Journal of Thermal Biology 45 (2014) 168–174

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Responses in acral and non-acral skin vasomotion and temperature during lowering of ambient temperature Maja Elstad a,n,1, Leif Vanggaard b,1, Astrid H Lossius c, Lars Walløe a, Tone Kristin Bergersen a,c a

Department of Physiology, Institute of Basic Medical Sciences, University of Oslo, Norway Arctic Institute, Denmark c Dermatologic Department, Oslo University Hospital, Norway b

art ic l e i nf o

a b s t r a c t

Article history: Received 22 April 2014 Received in revised form 15 September 2014 Accepted 15 September 2014 Available online 18 September 2014

Arteriovenous anastomoses (AVA) in acral skin (palms and soles) have a huge capacity to shunt blood directly from the arteries to the superficial venous plexus of the extremities. We hypothesized that acral skin, which supplies blood to the superficial venous plexus, has a stronger influence on blood flow adjustments during cooling in thermoneutral subjects than does non-acral skin. Thirteen healthy subjects were exposed to stepwise cooling from 32 °C to 25 °C and 17 °C in a climate chamber. Laser Doppler flux and skin temperature were measured simultaneously from the left and right third finger pulp and bilateral upper arm skin. Coherence and correlation analyses were performed of short-term fluctuations at each temperature interval. The flux from finger pulps showed the synchronous spontaneous fluctuations characteristic of skin areas containing AVAs. Fluctuation frequency, amplitude and synchronicity were all higher at 25 °C than at 32 °C and 17 °C (po 0.02). Bilateral flux from the upper arm skin showed an irregular, asynchronous vasomotor pattern with small amplitudes which were independent of ambient temperature. At 32 °C, ipsilateral median flux values from the right arm (95% confidence intervals) were 492 arbitrary units (au) (417, 537) in finger pulp and 43 au (35, 60) in upper arm skin. Flux values gradually decreased in finger pulp to 246 au (109, 363) at 25 °C, before an abrupt fall occurred at a median room temperature of 24 °C, resulting in a flux value of 79 au (31, 116) at 17 °C. In the upper arm skin a gradual fall throughout the cooling period to 21 au (13, 27) at 17 °C was observed. The fact that the response of blood flow to ambient cooling is stronger in acral skin than in non-acral skin suggests that AVAs have a greater capacity to adjust blood flow in thermoneutral zone than arterioles in non-acral skin. & 2014 Elsevier Ltd. All rights reserved.

Keywords: Arteriovenous anastomoses Skin perfusion Skin temperature Laser Doppler flux Temperature regulation

1. Introduction One important function of the skin is continuous regulation of central body temperature, which is kept within narrow limits by the hypothalamic feedback loop (Hodges and Johnson, 2009; Savage and Brengelman, 1996; Kingma et al., 2012). The autonomic nervous system controls skin blood flow by adjusting the frequency of the tonic impulses of the sympathetic vasoconstrictor Abbreviations: AVAs, arteriovenous anastomoses; CI, confidence interval; DAR, right dorsal upper arm; LDF, laser Doppler flux; LF, left third finger pulp; RF, right third finger pulp; T, skin temperature; TNZ, thermoneutral zone; VAL, left volar upper arm n Correspondence to: Department of Physiology, Institute of Basic Medical Sciences, University of Oslo, PO Box 1103 Blindern, N-0317 Oslo, Norway. E-mail address: [email protected] (M. Elstad). 1 Shared first authorship. http://dx.doi.org/10.1016/j.jtherbio.2014.09.003 0306-4565/& 2014 Elsevier Ltd. All rights reserved.

nerves (Bini et al., 1980; Johnson and Kellogg, 2010) and by activation of the vasodilator system during sweating (Johnson and Kellogg, 2010). There are large differences between acral and non-acral skin in neurovascular control and the types of resistance vessels that are present, and thus very different skin blood flow patterns in the two types of skin areas (Bergersen, 1993; Blair et al., 1960; Eriksen and Lossius, 1995; Romanovsky, 2014; Vanggaard et al., 2012). The range of ambient temperature that does not trigger shivering or sweating in a naked subject is termed the thermoneutral zone (TNZ) (Erikson et al., 1956; Lopez et al., 1994; Savage and Brengelman, 1996). Within the TNZ, body temperature is controlled solely by adjusting skin blood flow (Kingma et al., 2012), which accounts for 5–10% of cardiac output at rest (Rowell, 1977). During cooling, heat is conserved by an increase in skin vascular resistance, including constriction of the superficial veins.

M. Elstad et al. / Journal of Thermal Biology 45 (2014) 168–174

A decrease in superficial venous plexus blood volume is important in reducing heat dissipation because blood passes just below the skin surface of the extremities at low velocity (Rowell, 1977). The arterial side of the microvascular bed in skin consists of arterioles, pre-capillary sphincters, arterial capillaries and arteriovenous anastomoses (AVAs) (Higgins and Eady, 1981; Ryan, 1973). AVAs form direct links between arterioles and venules and are located in the skin of palms, soles, nose and external ear (Bergersen, 1993; Grant, 1930; Prichard and Daniel, 1956). They are not found in the skin of the back of the hand, arm, trunk or abdomen (Grant and Bland, 1931; Gray, 1989). AVAs have a thick muscular vessel wall and are more densely innervated than arterioles (Funk et al., 1994). Direct microscopic observations of the rabbit ear, where the density of AVAs is as high as in the human finger, show that the vasomotor activity of the AVAs is independent of the arterioles (Grant, 1930). With a diameter of up to 150 mm compared to the capillary diameter of 10 mm, the AVAs provide a low-resistance pathway (Jessen, 2001) and have a huge capacity to shunt blood directly from the arteries to the veins. They thus have a major impact on heat dissipation from skin surface of the extremities as a whole. A number of studies have suggested that the large synchronous blood velocity fluctuations in acral blood flow represent flow through the AVAs. Acral blood flow fluctuations are found to be closely correlated in parts of the body as widely separated as the finger of the right hand and the toe of the left foot (Burton, 1939). The frequency of blood flow fluctuations changes with the temperature balance of the subjects (Burton, 1939; Lossius et al., 1993; Thoresen and Walløe, 1980). In cool subjects (room temperature 15-18 °C), low finger blood flow is accompanied by a high frequency of sympathetic impulses, and in warm subjects (room temperature 35-42 °C), high finger blood flow is accompanied by a low frequency of sympathetic impulses. In thermoneutral subjects, the fluctuation frequency is two to three sympathetically induced vasoconstrictions per minute (Bini et al., 1980; Thoresen and Walløe, 1980). It has been suggested that the AVA rhythm is determined by the thermostat function of the hypothalamic center (Burton, 1939; Thoresen and Walløe, 1980). The frequency of spontaneous sympathetic activity in nerves to non-acral skin in thermoneutral subjects does not show a rhythmic pattern but is more random and low (Bini et al., 1980). Blood perfusion through the skin of the trunk and the back of the hand is low and shows an irregular non- synchronous pattern (Bergersen, 1993; Lossius et al., 1993). In the TNZ, the small increase or decrease in blood flow in non-acral skin is driven by similarly small changes in sympathetic vasoconstrictor activity to maintain homeothermy (Johnson et al., 2014). In acral skin of subjects in their TNZ, high flow through the shunt vessels of the AVAs will dominate over the low irregular perfusion through the arterioles, so that laser Doppler flux from the finger pulp mainly reflects the vasomotor activity of the AVAs (Eriksen and Lossius, 1995). We have recently shown distinct differences between the response of acral and non-acral skin temperature to a gradual reduction of ambient temperature from 32 °C to 13 °C (Vanggaard et al., 2012). The decrease in skin temperature during the cooling procedure occurred significantly later in acral than in non-acral skin. We interpreted this as a reflection of differences in vasomotor control between acral and non-acral skin. The majority of skin blood flow studies on thermoregulation have been performed on non-acral skin in areas such as the forearm and trunk (Hodges and Johnson, 2009; Kingma et al., 2012). This is the first study to use simultaneous recordings of blood flow in acral and non-acral skin during cooling in a thermoneutral environment. Our aim was to compare the vasomotor activity of AVAs in acral skin with that of arterioles in nonacral skin during cooling within the TNZ. We hypothesized that if

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acral skin has a stronger influence on blood flow adjustments during cooling within the TNZ than non-acral skin, then AVAs play a greater role in blood flow regulation during ambient cooling. We also intended to identify a suitable ambient temperature for investigating acral skin blood flow in thermoneutral conditions.

2. Material and methods 2.1. Subjects Thirteen healthy, non-smoking medical students (4 male, 9 female), aged between 19 and 23 years, were recruited to the study. They were asked not to drink coffee or tea or to undertake any exercise on the experimental day or eat for at least two hours before the start of each experiment. None of the subjects had any symptoms of cardiovascular disorder and none used any medication. Informed consent was obtained from all subjects. Their physical fitness was normal, and all took weekly exercise (median 6 h, range 2–8 h). The females were in the follicular phase of their menstrual cycle (days 2–14). 2.2. Protocol and instrumental set-up The experiments were carried out in a quiet climate chamber with the subject resting on a bench in a supine position, dressed in shorts and singlet. Each subject was acclimatized for 15–20 min at a room temperature of 32 °C and relative humidity of 20%. The room temperature was adjusted in accordance with the experimental protocol. After 15 min of recordings at 32 °C, the room temperature was gradually lowered, first from 32 to 25 °C and then from 25 to 17 °C. 15 min of recordings were made while the temperature was kept stable at 25 °C and 17 °C. The transition between the phases took around 20 min. In order to check probe position, sympathetic vasoconstriction episodes were induced by deep inspirations during the first and last 5 min of the recording. Laser Doppler flux (LDF) (DRT4, Moor Instruments, Devon, UK) and skin temperature (T) (YSI-401, YSI Inc., OH, USA) were recorded from acral skin (pulp of the right third finger (RF) and pulp of the left third finger (LF)) and from non-acral skin (volar side of the left upper arm (VAL) and dorsal side of the right upper arm (DAR)). Temperature probes were fastened 5–10 mm away from the LDF probes using surgical tape (3M Benderm, Micropore No). The noise-limiting filter on the Laser Doppler instrument was set at its highest level (21 kHz), and the emitted wavelength was 820 nm. The flux output signal was filtered at time constant 0.1 s. Instantaneous arterial blood pressure was obtained from the left fourth finger (Finometer). Three-lead ECG, tympanic temperature and room temperature were also recorded. All recordings were made continuously and simultaneously and the signals were sent to the computer for beat-by-beat averaging gated by ECG R waves. Tympanic temperature was recorded before cooling. The decrease in core temperature observed during previous cooling from 32 to 17 °C was 0.3 °C (Vanggaard et al., 2012) and thus not significant for our protocol. 2.3. Data analysis and statistics The correlation and cross-spectral analyses were made using 7.5 min recordings. Analyses were performed between flux recordings from RF and LF (acral skin), between flux recordings from DAR and VAL (non-acral skin) and between flux recordings from RF and DAR (acral and non-acral skin). Since fluctuation frequency in the finger pulp was lower at 32 °C than at 25 °C, a longer time interval was used at 32 °C to ensure that equal numbers of events were studied in the correlation analyses.

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Cross-spectral analysis provided the phase angles and coherence between the two signals approximately at 0.05 Hz (0.03– 0.07 Hz). A frequency interval of 0.03–0.07 Hz corresponds to fluctuations with periodicity between 14.2 and 33.3 s, covering the 2–3 AVA vasomotion episodes per minute (Thoresen and Walløe, 1980). Phase angles provided a measure of the phase shift between oscillating curves. The coherence estimate, which varies from 0 to 1, serves as a statistical measure of the reliability of the phase angle estimate and the linearity of the relationship. Since phase angles are on a closed circle, circular statistics were applied when estimating mean direction and variance. Averaged phase angles were computed by weighting the phase angles with their squared coherence, and standard deviations for the phase angles were calculated according to circular variance (Mardia, 1972). We considered two variables to be in phase if the phase angle between them was less than 45°, and to be in inverse phase if the phase angle was more than 135°. The Wilcoxon signed rank sum test against a two-sided alternative was used to test for differences between situations, with significance level p ¼0.05. The Wilcoxon median and upper and lower limits of the 95% confidence interval (CI) are reported, which corresponds to the non-parametric one-sample test (Hollander and Wolfe, 1999). The mean LDF values and range of the short-term velocity variations, estimated as 10 and 90% fractiles of the flux values measured, were calculated in a 745-s sliding window. Before averaging among the subjects, all LDF recordings were scaled to convert the 5-min mean of the 90% fractile before cooling to unity. Because there were small variations in the time interval required

to lower the room temperature, the time axes of the parts of the recordings made during temperature changes were scaled to be 15 min for all recordings before averaging among the subjects. The range of short-term variability estimated as the 10th and 90th percentile was calculated as the difference between the 90th and 10th percentile.

3. Results Ambient cooling from 32 to 17 °C (Fig. 1A) resulted in a reduction in skin temperature of approximately 10–12 °C for acral skin (RF and LF) and approximately 5 °C for non-acral skin (DAR and VAL) (Fig.1B). Beat-by-beat Laser Doppler flux (LDF) from the right and left arm are displayed in Fig. 1C and D, respectively. Table 1 summarises skin temperatures and mean LDF values at the three room temperature plateaus. Mean tympanic temperature before the protocol was started was 36.2 °C (95% CI: 35.9, 36.5). 3.1. Microcirculation in acral skin At 32 °C (Figs. 1C and D, upper tracings), LDF values in the pulp of the third finger of both hands were high (median 492 au RF and 421 au LF, Table 1) with large synchronous fluctuations (median phase angle 0.05 rad), high coherence (median 0.7) (Fig. 2) and high correlation (median r ¼0.87, 97.8% CI: 0.74, 0.94) seen in skin areas containing AVAs. The LDF fluctuations showed 1.7 vasoconstriction episodes per min (95% CI: 1.0, 2.3) and a median amplitude of 141 au (95% CI: 98, 209 au, Fig. 3B).

Skin Temperatures

Ambient temperature Temperature ( C)

Temperature ( C)

30 25 20 15

35 30 25 20 15

500 1000 1500 2000 2500 3000 3500 4000

500 1000 1500 2000 2500 3000 3500 4000

Time (s)

Time (s)

Laser Doppler Flux Right Side 600

Laser Doppler Flux Left Side LDF-RF LDF-DAR

400

200

0

LDF-LF LDF-VAL

600

Flux (au)

Flux (au)

T-VAL T-DAR T-RF T-LF

40

35

400

200

0

500 1000 1500 2000 2500 3000 3500 4000

Time (s)

500 1000 1500 2000 2500 3000 3500 4000

Time (s)

Fig. 1. Skin temperature and laser Doppler flux in one subject. Simultaneous bilateral recordings from pulp of the third fingers (acral skin) and upper arm skin (non-acral skin) in one subject during whole body surface cooling in a climate chamber from 32 °C to 17 °C (a).The 10-min plateau periods at 32 °C, 25 °C and 17 °C are indicated by vertical dotted lines. (b) Shows skin temperature, (c) laser Doppler flux right side, and (d) laser Doppler flux left side. T-VAL, skin temperature volar upper arm left; T-DAR, skin temperature dorsal upper arm right; T-RF, skin temperature right finger pulp; T-LF, skin temperature left finger pulp; LDF-VAL, laser Doppler flux volar upper arm left; LDF-DAR, laser Doppler flux dorsal upper arm right; LDF-RF, laser Doppler flux right finger pulp; LDF-LF, laser Doppler flux left finger pulp.

M. Elstad et al. / Journal of Thermal Biology 45 (2014) 168–174

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Table 1 Skin temperature, laser Doppler flux and heart rate at three room temperature plateaus. Variable

32 °C

25 °C

17 °C

T-ambient (°C) T-Dorsal upper arm right (°C) T-Volar upper arm left (°C) T-Right finger (°C) T-Left finger (°C) LDF-Dorsal upper arm right (au) LDF-Volar upper arm left (au) LDF-Right finger (au) LDF-Left finger (au) Heart rate (bpm)

32.7 [32.6. 32.9] 34.6 [34.2, 34.9]

25.6 [25.4, 25.8] 31.9 [31.3, 32.5]n

17.8 [17.5, 18.1] 29.3 [28.3, 30.2]n

þ

35.4 [35.0, 35.8]

33.1 [32.6, 33.7]n

30.4 [29.9, 31.4]n

þ

35.7 [35.3, 35.9] 35.5 [35.0, 35.9] 43.4 [34.6, 59.8]

31.7 [30.4, 33.3]n 31.2 [29.6, 33.0]n 32.0 [22.4, 42.6]¤

25.1 [23.5, 26.6]n þ 23.7 [21.6, 26.2]n þ 20.5 [13.0, 27.1]n#

41.6 [22.6, 78.2]

27.3 [15.5, 44.9]¤

14.9 [10.3, 22.3]¤#

491.6 [417.2, 536.8] 421.1 [322.1, 525.9] 66.6 [58.4, 74.7]

245.8 [108.7, 362.9]n 221.1 [93.5, 323.7]n 66.1 [59.7, 72.1]

78.7 [31.0, 116.3]n þ 58.4 [18.9, 92.4]n þ 65.4 [60.5, 72.8]#

Median and 95% confidence interval calculated by Hodges–Lehmann's estimate. T—temperature of skin or ambient temperature; LDF— laser Doppler flux. n

p¼ 0.0002 Significantly different from 32 °C. p¼ 0.0002 Significantly different from 25 °C. ¤ p o 0.05 Significantly different from 32 °C. # p o 0.05 Significantly different from 25 °C. þ

90 180

90 0 180

270

90 0 180

270

0 270

1.0

Coherence

0.8 0.6

25 °C than at 32 °C (median 243 au (95% CI: 197, 295 au, p ¼0.017, Fig. 3B). During further cooling from 25 °C to 17 °C, the bilateral maximum LDF values in the finger pulps decreased gradually until sustained vasoconstriction was seen at 17 °C with low LDF values (median 79 au RF and 58 au LF, Table 1). At 17 °C, the strength of the synchronicity (median phase angle –0.13) between LDF fluctuations in RF and LF was significantly lower than at 25 °C (coherence 0.68, p ¼0.02, Fig. 2, and correlation median r ¼0.82, 97.8% CI: 0.67, 0.86, p¼ 0.02). Maximum finger pulp flux dropped to the 50-percentile of the maximum values calculated at 32 °C at a median room temperature of 24.2 °C (95% CI:21.8 °C, 27 °C). 3.2. Microcirculation in non-acral skin

0.4 0.2 0.0

32

25

17

Temperature ( C) Fig. 2. Phase angles and coherence between laser Doppler flux in the right and left finger pulp at three room temperatures. The upper three circles show the individual phase angles between laser Doppler flux in the right and left finger pulp (open small circle indicates coherence below 0.5). The lines indicate the median phase angle. The three lower graphs indicate the individual coherence between laser Doppler flux in the right and left finger pulp. The horizontal lines indicate the median coherence.

At 25 °C, the strength of the synchronicity (median phase angle 0.04 rad) between the LDF values of RF and LF was higher than at 32 C. The coherence was higher than at 32 °C (median 0.9, p ¼0.002, Fig. 2), and correlation remained high (median r ¼0.92, 97.8% CI: 0.85, 0.96, p ¼0.1). The LDF fluctuations showed 2.5 vasoconstriction episodes per min (95% CI: 1.9, 2.9), which was significantly higher than observed at 32 °C (p ¼0.005). At 25 °C, both the maximum and minimum LDF values gradually decreased to half the value at 32 °C (Fig., 3B, Table 1). However, the LDF fluctuation amplitude in the finger pulp was significantly higher at

At 32 °C (Figs. 1C and D, lower tracings), LDF patterns in the upper dorsal right arm (DAR) and upper volar left arm (VAL) were different from those recorded from the finger pulps. The LDF values were much lower (median DAR 43.4, median VAL 41.6, Table 1) than those measured in the finger pulp and the fluctuation amplitudes were much smaller (Fig. 3C). There was low synchronicity between the bilateral upper arm LDF values (median coherence 0.29, median correlation r ¼0.34, 97.8% CI: 0.28, 0.50) and no synchronicity between RF and DAR (median coherence 0.13, median correlation r¼ –0.02, 97.8% CI: –0.05, 0.29). Because of the low coherence between DAR and VAL, phase angles were not calculated. During cooling from 32 °C to 17 °C, LDF values at all temperatures were lower than the minimum LDF values in the finger pulp (Table 1). The LDF values of the skin of the upper arm showed a gradual small decrease to median DAR 21 au and median VAL 15 au (Table 1), with little change in LDF fluctuation amplitude compared to the changes in LDF amplitude of finger pulp (Table 1, Fig. 1C and D, Fig. 3B and C). The fluctuation pattern was similar at 25 °C and 17 °C, with median coherence below 0.4 for all three plateaus (Figs. 1C and D, lower traces, Table 1). 3.3. Skin temperature Skin temperature is influenced both by ambient temperature and by skin blood flow. When room temperature was lowered from 32 °C to 25 °C, the temperature of non-acral and acral skin decreased at the same rate,  0.3 °C decrease per 1 °C decrease in room temperature (mean decrease in skin temperature per 1 °C

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Laser Doppler Flux Right Finger

Ambient temperature 600

30

Total flux (au)

Temperature ( C)

35

25 20

400

200

0

15 500

1000 1500

500

3000 3500 4000

1000 1500

3000 3500 4000

Time (s)

Time (s)

Laser Doppler Flux Right Upper Arm

Flux (au)

60

40

20

0 500

1000 1500

3000 3500 4000

Time(s) Fig. 3. Amplitude range of laser Doppler flux on the right side. The lower graphs show mean of responses in finger and arm skin on cooling from 32 °C to 17 °C displayed as 7 45 s sliding window average (middle trace) and 10th and 90th percentiles of flux samples in the window. The amplitude of the fluctuations in laser Doppler flux of the finger pulp is higher at 25 °C than at 32 °C and drops again at 17 °C.

decrease in room temperature with 95% CI: T-RF 0.41 (0.29, 0.54), T-LF 0.48 (0.35, 0.63), T-DAR 0.27 (0.24, 0.32) and T-VAL 0.37 (0.30, 0.43)). During further cooling, acral and non-acral skin showed different responses. 3.3.1. Acral skin Further cooling from 25 °C to 17 °C gave a larger drop in the skin temperature of the finger pulp than the first cooling step (mean decrease in skin temperature per 1 °C decrease in room temperature and 95% CI: T-RF 0.61 (0.53, 0.71), T-LF 0.75 (0.61, 0.913), p o0.01). The drop in finger pulp temperature during cooling from 25 to 17 °C was 6.6 °C (Table 1, Fig. 1), as compared with a median drop of 4.0 °C (Table 1, Fig. 1) from 32 to 25 °C. It was correlated with the cessation of LDF fluctuations in the finger pulp and occurred at a skin temperature of 30.9 °C (95% CI: 29.8 °C, 32.0 °C) in the right finger pulp and 30.8 °C (95% CI: 28.8 °C, 32.4 °C) in the left finger pulp. Vasoconstriction occurred when the skin temperature of the finger pulp was 6.8 °C (95% CI: 4.2 °C, 8.3 °C, right) or 6.1 °C (95% CI: 3.4 °C, 7.6 °C, left) higher than room temperature. 3.3.2. Non-acral skin The gradual fall in LDF values recorded from the upper left volar arm and upper right dorsal arm were correlated with a

gradual fall in skin temperature at the same sites (Fig. 1). The change in skin temperature was similar during cooling from 32 to 25 °C (DAR 2.7°, VAL 2.3 °C, Table 1) and from 25 to 17 °C (DAR 2.6 °C, VAL 2.7 °C, Table 1). (Mean decrease in skin temperature per 1 °C decrease in room temperature with 95% CI: T-DAR 0.25 (0.19, 0.30) and T-VAL 0.36 (0.29, 0.42)).

4. Discussion This paper presents the first study of the impact of ambient cooling on skin blood flow measured by laser Doppler in acral and non-acral skin in thermoneutral subjects. The large fluctuations in acral skin blood flow are compared with blood flow fluctuations in non-acral skin. The highest values for the frequency, amplitude and synchronicity of blood flow fluctuations in acral skin were found at a room temperature between 32 and 24 °C. The nonsynchronous pattern of blood flow in non-acral skin was unaffected by the cooling procedure. The main difference between the microcirculation of acral and non-acral skin is the presence of AVAs in acral skin. The characteristic pattern of blood flow fluctuations in acral skin is caused by synchronous vasomotor activity of the AVAs. The AVAs are capable of making large adjustments in blood flow through synchronous activation and

M. Elstad et al. / Journal of Thermal Biology 45 (2014) 168–174

this, combined with their capacity to supply the venous plexus of the extremities, means that acral skin has a stronger influence on blood flow within the TNZ than arterioles in non-acral skin. The blood volume in the venous plexus is the main source of heat dissipation from the extremities (Rowell, 1977).

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loss of regulation of heat dissipation from the extremities. Standardised investigations of AVA function could probably provide useful information on a number of diseases of the skin, microcirculation and nervous system. 4.3. Limitations

4.1. The AVA organ The present study supports the hypothesis that acral and nonacral skin play different roles in temperature regulation (Romanovsky, 2014). Our results show low LDF values with a gradual fall in non-acral skin blood flow during cooling, as also found by others (Johnson et al., 2014; Roddie and Shepherd, 1957; Savage and Brengelman, 1996). In acral skin, high fluctuating values were seen until the AVAs closed and LDF values dropped by 83%. Closure of the AVAs leads to low velocities in the supplying arteries of the extremities (Thoresen and Walløe, 1980). Heat conservation mechanisms in the skin of the extremities are governed both by the AVAs and by the skin arterioles. Closure of the AVAs and arterioles together with constriction of superficial veins directs blood flow to the deep veins, where heat conservation occurs through a countercurrent mechanism between arteries and the deep veins (Jessen, 2001). The low blood velocity in veins and arteries results in heat transfer from the arterial blood to the blood in the deep veins and back to the body core (Jessen, 2001). The AVAs and the superficial veins can be considered to form an entity, called the AVA organ (Vanggaard et al., 2012), which covers approximately 50% of the skin surface (the surface of the full length of the four limbs (Rhodes et al., 2013)). Most heat dissipates from the slow-moving blood in the superficial veins of the extremities (Rowell, 1977). Thus, their ability to shunt blood from the arteries to the veins gives the AVAs a central role in adjusting heat dissipation from the extremities during cooling. In addition, the arterioles of the skin contribute to heat conservation. Although their capacity to adjust blood flow within the TNZ is relatively small, they cover a large skin surface area. In the present study a rapid drop in skin temperature occurred when room temperature was lowered from 25 °C to 17 °C (median 24.2 °C). We suggest that the rapid drop in skin temperature is caused by rather abrupt closing of the AVAs. However, the 95% CI was rather broad, from 21.8 °C to 27 °C, reflecting large interindividual differences in the TNZ. 4.2. Room temperature during clinical and experimental investigations Our most important finding is that the blood flow fluctuations in AVA skin of the right and left hand show the largest amplitude range, highest fluctuation frequency and greatest synchronicity at room temperatures between 32 and 24 °C (Bergersen et al., 1995; Burton, 1939; Lossius and Eriksen, 1995; Thoresen and Walløe, 1980). Recently, AVA fluctuations have been suggested as an objective criterion for determining a subject′s TNZ (Kingma et al., 2012). Our study supports this suggestion, and we propose that clinical and experimental investigations in which thermoneutral state is important should be performed in room temperatures above 24 °C. If investigations of AVA vasomotion are performed at temperatures below 24 °C, most of the AVA fluctuation is eliminated. Within the TNZ, AVA fluctuations are an important mechanism for regulation of heat dissipation from the extremities. Thus a high fluctuation frequency reflects standard thermoneutral conditions, and it is important to identify these conditions in studies of skin blood flow. We have recently documented that the AVA vasomotion pattern is lost in systemic sclerosis patients with digital ulcers (Bergersen et al., 2014). In acral vascular syndromes, the microcirculation of palms and soles is disturbed, leading to

In the study protocol we used only three room temperature plateaus, separated by 7 °C. In order to define an optimum between 25 °C and 32 °C we would need finer ambient temperature resolution. The vasomotor activity of the AVAs is measured indirectly by changes in blood flow measured by laser Doppler flux. 4.4. Clinical implications In physiological and clinical investigations where AVA function may be of importance, room temperature needs to be within the TNZ. Skin circulation influenced by the TNZ should be investigated at room temperatures above 24 °C. A room temperature of around 25 °C (above 24 °C) is better for studying AVA function than a temperature of around 32 °C because amplitude range, synchronicity and fluctuation frequency are all higher at 25 °C.

5. Conclusion We have shown that the response of vasomotor activity to ambient cooling is stronger in acral skin than in non-acral skin in subjects within their TNZ. We found that AVAs in acral skin have a greater capacity to adjust blood flow during cooling within the TNZ than the arterioles in the non-acral skin of the extremities. In addition, we propose that the AVA fluctuation pattern could be used to define the TNZ. AVA fluctuation frequency and synchronicity are higher at ambient temperatures above 24 °C. Thus, studies of acral blood flow should be performed at ambient temperatures above 24 °C.

Acknowledgements Maja Elstad was funded by the Research Council of Norway (Grant no. 230354).

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