Scand J Med Sci Sports 2015: 25: 650–660 doi: 10.1111/sms.12291

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Hand temperature responses to local cooling after a 10-day confinement to normobaric hypoxia with and without exercise M. E. Keramidas1, R. Kölegård1, I. B. Mekjavic2, O. Eiken1 1

Department of Environmental Physiology, School of Technology and Health, Royal Institute of Technology, Stockholm, Sweden, Department of Automation, Biocybernetics and Robotics, Jozef Stefan Institute, Ljubljana, Slovenia Corresponding author: Michail E. Keramidas, MSc, PhD, Department of Environmental Physiology, School of Technology and Health, Royal Institute of Technology, Berzelius väg 13, Solna SE-171 65, Sweden. Tel: +46 8 524 839 69, Fax: +46 8 330923, E-mail: [email protected] 2

Accepted for publication 17 June 2014

The study examined the effects of a 10-day normobaric hypoxic confinement (FiO2: 0.14), with [hypoxic exercise training (HT); n = 8)] or without [hypoxic ambulatory (HA; n = 6)] exercise, on the hand temperature responses during and after local cold stress. Before and after the confinement, subjects immersed their right hand for 30 min in 8 °C water [cold water immersion (CWI)], followed by a 15-min spontaneous rewarming (RW), while breathing either room air (AIR), or a hypoxic gas mixture (HYPO). The hand temperature responses were monitored with thermocouples and infrared thermography. The confinement did not influence the hand temperature responses of the HA group during the AIR and HYPO

CWI and the HYPO RW phases; but it impaired the AIR RW response (−1.3 °C; P = 0.05). After the confinement, the hand temperature responses were unaltered in the HT group throughout the AIR trial. However, the average hand temperature was increased during the HYPO CWI (+0.5 °C; P ≤ 0.05) and RW (+2.4 °C; P ≤ 0.001) phases. Accordingly, present findings suggest that prolonged exposure to normobaric hypoxia per se does not alter the hand temperature responses to local cooling; yet, it impairs the normoxic RW response. Conversely, the combined stimuli of continuous hypoxia and exercise enhance the finger cold-induced vasodilatation and hand RW responses, specifically, under hypoxic conditions.

Exposure to high altitude is commonly considered a predisposing environmental factor for local cold injury (Harirchi et al., 2005).Yet, the findings derived from crosssectional studies examining the peripheral vasomotor responses to local cooling, assumed to reflect risk of local cold injury, during and after prolonged hypoxia are equivocal; a few studies have observed an enhancement of local cold tolerance (Nair et al., 1973; Mathew et al., 1977; Rai et al., 1978; Felicijan et al., 2008; Amon et al., 2012), while others have reported either an impairment (Nair et al., 1973; Savourey et al., 1997; Purkayastha et al., 1999; Castellani et al., 2002) or no change (Nair et al., 1973; Mathew et al., 1977; Daanen & van Ruiten, 2000). At high altitude, hypoxia coexists with other environmental and behavioral stressors, mainly cold and exercise, which, independently or synergistically, determine the nature and the level of acclimatization to the environment. Specifically, long-term exposure to cold environment might increase local cold tolerance (Purkayastha et al., 1992; Brandstrom et al., 2008), although conversely an exaggerated cold-induced peripheral vasoconstriction has also been observed (Livingstone, 1976; Livingstone et al., 1996). In addition, regular physical activity enhances the cold-induced vasodilatation (CIVD) response in the fingers (Keramidas et al., 2010), an effect that is more

pronounced when the exercise training is combined with long-term, intermittent hypoxic exposures (i.e., sleep-high train-low protocol; Amon et al., 2012). Hence, whether the direction and the magnitude of the peripheral vasomotor responses to local cooling after a period of altitude acclimatization are dependent upon the distinct effect of hypoxia, cold, and exercise, or upon the interference of these components, still remains uncertain. Accordingly, the purpose of the present study was to examine the effects of a 10-day confinement to normobaric hypoxia on the hand temperature responses of healthy male lowlanders during and after local cold stress. To separately determine the contribution of the hypoxic stimulus per se, and its interaction with exercise training to the acclimation process, subjects were divided into two groups: (a) the hypoxic ambulatory (HA) group; and (b) the hypoxic exercise training (HT) group. We hypothesized that, due to hypoxia-induced peripheral vasoconstriction (Durand et al., 1969; Passino et al., 1996), a 10-day period of continuous hypoxia would increase cutaneous vasoconstriction in the hand during and after local cold provocation. However, based on the recurrent evidence that regular exercise enhances local cold tolerance (Keramidas et al., 2010; Amon et al., 2012), we hypothesized that the synergy of

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Hypoxic acclimation and local cooling hypoxia and exercise would augment the finger CIVD and the hand rewarming (RW) responses. Materials and methods The study was conducted at the Olympic Sports Center Planica (Ratecˇe, Slovenia) that is situated at an altitude of 940 m. The study was part of a larger project investigating the effects of a 10-day hypoxic confinement on several functions of the cardiovascular, respiratory, and thermoregulatory systems. Results from the other investigations have been (Debevec et al., 2014) or will be reported elsewhere.

Subjects Sixteen healthy men participated in the study. However, two of the subjects withdrew during the intervening period (one subject for personal reasons, and the other for medical reasons), and hence 14 subjects were included in the final analysis. All subjects were near-sea level residents, and had not been exposed to altitude > 500 m during the month preceding the experiments. They were nonsmokers, and had no history of any cardiovascular or pulmonary disease. All of them were physically active on a recreational basis, and had no or very limited previous experience with cold exposure experiments. The subjects were informed in detail about the experimental procedures, and gave their written consent. The experimental protocol was approved by the National Committee for Medical Ethics at the Ministry of Health of the Republic of Slovenia and conformed to the Declaration of Helsinki.

Experimental protocol Before the main experimental testing sessions, all subjects performed an incremental exercise test to exhaustion on a cycle 2 max ) , ergometer to determine their maximal oxygen uptake ( VO which was measured online with a metabolic cart (Quark CPET; Cosmed, Rome, Italy). Following that, subjects were balanced for  2 max , and assigned to either the HA (n = 6, age: 25 ± 3 age and VO years, height: 177.7 ± 3.5 cm, body mass: 70.4 ± 10 kg, body fat:  2 max : 3.0 ± 0.6 L/min) or the HT (n = 8, age: 21.9 ± 4.7%, VO 26 ± 2 years, height: 182.7 ± 5.9 cm, body mass: 76.6 ± 6.3 kg,  2 max : 3.2 ± 0.3 L/min) group. body fat: 22.8 ± 6.2%, VO The overall experimental protocol is presented in Fig. 1. Both groups were continuously confined for 10 days on one floor of the Olympic Sports Center, which was always maintained in hypoxic conditions (FiO2: 0.14; ambient simulated altitude of ∼ 4100 m). Before and after the hypoxic confinement period, all subjects conducted a blood examination and a hypoxic incremental exercise test to exhaustion. On a separate day, they performed two cold water hand immersion trials, during which they were inspiring either room air (AIR) or a hypoxic gas mixture (HYPO; FiO2: 0.14).

were confined to a total area of ∼ 360 m2, which included eight double bedrooms and a living room. Hypoxia on the floor was achieved using an O2 dilution system (b-Cat, Tiel, the Netherlands), based on the vacuum pressure swing adsorption principle; the O2 level was monitored continuously with O2 sensors (PGM1100; Rae Systems, San Jose, California, USA). The mean ambient temperature, relative humidity, and barometric pressure in the living area were 23.1 ± 1.0 °C, 56 ± 8%, and 682 ± 4 mmHg, respectively. All subjects consumed the same standardized diet on each day, and were allowed to drink water and tea ad libitum. During the 10-day period, the HA group was requested not to engage in any strenuous activity, and to limit their physical activity to slow walks in the living area. The HT group, in contrast, exercised twice daily, once in the morning and once in the afternoon, in a designated hypoxic laboratory (FiO2: 0.14). Each training session consisted of a 60-min cycling at 50% of the hypoxic peak power output (PPO) determined prior to the onset of the confinement. Thus, the exercise intensity was maintained so that the heart rate (HR) corresponded to the HR measured at 50% of PPO during the hypoxic incremental exercise test. HR (iBody; Wahoo Fitness, Atlanta, Georgia, USA) and capillary oxyhemoglobin saturation (SpO2, 3100 WristOx; Nonin Medicals, Plymouth, Minnesota, USA) were monitored and recorded continuously during the exercise sessions. The mean HR and SpO2 values obtained during the training sessions were 142 ± 11 bpm and 85 ± 3%, respectively.

Testing sessions Hematological analyses After an overnight fast, venous blood samples (2 mL) were collected from the antecubital vein of the subjects to determine their hemoglobin concentration ([Hb]), erythrocyte volume fraction (EVF), and red blood cell (RBC) concentration. The analyses were obtained with an automated laser-based hematology analyzer (ADVIA 2120; Siemens, Munich, Germany) within 6 h after the blood sampling.

Exercise testing All subjects performed an incremental exercise test to exhaustion on an electrically braked cycle-ergometer (Daum Electronic, Furth, Germany), during which they were breathing a hypoxic gas mixture (FiO2: 0.14). The exercise load was increased by 25 W/min until exhaustion, which was defined by the inability to maintain exercise at a given workload (cycling cadence < 60 rpm) and a subjective rating of perception of effort at or near maximal. PPO was calculated from the last completed workload by adding to it the fraction of time spent at the final non-completed workload multiplied by 25 W.

Hypoxic confinement

Cold water hand immersion trials

The hypoxic confinement period commenced at 09:00 h and finished 10 days later at 09:00 h. Throughout this period, both groups

The AIR and HYPO cold water hand immersion trials were conducted in a counterbalanced order and separated by a 60-min

Fig. 1. Schematic representation of the overall study protocol.

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Keramidas et al. interval. The sequence of the trials was the same for each subject during both testing periods. All the trials were performed in the morning to ensure that the effect of diurnal variations was similar. Subjects were dressed in a T-shirt, short trousers, and socks, and remained in a sitting position on a chair. Prior to the start of each trial, subjects were accustomed to the laboratory conditions for ∼ 20 min. The mean temperature and relative humidity in the laboratory were 21.0 ± 0.8 °C and 38 ± 6%, respectively. Throughout the HYPO trial, subjects breathed through a lowresistance two-way respiratory valve (Model 2, 700 T-Shape; Hans Rudolph, Inc., Shawnee, Oklahoma. USA). The inspiratory side of the valve was connected via a respiratory corrugated tubing to a 200-L Douglas bag filled with the premixed humidified breathing mixture. Both trials commenced with a 5-min baseline phase, during which subjects rested with both hands on an emissivity neutral, customized hand support at the level of their hips. After that, the right hand was covered with a thin plastic bag (thickness of 0.025 mm) sealed with air-permeable tape to the skin (∼ 10 cm above the wrist), and the hand was then immersed up to the ulnar and radial styloids in warm water (35 °C) for 5 min. Subsequently, the hand was removed from the warm water tank, and placed without the plastic bag on the hand support for ∼ 1 min, during which infrared thermal images were obtained. Thereafter, the hand was covered with a new plastic bag and was immersed in a tank containing cold water (8 °C) for 30 min [cold water immersion (CWI)]. The temperature of the water was maintained by means of a cooling system (Haake, Karlsruhe, Germany), and a small impeller continuously stirred the water inside the tank. After the completion of the CWI phase, the hand was removed from the water, dried with a towel, if necessary, and a 15-min spontaneous RW phase ensued, during which both hands were resting on the hand support as in the baseline phase. Throughout each trial, subjects were instructed to keep the non-immersed hand immobile on the hand support at the level of the hip. The finger skin temperatures of the immersed hand were measured with copper-constantan (T-type) thermocouple (each conductor was 0.2 mm in diameter) probes (Physitemp Instruments Inc, Clifton, New Jersey, USA), which were attached to the skin in the middle of the palmar side of the distal phalanx of each finger. The primary insulation of the thermocouples was polytetrafluoroethylene; the non-insulated welded junctions of the thermocouples were attached directly to the skin with thin airpermeable tape (Tegaderm; 3M Healthcare, St. Paul, Minnesota, USA). All skin temperatures were sampled every second with a NI USB-6215 (National Instruments, Austin, Texas, USA) data acquisition system, processed with TestPoint software (TestPoint v7®, Norton, Massachusetts, USA) and stored in a PC (Dell, Round Rock, Texas, USA) for further analysis. Following a manual check of the raw data, a computer program written in TestPoint (Massachusetts, USA) was used to calculate the average temperature (Tavg) of each finger obtained during every phase, as well as the minimum (Tmin) and maximum (Tmax) temperatures reached during the CWI and RW phases. The same program was also used to detect any finger CIVD response, defined as a local skin temperature wave (N) in terms of > 1 °C increase lasting for a minimum duration of 3 min. In case of a CIVD response, the following parameters were determined: (a) the temperature amplitude (ΔT), which was the difference between the lowest temperature recorded just before the CIVD and the highest temperature reached during the CIVD; and (b) the duration of a local skin temperature wave (Δt). Moreover, Tavg of the distal phalanx of the thumb of the left non-immersed hand was also measured with a thermocouple probe (National Instruments). At the end of the baseline phase, immediately after the warm water phase, and during the RW phase (at minutes 1, 2, 3, 4, 5, 10, and 15), the skin temperature from the dorsal and ventral sides of the immersed hand was recorded with an infrared camera

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(T365; FLIR Systems AB, Täby, Sweden), which was calibrated automatically. The distance between the camera and the hand was ∼ 60 cm. After the completion of the test, the thermal images were transferred to a PC (Dell) via a USB cable, and subsequently analyzed using the ThermaCam Researcher PRO software (version 2.10; FLIR Systems). Tavg was determined for the following anatomical areas of the right hand: (a) the palmar (Fp-d) and the dorsal (Fd-d) sides of the distal phalanx of each finger; (b) the palmar (Fp-p) and the dorsal (Fd-p) sides of the proximal phalanx of each finger; (c) the total palm; and (d) opisthenar areas. During the baseline and RW phases, the infrared tympanic temperature (Ttympanic) was measured using an infrared thermometer (ThermoScan IRT 3020; Braun, Kronberg, Germany). Two consecutive measurements were obtained each time and the higher of the two values was used for subsequent analysis. Throughout the trials, HR was recorded using a HR monitor (S800CX; Polar, Kempele, Finland). Systolic arterial pressure (SAP) and diastolic arterial pressure (DAP) were measured at 5-min intervals with a noninvasive oscillometric automated sphygmomanometer (Omron M6, Kyoto, Japan) positioned on the left upper arm. SpO2 was monitored with a finger pulse oxymeter (BCI 3301, Waukesha, Wisconsin, USA) on the left index finger. During the baseline, warm water, CWI (at minutes 1, 2, 3, 4, and 5, and every 5 min thereafter), and RW phases, the subjects were requested to provide ratings of their thermal sensation on a 7-point scale (from 1 = cold to 7 = hot), thermal comfort on a 4-point scale (from 1 = comfortable to 4 = very uncomfortable), and local pain on a 10-point scale (from 0 = no pain to 10 = unbearable pain). All scales were explained to the subjects by the same investigator prior to each test.

Statistical analysis Statistical analyses were performed using Statistica 5.0 (StatSoft, Inc., Tulsa, Oklahoma, USA). All variables are presented as mean ± SD, unless otherwise indicated. Considering the high interindividual variability of the CIVD response (O’Brien, 2005), and due to the unequal group sizes (cf. Roberts & Russo, 2006), we reasoned that it was appropriate to examine the effects of the two experimental interventions separately by comparing the post-confinement responses to the pre-confinement responses. Accordingly, a two-way analysis of variance (ANOVA) with repeated measures was used to evaluate the temperature (testing period × digit/hand region) and hemodynamic (testing period × phase) responses in each group. The Tukey post-hoc test was employed to identify specific differences between means when ANOVAs revealed significant F-ratio for main effects. Differences in N, thermal comfort, thermal sensation, and local pain were evaluated with a Wilcoxon-matched pairs nonparametric test. The statistical significance of PPO and hematological responses were assessed with a paired t-test analysis. The alpha level of significance was set a priori at 0.05.

Results PPO and hematological responses Following the 10-day confinement period, PPO was increased in the HT group (pre = 259 ± 34 W, post = 276 ± 34 W; P = 0.002), but remained unchanged in the HA group (pre = 244 ± 37 W, post = 247 ± 41 W; P = 0.41). In both groups, there was a significant increase in [Hb] (HA group: pre = 155 ± 7 g/L, post = 166 ± 8 g/L; HT group: pre = 151 ± 8 g/L, post = 162 ± 15 g/L), EVF (HA group: pre = 0.45 ± 0.02, post = 0.48 ± 0.03; HT group: pre = 0.44 ± 0.03, post = 0.47 ± 0.04), and RBCs

Hypoxic acclimation and local cooling (HA group: pre = 5.2 ± 0.3 1012·L−1, post = 5.6 ± 0.3 1012·L−1; HT group: pre = 5.0 ± 0.2 1012·L−1, post = 5.3 ± 0.5 1012·L−1) after the hypoxic confinement (P ≤ 0.05).

Cold water hand immersion trial Skin temperature responses AIR trial. The confinement period tended to decrease baseline Tavg in the HA group (∼ 1 °C; Fig. 2(a)), and to increase it in the HT group (∼ 1.3 °C; Fig. 2(b)), albeit the differences between the pre- and post-trials were not statistically significant (HA group: P = 0.35; HT group: P = 0.55). During the warm water immersion phase, both the HA (pre = 31.1 ± 1.2 °C, post = 30.9 ± 1.0 °C; P = 0.34) and the HT (pre = 31.2 ± 0.8 °C, post = 31.1 ± 0.8 °C; P = 0.87) groups had similar hand Tavg in the pre- and post-trials.

Each group’s mean time series for Tavg of the index finger during the cold water immersion trial are depicted in Fig. 3(a) and (b). During the CWI phase, Tavg, Tmin, and Tmax of the fingers did not differ between the testing periods for any of the groups (P > 0.05; Tables 1 and 2). Likewise, no differences were observed between the testing periods in any of the other CIVD parameters, such as N, ΔT, and Δt (P > 0.05; Tables 1 and 2). Throughout the CWI phase, Tavg of the non-immersed thumb was unaltered by the confinement period both in the HA group (pre = 29.2 ± 3.1 °C, post = 30.4 ± 3.4 °C; P = 0.29) and the HT group (pre = 30.7 ± 3.0 °C, post = 31.3 ± 3.4 °C; P = 0.68). During the RW phase, the finger Tmin was similar in the two testing periods for both groups (HA group: pre = 9.1 ± 0.7 °C, post = 9.4 ± 0.9 °C; HT group: pre = 9.1 ± 1.0 °C, post = 9.1 ± 0.3 °C). However, the response of the hand during the post-RW phase was not the same in the

Fig. 2. Average temperature (Tavg) of each segment of the right hand and of the overall hand during the 5-min baseline phase in the control (AIR) and hypoxic (HYPO) trial before and after the 10-day confinement period for the hypoxic ambulatory (HA; a, c) and hypoxic training (HT; b, d) groups. Values are mean ± SD. *Significantly different from the pre-trial (P ≤ 0.05). Opisth: total opisthenar area; Fp-d: palmar side of the distal phalanx of the fingers; Fp-p: palmar side of the proximal phalanx of the fingers; Fd-d: dorsal side of the distal phalanx of the fingers; Fd-p: dorsal side of the proximal phalanx of the fingers.

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Fig. 3. Average temperature (Tavg) of the palmar side of the distal phalanx of the right index during the control (AIR) and the hypoxic (HYPO) cold water hand immersion trial before and after the 10-day confinement period for the hypoxic ambulatory (HA; a, c) and hypoxic training (HT; b, d) groups. Values are mean ± SD.

Table 1. Average temperature (Tavg), minimum temperature (Tmin), maximum temperature (Tmax), number of local skin temperature waves (N ), temperature amplitude of the wave (ΔT ), and duration of the wave (Δt) on the palmar side of the distal phalanx of each finger of the right hand obtained during the 30-min cold water immersion phase in the control (AIR) trial in the hypoxic ambulatory group (HA, n = 6)

Pre

Tavg (°C) Tmin (°C) Tmax (°C) N ΔT (°C) Δt (min)

Post

I

II

III

IV

V

I

II

III

IV

V

10.6 ± 1.0 8.8 ± 0.6 12.3 ± 1.2 0 (0–2) 0.5 ± 0.8 2.3 ± 3.6

11.0 ± 0.5 9.1 ± 0.5 13.0 ± 1.6 1 (1–2) 3.2 ± 2.4 9.5 ± 2.4

10.7 ± 0.8 8.8 ± 0.5 13.0 ± 3.0 1 (0–2) 2.6 ± 2.5 8.3 ± 5.8

10.3 ± 0.4 8.7 ± 0.4 12.4 ± 1.1 1 (1–3) 2.7 ± 1.6 7.0 ± 0.7

10.1 ± 0.6 8.5 ± 0.5 11.7 ± 1.6 1 (0–2) 2.5 ± 2.2 8.3 ± 5.3

10.3 ± 1.4 8.7 ± 0.4 11.5 ± 1.4 0 (0–1) 0.5 ± 1.2 2.6 ± 5.9

11.0 ± 1.6 8.8 ± .0.4 13.7 ± 4.2 1 (0–2) 2.8 ± 2.9 5.3 ± 5.4

10.5 ± 1.5 8.6 ± 0.5 13.1 ± 3.9 1 (0–1) 3.1 ± 3.8 6.6 ± 5.9

10.4 ± 1.1 8.5 ± 0.4 12.8 ± 3.2 1 (0–2) 2.8 ± 3.3 6.2 ± 5.6

10.3 ± 1.1 8.5 ± 0.4 13.1 ± 3.2 1 (0–2) 3.1 ± 2.9 6.0 ± 4.9

Values are mean ± SD. Values are median (range) for N. Finger I, thumb; Finger II, index; Finger III, middle; Finger IV, ring; Finger V, small.

two groups after the confinement (Fig. 4(a) and (b)). In particular, in the HA group, the confinement impaired the RW response in almost all regions (P = 0.05; Fig. 5(a)). On the contrary, in the HT group, the confinement did not affect the RW Tavg of the hand (P = 0.99; Fig. 5(b)).

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HYPO trial. The hand Tavg in the HA group was similar in the two testing periods both during the baseline (P = 0.99; Fig. 2(c)) and the warm water immersion (pre = 31.1 ± 0.9 °C, post = 31.0 ± 1.2 °C; P = 0.84) phase. Conversely, in the HT group, the baseline hand

Hypoxic acclimation and local cooling Table 2. Average temperature (Tavg), minimum temperature (Tmin), maximum temperature (Tmax), number of local skin temperature waves (N ), temperature amplitude of the wave (ΔT ), and duration of the wave (Δt) on the palmar side of the distal phalanx of each finger of the right hand obtained during the 30-min cold water immersion phase in the control (AIR) trial in the hypoxic training group (HT, n = 8)

Pre

Tavg (°C) Tmin (°C) Tmax (°C) N ΔT (°C) Δt (min)

Post

I

II

III

IV

V

I

II

III

IV

V

10.2 ± 0.6 8.6 ± 0.7 12.3 ± 1.3 1 (0–1) 0.8 ± 1.0 3.6 ± 3.9

10.1 ± 0.5 8.6 ± 0.6 11.6 ± 0.8 1 (0–2) 1.7 ± 1.1 8.2 ± 6.4

10.0 ± 0.9 8.3 ± 0.6 11.8 ± 0.5 1 (0–2) 1.7 ± 1.9 6.3 ± 6.0

9.8 ± 0.6 8.4 ± 0.5 11.2 ± 0.7 1 (1–2) 1.6 ± 1.1 6.8 ± 5.4

9.4 ± 0.3 8.2 ± 0.4 10.6 ± 0.5 1 (0–2) 1.3 ± 1.0 6.8 ± 5.2

10.2 ± 0.6 8.8 ± 0.4 11.8 ± 1.3 0 (0–1) 0.4 ± 0.8 1.4 ± 2.4

10.5 ± 0.7 9.1 ± .0.5 11.6 ± 1.1 1 (0–2) 1.6 ± 1.4 7.5 ± 6.1

10.0 ± 1.0 8.6 ± 0.7 11.5 ± 1.7 1 (0–1) 1.8 ± 1.9 5.4 ± 5.4

9.9 ± 0.9 8.7 ± 0.7 11.1 ± 1.3 1 (0–2) 1.5 ± 1.4 5.1 ± 4.8

9.7 ± 0.7 8.5 ± 0.6 10.8 ± 1.1 1 (0–1) 1.3 ± 1.3 5.3 ± 5.0

Values are mean ± SD. Values are median (range) for N. Finger I, thumb; Finger II, index; Finger III, middle; Finger IV, ring; Finger V, small.

Fig. 4. Representative infrared thermal images from one subject of the hypoxic ambulatory (HA; a, c) group and one subject of the hypoxic training (HT; b, d) group at the 15th minute of the rewarming phase in the control (AIR) and hypoxic (HYPO) trial before and after the 10-day confinement period.

Tavg was ∼ 2.2 °C warmer in the post- than the pre-trial (P = 0.04; Fig. 2(d)), whereas there were no differences in Tavg between the testing periods during the warm water immersion phase (pre = 31.1 ± 0.9 °C, post = 31.6 ± 0.8 °C; P = 0.13); but the Tavg of the palmar side of the distal phalanx of the fingers was significantly higher after the confinement (pre = 33.8 ± 2.3 °C, post = 35.2 ± 0.9 °C; P = 0.04). In the HA group, there were no differences between the testing periods neither in Tavg nor in any of the other CIVD parameters during the CWI phase (P > 0.05; Table 3, Fig. 3(c)). In the HT group, the confinement increased Tavg of the thumb, index, and small fingers during the CWI phase (Table 4, Fig. 3(d)), but it did not affect any of the CIVD parameters (P > 0.05; Table 4).

In the HA group, there was a statistical tendency for confinement to increase Tavg of the non-immersed thumb during the post-CWI phase (pre = 29.3 ± 3.8 °C, post = 30.8 ± 3.1 °C; P = 0.07). In the HT group, Tavg of the non-immersed thumb during the CWI phase was significantly higher post- than pre-confinement (pre = 31.1 ± 2.1 °C, post = 33.0 ± 1.9 °C; P = 0.02). The RW Tmin of the fingers was not altered by the confinement in any of the groups (HA group: pre = 9.8 ± 0.5 °C, post = 9.5 ± 1.0 °C; HT group: pre = 9.1 ± 0.7 °C, post = 9.4 ± 0.9 °C; P > 0.05). In the HA group, the Tavg hand RW response was unchanged by the confinement (P = 0.40; Figs. 4(c) and 5(c)), whereas in the HT group, the hand Tavg was markedly increased during the post-RW phase (P = 0.001; Figs. 4(d) and 5(d)).

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Fig. 5. Average temperature (Tavg) of each segment of the right hand and of the overall hand during the last 5 min of the rewarming (RW) phase in the control (AIR) and hypoxic (HYPO) trial before and after the 10-day confinement period for the hypoxic ambulatory (HA; a, c) and hypoxic training (HT; b, d) groups. Values are mean ± SD. *Significantly different from the pre-trial (P ≤ 0.05). Opisth: total opisthenar area; Fp-d: palmar side of the distal phalanx of the fingers; Fp-p: palmar side of the proximal phalanx of the fingers; Fd-d: dorsal side of the distal phalanx of the fingers; Fd-p: dorsal side of the proximal phalanx of the fingers. Table 3. Average temperature (Tavg), minimum temperature (Tmin), maximum temperature (Tmax), number of local skin temperature waves (N ), temperature amplitude of the wave (ΔT ), and duration of the wave (Δt) on the palmar side of the distal phalanx of each finger of the right hand obtained during the 30-min cold water immersion phase in the hypoxic (HYPO) trial in the hypoxic ambulatory group (HA, n = 6)

Pre

Tavg (°C) Tmin (°C) Tmax (°C) N ΔT (°C) Δt (min)

Post

I

II

III

IV

V

I

II

III

IV

V

10.2 ± 0.5 8.9 ± 0.8 11.8 ± 0.7 0 (0–1) 0.5 ± 0.8 1.8 ± 3.6

10.7 ± 0.4 9.2 ± 0.7 12.2 ± 0.9 1 (0–2) 2.3 ± 1.7 6.2 ± 4.1

10.4 ± 0.5 8.8 ± 0.7 11.7 ± 0.8 1 (0–2) 1.8 ± 1.5 5.2 ± 4.8

10.0 ± 0.6 8.6 ± 0.6 11.5 ± 1.5 1 (1–2) 2.0 ± 2.3 6.3 ± 4.3

10.1 ± 0.3 8.6 ± 0.6 11.5 ± 1.0 1 (0–2) 1.9 ± 1.9 6.0 ± 2.7

10.4 ± 1.0 8.9 ± 0.6 12.0 ± 1.2 0 (0–2) 0.6 ± 1.0 2.5 ± 4.8

10.6 ± 0.7 8.9 ± .0.5 12.3 ± 1.7 1 (0–2) 2.4 ± 2.4 9.4 ± 5.0

10.2 ± 0.5 8.6 ± 0.5 11.9 ± 1.2 1 (0–1) 2.6 ± 1.8 4.5 ± 6.3

10.3 ± 0.5 8.7 ± 0.5 12.0 ± 1.2 1 (0–2) 2.6 ± 1.7 4.7 ± 5.9

10.2 ± 0.6 8.6 ± 0.6 12.0 ± 1.0 1 (0–2) 1.6 ± 1.8 4.0 ± 5.0

Values are mean ± SD. Values are median (range) for N. Finger I, thumb; Finger II, index; Finger III, middle; Finger IV, ring; Finger V, small.

Ttympanic and hemodynamic responses AIR trial. Ttympanic did not vary during the course of the pre-trial (baseline phase: 36.4 ± 0.4 °C, RW phase = 36.3 ± 0.5 °C; P = 0.23) or differ between the

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testing periods (pre = 36.4 ± 0.5 °C, post = 36.3 ± 0.4 °C; P = 0.11) in any of the groups. In the HA group, no differences were observed between the testing periods for HR (P = 0.74), SAP (P = 0.10), or DAP

Hypoxic acclimation and local cooling Table 4. Average temperature (Tavg), minimum temperature (Tmin), maximum temperature (Tmax), number of local skin temperature waves (N ), temperature amplitude of the wave (ΔT ), and duration of the wave (Δt) on the palmar side of the distal phalanx of each finger of the right hand obtained during the 30-min cold water immersion phase in the hypoxic (HYPO) trial in the hypoxic training group (HT, n = 8)

Pre

Tavg (°C) Tmin (°C) Tmax (°C) N ΔT (°C) Δt (min)

Post

I

II

III

IV

V

I

II

III

IV

V

9.9 ± 0.6 8.6 ± 0.6 11.3 ± 0.9 0 (0–2) 0.3 ± 0.5 1.8 ± 3.6

10.2 ± 0.8 8.8 ± 0.8 11.5 ± 0.7 1 (0–3) 1.5 ± 1.1 6.2 ± 4.1

9.9 ± 0.7 8.4 ± 0.7 11.5 ± 0.4 1 (0–2) 1.2 ± 1.1 5.2 ± 4.8

9.9 ± 0.7 8.6 ± 0.7 11.0 ± 0.6 1 (0–3) 1.3 ± 0.9 6.3 ± 4.3

9.3 ± 0.6 8.2 ± 0.5 10.1 ± 0.5 1 (0–2) 1.3 ± 0.7 6.0 ± 2.7

10.5 ± 1.0* 8.7 ± 0.4 12.3 ± 1.5 0 (0–1) 0.8 ± 1.4 2.5 ± 4.8

10.8 ± 1.2* 9.0 ± 0.5 12.7 ± 3 1 (0–2) 2.8 ± 3.1 9.4 ± 5.0

10.1 ± 0.9 8.5 ± 0.5 11.9 ± 2.2 1 (0–1) 1.4 ± 2.7 4.5 ± 6.3

10.1 ± 1.2 8.7 ± 0.6 11.6 ± 2.2 1 (0–2) 1.5 ± 2.6 4.7 ± 5.9

9.9 ± 1.1* 8.5 ± 0.4* 11.3 ± 2.6 1 (0–2) 1.5 ± 2.4 4.0 ± 5.0

*Significant difference from the pre-trial (P ≤ 0.05). Values are mean ± SD. Values are median (range) for N. Finger I, thumb; Finger II, index; Finger III, middle; Finger IV, ring; Finger V, small. Table 5. Mean heart rate (HR), systolic arterial pressure (SAP), and diastolic arterial pressure (DAP) obtained during the control (AIR) and hypoxic (HYPO) cold water immersion trials for the hypoxic ambulatory (HA) and hypoxic training (HT) groups before and after the 10-day confinement period

HA group

HT group

Pre B

Post CWI

RW

B

Pre CWI

RW

B

Post CWI

RW

B

CWI

RW

AIR test HR (bpm) 73 ± 5 72 ± 6 67 ± 6 75 ± 12 76 ± 10 71 ± 11 78 ± 7 74 ± 12 72 ± 11 68 ± 11* 69 ± 10* 66 ± 10* SAP (mmHg) 118 ± 4 129 ± 13 119 ± 11 115 ± 11 122 ± 9 119 ± 14 122 ± 11 128 ± 12 124 ± 11 122 ± 10 128 ± 8 122 ± 5 DAP (mmHg) 72 ± 16 89 ± 11 83 ± 11 78 ± 3 84 ± 6 84 ± 11 77 ± 12 87 ± 11 84 ± 8 77 ± 4 86 ± 10 85 ± 8 HYPO test HR (bpm) 77 ± 8 74 ± 10 68 ± 9 78 ± 14 77 ± 12 72 ± 7 84 ± 10 84 ± 10 79 ± 9 82 ± 12 80 ± 11* 74 ± 9* SAP (mmHg) 113 ± 12 126 ± 11 121 ± 14 120 ± 10 126 ± 10 121 ± 11 124 ± 6 131 ± 13 127 ± 14 124 ± 7 130 ± 9 123 ± 9 DAP (mmHg) 73 ± 12 84 ± 10 80 ± 9 79 ± 4 87 ± 6 84 ± 11 79 ± 14 86 ± 11 85 ± 14 83 ± 8 85 ± 8 83 ± 8 *Significant difference from the pre-trial (P ≤ 0.05). Values are mean ± SD. B, a 5-min baseline phase; CWI, a 30-min cold water hand immersion phase; RW, a 15-min rewarming phase. SAP and DAP during CWI phase were significantly different from B and RW values (P ≤ 0.05).

(P = 0.62) (Table 5). In the HT group, HR was significantly lower throughout the post-trial than during the pre-trial (P = 0.02); there were no changes between the testing periods for SAP (P = 0.86) and DAP (P = 0.93; Table 5). SpO2 was similar in the two testing periods (pre = 97 ± 1%, post = 97 ± 1%; P = 0.65).

of the groups with regard to thermal sensation [HA group: pre = 1 (1–4), post = 1 (1–3); HT group: pre = 1 (1–3), post = 2 (1–3)], local pain [HA group: pre = 2 (1–4), post = 2 (1–4); HT group: pre = 2 (1–5), post = 2 (1–5)], or thermal comfort [HA group: pre = 2 (1–3), post = 2 (1–4); HT group: pre = 2 (1–4), post = 2 (2–4)].

HYPO trial. Ttympanic was unaltered during the pre-trial (baseline phase: 36.4 ± 0.5 °C, RW phase = 36.3 ± 0.5 °C; P = 0.16) and did not differ between the testing periods (pre = 36.4 ± 0.5 °C, post = 36.2 ± 0.4 °C; P = 0.10) in any of the groups. In the HA group, there were no changes between the testing periods for HR (P = 0.68), SAP (P = 0.60), or DAP (P = 0.62) (Table 5). In the HT group, the confinement decreased HR during the CWI (P = 0.05) and RW (P = 0.003) phases, but did not alter SAP (P = 0.55) and DAP (P = 0.78) (Table 5). SpO2 was higher at the post-trials (pre = 89 ± 2%, post = 91 ± 1%; P = 0.01).

HYPO test. During the CWI phase, the thermal sensation [HA group: pre = 1 (1–4), post = 1 (1–4); HT group: pre = 2 (1–3), post = 1 (1–3)] and the local pain [HA group: pre = 3 (1–7), post = 3 (1–4); HT group: pre = 3 (1–4), post = 2 (1–5)] were not changed after the confinement in any of the groups (P > 0.05). The thermal comfort was unaltered in the HA group [(pre = 2 (1–4), post = 2 (2–3); P > 0.05]. However, the HT group felt less uncomfortable at the 5th and 10th minutes of the post-CWI phase [(pre = 3 (2–4), post = 2 (2–4); P = 0.05]; no other differences were observed at any other time point (P > 0.05).

Psychometric responses

Discussion

AIR test. During the CWI phase, there were no differences (P > 0.05) between the testing periods for any

The discrepancy between the findings of previous studies that have reported either an enhancement (Nair

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Keramidas et al. et al., 1973; Mathew et al., 1977; Felicijan et al., 2008), impairment (Nair et al., 1973; Savourey et al., 1997; Purkayastha et al., 1999; Castellani et al., 2002), or no change (Nair et al., 1973; Mathew et al., 1977; Daanen & van Ruiten, 2000) in the effects of prolonged hypoxia on local cold tolerance, during and/or after a sojourn at high altitude, may be attributed to different environmental conditions encountered and the varying impact of each high-altitude stressor (i.e., hypoxia, cold, and exercise) on the acclimatization process. In the present study, wherein all subjects were confined to a strictly controlled environment, the effects of continuous hypoxia alone, and in combination with moderate-intensity aerobic exercise training, were discerned. The main finding of the study is that a 10-day exposure to normobaric hypoxia per se did not alter the hand temperature responses of healthy men during local cooling, but diminished the RW response after the AIR cold provocation. The continuous exposure to hypoxia combined with regular exercise did not significantly influence the hand temperature responses during the AIR trial, but led to an enhancement of the CIVD and RW responses in the HYPO trial. Notably, during the post-HYPO trial, the enhanced finger CIVD and hand RW responses in the HT group were preceded by a considerably higher baseline Tavg than in the pre-HYPO trial; whereas in the post-AIR trial, during which the RW responses of the HA group were impaired, the baseline Tavg was ∼ 1 °C lower than in the pre-AIR trial. That being the case, the confinementmediated alterations of the basal vasomotor tone should be taken into account in the evaluation of the temperature reactions during and after local cooling, given that they are dependent, at least to some degree, on the preimmersion hand temperatures (cf. Greenfield et al., 1952; Keramidas et al. 2014b; Yamazaki, 2010). The underlying mechanisms for the vasomotor responses of each group after the hypoxic confinement are difficult to discern from the current results. Following the 10-day confinement in normobaric hypoxia, both groups reached a similar degree of acclimation, as the elevated postvalues in SpO2 and blood O2 content indicated. Therefore, the differences observed between the groups in hand vasomotor responses to local cooling could not be ascribed to the level of hypoxic acclimation, and especially to the levels of blood viscosity (similar increase in EVF). Presumably, the increased hand temperature responses in the HT group were wholly, or in part, attributable to a reduction of sympathetic activity, as suggested by the lower HR responses in the post-trials. This effect was likely induced by the aerobic training (Schmitt et al., 2006). It is noteworthy that in the HA group, the HR was mainly unchanged during the post-trials. Yet, HR was slightly increased (∼ 4 bpm), albeit not statistically significant, in the RW phase of the post-AIR trial, wherein an impaired RW response was elicited. Indeed, after a sustained period of hypoxia, a long-lasting sympathetic

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overactivity prevails (Passino et al., 1996; Kanstrup et al., 1999; Hansen & Sander, 2003), often resulting in a spillover of catecholamines (cf. Rostrup, 1998). Apart from decreasing the neurovascular drive, the HT regimen may have increased local CIVD and RW responses by inducing adaptations within the cutaneous microvasculature, namely by enhancing endotheliumdependent vasomotor function (Vassalle et al., 2003; Lenasi & Strucl, 2004; Wang, 2005). It has been suggested that the greater microvascular endothelial function induced by physical training is attributable to increased antioxidant defense (Franzoni et al., 2004). Yamazaki (2010) has also demonstrated that topical antioxidant supplementation on the nonglabrous skin inhibits the cutaneous vasoconstrictor response to local cooling through adrenoceptor mechanisms. Interestingly, based on the data of a recent study (Debevec et al., 2014) that is part of the same cohort as the present study, a substantial increase in oxidative stress was detected in the HA group; whereas in the HT group, the oxidative stress was blunted via antioxidant defense improvement. In a like manner, Purkayastha et al. (1999) found that vitamin C supplementation at altitude improved the finger CIVD response. Still, the underlying mechanisms of the altered peripheral vasomotor activity to local cooling following a prolonged period of normobaric hypoxia remain speculative, and need to be further investigated. In both groups, a hypoxia-specific adaptation was indicated, given that after the confinement period, their hand vasomotor reactions were, in relative terms, better in the HYPO than the AIR trials. Amon et al. (2012) observed enhanced finger CIVD response in both testing conditions following a 28-day sleep-high train-low regimen. Whether the present specific adaptation was due to the continuous mode of hypoxic stimulus or to the shorter duration of the exposure remains to be settled. As regards the latter possibility, Mathew et al. (1977) have reported that the lowest finger temperature during local cooling was obtained after 2 weeks at high altitude, and that a greater CIVD response was elicited after a 1-year sojourn at altitude. A reduction in skin finger arteriolar blood flow variability after a 7-day high-altitude acclimatization has also been demonstrated (Passino et al., 1996). Of interest in this regard is the notion of a developmental acclimatization in highlanders relating to their local cold tolerance; a notion that is based on the finding that theAndean Indian youths had lower finger temperatures than adult Indians during a local cooling test (Little & Hanna, 1977). Hence, a time–response relationship may exist, and full adaptation might require longer periods of hypoxic exposure. Yet, present results postulate that daily exercise accelerates the acclimation process. Methodological considerations A potential limitation of the present study is the relatively small sample size. Still, we were able to detect

Hypoxic acclimation and local cooling statistically significant changes, which a posteriori reveal an adequate statistical power. The lack of a normoxic ambulatory group could also be debated; however, given the reproducibility of the CIVD (O’Brien, 2005) and the RW (Cleophas et al., 1982) responses, the inclusion of a normoxic group might not be essential. The two cold water hand immersion trials were separated by a 60-min interval that might be considered a short period for full recovery from the initial cold stimulus. However, the Tavg of the fingers did not differ between the first and the second trials, neither during the baseline (trial A: 30.8 ± 3.9 °C, trial B: 30.2 ± 4.6 °C; P = 0.59) nor during the CWI phase (trial A: 10.2 ± 0.8 °C, trial B: 10.2 ± 0.8 °C; P = 0.79). Furthermore, the counterbalanced order of the trials and the same sequence of the trials in the two testing periods minimized the risk of any order effect. It has been previously shown that exercise might diminish the hypoxia-induced drop of plasma volume (Withey et al., 1983), a response that might account for the differences in vasomotor activity between the groups. However, both cold water hand immersion trials were performed 2 days after the hypoxic confinement period, during which plasma volume was most likely restored (Robach et al., 2002). Notably, on that day, Hb and EVF had returned to the pre-confinement levels in both groups as presented in detail by Debevec et al., (2014). Nevertheless, O’Brien and Montain (2003) have shown that small-to-moderate changes in plasma volume do not blunt the finger CIVD response. Considering the adverse effects of physical inactivity on the temperature responses of the extremities during local cold provocation (Keramidas et al., 2014a), it might be argued that the limited amount of physical activity of the HA group could have led to a vascular deconditioning, which in turn contributed to the impaired hand vasomotor function. However, the similar pre- and post-PPO values, and the supporting evidence of a previous study with the same experimental design (Kounalakis et al., 2013) appear to eliminate the possibility of such a deconditioning effect. Moreover, although no differences exist between acute normobaric and hypobaric hypoxia as regards the hand temperature responses during local cooling (Meeuwsen et al., 2009), it remains to be settled whether the peripheral vasomotor responses after a sustained exposure to a hypobaric environment would be similar with those reported herein.

In conclusion, the present findings demonstrate that prolonged exposure to normobaric hypoxia per se does not alter the hand temperature responses to local cooling in male lowlanders; however, it impairs the hand RW response during a normoxic test. Conversely, the combined stimuli of a continuous exposure to normobaric hypoxia and regular exercise lead to an enhancement of the finger CIVD and hand RW responses, specifically, under hypoxic conditions.

Perspectives It has been demonstrated that exposure to high altitude is associated with high incidence of local cold injury (Pichotka et al., 1951; Harirchi et al., 2005). According to the World Health Organization, approximately 35 million people travel annually to places at altitudes > 3000 m for job-related (e.g., military personnel, miners) or recreational (e.g., skiers, mountaineers) purposes (cf. Dumont et al., 2005). Hence, we reasoned that it might be of occupational and clinical relevance to investigate the effects of prolonged exposure to hypoxia on local cold tolerance. Although the current study does not examine the causality between sustained hypoxia and local cold injury, in view of the premises that the finger CIVD response might have a cryoprotective function (Mathew et al., 1973, 1974) and that the ability of an efficient hand RW response might reduce the cold injury susceptibility (Francis & Golden, 1985), current findings suggest that a short-term acclimation protocol, including both hypoxia and exercise stimuli, might reduce the risk for local cold injury at high altitude. Key words: CIVD, cold injury, cold tolerance, crossadaptation, high altitude, rewarming.

Acknowledgements The current project was supported by grants from the Swedish Armed Forces (No. 922: 0905) and the Slovene Research Agency. The study was also supported, in part, by the European Union’s Framework Programme (2007–2013) under grant Agreement No. 284438. We are grateful to all the subjects for their participation. We would also like to thank the personnel at the Olympic Sports Center of Planica.

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