (16) first demonstrated improvements in cutaneous vasodilation following a long-term repeated local heating protocol in which shear stress was manipulated. They performed bilateral heating of the forearms via warm water immersion (42°C), three times a week for 30 min, for 8 wk. During immersion, shear stress was manipulated in one of the arms via inflation of a pneumatic cuff to 100 mmHg, while the other arm was not cuffed. In the uncuffed forearm, there were significant differences between week 0 and 4 at the peak (42°C) of an incremental local heating protocol that remained elevated at week 8. These improvements were not observed in the cuffed forearm, highlighting the role of episodic shear stress, and not heat per se, in the peripheral vascular adaptation. This is consistent with other studies that have demonstrated that vascular improvements in NO-mediated endothelial function following exercise are dependent on shear stress (2, 17, 46).

While Green et al. (16) concluded that shear stress was a necessary stimulus for microvascular adaptation following repeated forearm heat training, they were unable to distinguish between the role of shear stress and/or skin temperature in driving the adaptations. More recently, Carter et al. (8) demonstrated that 8 wk of heat acclimation, using lower body warm water immersion, improved cutaneous vascular responsiveness to local heating of the skin in the forearm. However, when forearm skin temperature was clamped at 30°C across the 8 wk, improvements in cutaneous vascular function were no longer observed. Additionally, when both SkBF and temperature were clamped by placement of a pneumatic cuff, no adaptations occurred. It was concluded that cutaneous microvascular adaptations following 8 wk of heat acclimation are dependent on repeated episodic shear stress and that skin temperature can modulate this response.

It is possible that the rise in SkBF during hyperthermia and the attendant rise in shear stress could have elicited the microvascular changes observed by Lorenzo and Minson (28) during exercise heat acclimation; however, no improvement in NO-dependent SkBF (rapid local heating) was observed. Furthermore, in the present study we did not observe microvascular adaptations, despite a larger shear stimulus/blood flow over the same time course used by Lorenzo and Minson. When considered with the aforementioned studies, this would suggest that vascular adaptations to these stimuli may require >10 days. Alternatively, humoral factors associated with an elevated core body temperature during heat acclimation sessions could have contributed. For example, Hsp90 is an essential cofactor of NOS that is upregulated by heat and can augment activity independent of NOS abundance (18, 36). In another study, in vitro heat shock at 43.5°C resulted in increased Hsp90 expression in human umbilical vein endothelial cells, which is associated with increased basal endothelial NOS activity as well as increased NO bioavailability (41). Conversely, in vivo exposure to 42°C for 15 min has been shown to upregulate Hsp70 expression in rats (1, 27). This suggests that there may be additional factors in vivo that lower the necessary threshold for Hsp expression. It is therefore feasible that repeated forearm heating to 42°C may have provided an adequate stimulus to upregulate Hsps locally in the vasculature and may account for some of the NO-mediated adaptations attributed to shear stress by Green et al. (16) but that these adaptations require >10 days when heat is not locally applied in the presence of whole body heat stress.

To our knowledge, no attempt has been made to quantify maximal SkBF following repeated local heating of the forearm. In the present study this was assessed by local heating of the forearm at 42°C with a warm water-spray device for 45 min, which has previously been reported to achieve maximal forearm SkBF (29, 45). Assessment of maximal forearm SkBF in the present study showed no changes after repeated local forearm heating (Fig. 3). It can be concluded that 10 days of forearm heating does not alter maximal forearm blood flow, the majority of which is blood flow to the skin. However, because of differences in methodology (10 days vs. 8 wk), it cannot be reasonably concluded that maximal SkBF is not affected by more prolonged repeated heating of the forearm.

There are a few limitations to this study. We observed a main effect of time at 10 mM ACh due to a slight increase in the control and experimental arms. However, we did not observe a statistically significant interaction with seven subjects. It is important to note that the preheating intervention values to 10 mM ACh in the experimental arm were lower on average than the preintervention values in the control arm (Fig. 2). Even if we had observed a statistically significant interaction, it is unlikely to be physiologically meaningful, as the postintervention values were similar in the two arms. Furthermore, sample size calculations for local heating and FVC reveal that ~50 subjects would be required to achieve a power of 0.80 (α = 0.05). This suggests that any adaptation that did occur would be physiologically negligible.

In summary, our results provide evidence that >10 days may be required for meaningful peripheral vascular adaptations from repeated forearm heating in young healthy individuals. Furthermore, our findings, compared with those of Lorenzo and Minson (28), suggest that whole body heat acclimation confers peripheral adaptations that are secondary to changes in internal temperature. It may also be that individuals from various disease populations demonstrate peripheral adaptations that cannot be observed in young healthy individuals. For example, acute lower limb heating improved macrovascular and microvascular function assessed after heating in aged adults, but not in young healthy individuals (39). While the aforementioned study is acute and resulted in a rise in core body temperature, it may be that a ceiling effect in young healthy subjects is masking potential peripheral adaptations observed in at-risk populations. Additional studies are needed to characterize the mechanisms that underlie peripheral adaptation following long-term repeated local heating and to further elucidate the minimum exposures required for shorter-term repeated local heating.

GRANTS

This work was supported by the Kenneth and Kenda Singer Endowed Professorship in Human Physiology.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

M.A.F., V.E.B., and K.N.J. performed experiments; M.A.F. and V.E.B. analyzed data; M.A.F., V.E.B., and C.T.M. interpreted results of experiments; M.A.F. prepared figures; M.A.F. drafted manuscript; M.A.F., V.E.B., S.L., and C.T.M. edited and revised manuscript; M.A.F., V.E.B., S.L., and C.T.M. approved final version of manuscript; V.E.B., K.N.J., S.L., and C.T.M. conceived and designed research.