Cryobiology xxx (2014) xxx–xxx
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Gender-specific cold responses induce a similar body-cooling rate but different neuroendocrine and immune responses q Solianik Rima a,⇑, Albertas Skurvydas a, Astra Vitkauskiene˙ b, Marius Brazaitis a a b
Sports Research and Innovation Institute, Lithuanian Sports University, Sporto str. 6, LT-44221 Kaunas, Lithuania Department of Laboratory Medicine, Medical Academy, Lithuanian University of Health Sciences, Eiveniu str. 2, LT-50028 Kaunas, Lithuania
a r t i c l e
i n f o
Article history: Received 23 December 2013 Accepted 24 April 2014 Available online xxxx Keywords: Acute cold stress Cold response strategies Thermoregulation
a b s t r a c t This study investigated whether there are any gender differences in body-heating strategies during cold stress and whether the immune and neuroendocrine responses to physiological stress differ between men and women. Thirty-two participants (18 men and 14 women) were exposed to acute cold stress by immersion to the manubrium level in 14 °C water. The cold stress continued until rectal temperature (TRE) reached 35.5 °C or for a maximum of 170 min. The responses to cold stress of various indicators of body temperature, insulation, metabolism, shivering, stress, and endocrine and immune function were compared between men and women. During cold stress, TRE and muscle and mean skin temperatures decreased in all subjects (P < 0.001). These variables and the TRE cooling rate did not differ between men and women. The insulative response was greater in women (P < 0.05), whereas metabolic heat production and shivering were greater (P < 0.05) in men. Indicators of cold strain did not differ between men and women, but men exhibited larger changes in the indicators of neuroendocrine (epinephrine level) and in immune (tumor necrosis factor-a level) responses (both P < 0.05). The results of the present study indicated that men exhibited a greater metabolic response and shivering thermogenesis during acute cold stress, whereas women exhibited a greater insulative response. Despite the similar experience of cold strain in men and women, the neuroendocrine and immune responses were larger in men. Contrary to our expectations, the cooling rate was similar in men and women. Ó 2014 Elsevier Inc. All rights reserved.
Introduction The thermal stress encountered in many cold work environments may negatively affect various aspects of human performance and behavior [1]. Both laboratory and field studies can be found on this subject. It is well established that acute cold exposure increases the levels of catecholamine [16,31] and other stress hormones [16,27], and impairs motor [3,30] and cognitive performance [24]. There is no current consensus about the responses of the immune system to cold, in particular whether these changes lead to suppression [16,32] or activation [4,28,44] of the immune system. Despite the common opinion that gender affects the body’s response to cold [15,19], most thermal research has been conducted in men, and thus our general understanding of the responses to cold stress is based on the men. Women exhibit a faster body-cooling rate during cold stress [18,20,34,37], and this may increase the immune and physiological responses in women.
q Statement of funding: This research received no grant from any funding agency in the public, commercial or not-for-profit sectors. ⇑ Corresponding author. Fax: +370 37 204 515. E-mail address: [email protected] (R. Solianik).
The main cold responses can be insulative (a reduction in skin temperature relative to core temperature) or metabolic (increase in energy expenditure by shivering or nonshivering thermogenesis) [19,42]. The evidence suggests that differences in thermoregulation between men and women are related mainly to anthropometric differences. Adipose tissue and vasoconstricted skeletal muscle are responsible for thermal insulation [35]. Women have more fat mass [34], whereas men have more skeletal muscle mass [12]. During shivering, insulative muscle function is lost. According to van der Lans et al. [40], the greater reliance of shivering in men should increase metabolic heat production (MHP). We hypothesized that women would exhibit a greater insulative response (more tissue insulation) and men would exhibit a greater metabolic response (more MHP and shivering). The body surface area to mass ratio has an important influence on heat loss and body-cooling rate during exposure to the cold [35]. This ratio is larger for women, thereby facilitating core temperature cooling [7]. Current evidence suggests the importance of the increased contribution of brown adipose tissue (BAT) to nonshivering thermogenesis during cold exposure [29,40,42]. BAT in humans is
http://dx.doi.org/10.1016/j.cryobiol.2014.04.015 0011-2240/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: R. Solianik et al., Gender-specific cold responses induce a similar body-cooling rate but different neuroendocrine and immune responses, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.04.015
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R. Solianik et al. / Cryobiology xxx (2014) xxx–xxx
activated by elevated plasma catecholamine level [33,45]. The probability of detecting BAT increases with norepinephrine level [11] and is higher for women than men [8,26]. It has been thought that BAT is unnecessary in adults, who have a higher basal metabolic rate and greater muscle mass for shivering [8]. Thus, it is not surprising that Cypess et al. [8] observed a greater capacity to activate and increase BAT mass in women. BAT plays an important role in cold-induced nonshivering thermogenesis, and there is an inverse relationship between BAT activity and shivering [23]. The greater shivering ability in men is a function of their larger muscle mass [12] and thus their BAT activity during cold exposure is lower than in women. The main aim of this study was to compare the physiological and immune response to cold stress between men and women. No studies have reported gender-related differences in cold-response strategies, in particular the responses of the neuroendocrine and immune systems. Considering the anthropometric differences mentioned above, we hypothesized that, in response to cold stress, men will use metabolic strategies, including shivering thermogenesis, and that women would use insulative strategies. Considering the findings of Moran et al. [21] showing greater cold strain with faster cooling of core temperature in response to cold stress, we hypothesized that, during controlled cold stress, women will experience a greater stress level and thus larger changes in markers of the neuroendocrine and immune systems.
Methods Participants Fifty participants were assessed for eligibility. Subjects with Raynaud’s syndrome, asthma, neurological pathology, or conditions that could be worsened by exposure to cold water were excluded from this study. The criteria for inclusion were: (1) age 18–25 years; (2) no excessive sport activities, i.e., 8 h the night before the experiment and to refrain from alcohol, heavy exercise and caffeine for at least 24 h before the experiment. To avoid an effect of diet-induced thermogenesis, the subjects fasted from 10 h before the start of the experimental trial until it ended [43]. The experimental acute cold stress procedure was performed in the morning (07:00–11:00). The subject dressed in a swimsuit and self-inserted the rectal probe, and then the strap for recording heart rate (HR) was attached to the chest. The subject was then asked to lie down in a semirecumbent position for 30 min at an ambient temperature of 22 °C and 60% relative humidity. Resting pulmonary gas exchange and HR were recorded during the last 20 min. The resting (control) skin, muscle (TMU), and rectal (TRE) temperatures were measured, and blood samples were taken and stored for later analysis. The subject then underwent the cold stress procedure. The participant was immersed in a semirecumbent position up to the level of the manubrium in a 14 °C water bath. After 20 min of immersion, the subject exited from the bath and rested for 10 min by sitting in the room environment, and then the same procedure was repeated. The cold stress continued until TRE decreased to 35.5 °C or until 170 min in total (120 min maximum total immersion time), at which time the immersion ended regardless of the TRE. The exposure time until the TRE was achieved was recorded. TRE, HR, and subjective rating of shivering were recorded every 5 min throughout the cooling experiment. Pulmonary gas exchange was recorded only during each 20 min water immersion. Immediately after the cold stress experiment, the subject was towel dried, the skin, TRE and TMU were measured, and blood samples were taken.
Experimental measurements Anthropometric measurements The subject’s weight (Wt, in kg), fat-free mass (FFM, in kg), body fat (%BF, in percent) (TBF-300 body composition scale; Tanita, UK Ltd., West Drayton, UK), and height (in cm) were estimated during the familiarization. The subject’s body surface area (BSA, in m2) was estimated using the following best-fit equations: BSA = 0.01281 weight0.44 height0.60 for men and BSA = 0.01474 weight0.47 height0.55 for women [38]. Body mass index (BMI, in kg m2) and BSA/Wt ratio (%BSA/Wt, in percent) were calculated. Skinfold thickness (in mm) was measured using a skinfold caliper (SH5020, Saehan, Masan, Korea) at 10 sites: chin,
Table 1 Baseline characteristics of study participants. Men (n = 18)
Age, year Height, cm Mass, kg Body mass index, kg m2 Fat free mass, kg Body fat, % Mean skinfold thickness, mm Body surface area, m2 Surface to mass ratio, %
Women (n = 14)
x
SD
95% CI
x
SD
95% CI
20.72 181.72 77.98 23.61 66.14 14.92 12.06 1.97 2.55
0.96 5.96 8.61 2.47 5.65 3.43 4.14 0.12 0.15
20.25–21.20 178.76–184.69 73.70–82.27 22.38–24.83 63.33–68.95 13.21–16.62 9.94–14.19 1.92–2.03 2.47–2.62
21.36 170.64§,a 63.34§,a 21.74 45.65§,a 27.11§,a 17.89§,b 1.75§,a 2.78§,a
2.47 6.94 9.87 3.06 3.86 5.93 5.30 0.15 0.21
19.93–22.78 166.64–174.65 57.64–69.03 19.98–23.51 43.42–47.88 23.69–30.54 14.83–20.96 1.66–1.83 2.66–2.90
Data are presented as mean ð xÞ, standard deviation (SD) and 95% confidence interval (95% CI). a,bDenote significant differences of P < 0.001 and P < 0.05, respectively between men and women (§).
Please cite this article in press as: R. Solianik et al., Gender-specific cold responses induce a similar body-cooling rate but different neuroendocrine and immune responses, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.04.015
R. Solianik et al. / Cryobiology xxx (2014) xxx–xxx
subscapular, pectoral, suprailium, midaxillary, abdomen, triceps, anterior thigh, medial collateral ligament, and medial calf, and the mean skinfold thickness was calculated [20].
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ments of VO2 and the respiratory exchange ratio (RER = VCO2/VO2) according to Péronnet and Massicotte [25] as follows: MHP = (281.65 + 80.65 RER) VO2. For comparative purposes, MHP was normalized against Wt and BSA.
Temperature recording Measurement of shivering
TRE was monitored throughout the experimental trial with a thermocouple (Rectal Probe, Ellab, Hvidovre, Denmark; accuracy ± 0.01 °C) inserted to a minimum of 12 cm past the anal sphincter. The data for the TRE baseline and the end point of cold stress were used for analysis. The TRE cooling rate per hour was calculated. The TMU and skin temperature were measured before and at the end of the cold stress procedure. The TMU was measured with a needle microprobe (MKA, Ellab; accuracy ± 0.01 °C) inserted at a depth of 3 cm under the skin covering the gastrocnemius lateralis muscle in the right leg. No local anesthesia was administered before needle insertion. After the first measurement, the insertion area was marked with a sterile skin marker to ensure repeatability of needle insertion before and after the cooling exposure. Back, thigh, and forearm skin temperatures were monitored by surface thermistors (DM852, Ellab; accuracy ± 0.01 °C). The mean skin temperature (TSK, in °C) was calculated by the Burton [5] equation as: TSK = 0.5Back + 0.36Thigh + 0.14Forearm.
The method to measure subjective shivering for the whole body has been described elsewhere [2]. Briefly, the shivering sensation ranged from 1 (vigorously shivering) to 4 (not at all). The subject was instructed to relate his or her sensations at the time of reporting every 5 min throughout the cold stress procedure. A mean rating score was calculated for each session. As the sites of body temperature measurements reflect the core and skin temperatures, predictions of shivering (Mshiv, in W m2) are based largely on the data from these sites. A predictive formula to estimate metabolic rate due to shivering metabolism has been proposed [36] as: Mshiv = (155.5 (37 TRE) + 47.9 (33 TSK) 1.57 (33 TSK)2)/(%BF)0.5, where 37 °C – TRE set point; 33 °C – TSK set point. The value of Mshiv was determined from the difference between the resting and the end of the cold stress procedure, when TRE and TSK were the lowest.
Cold strain index calculation
Thermal insulation calculation
The cold strain index (CSI) was based on TRE and TSK as a rating of cold stress on a universal scale of 0–10 with 1–2 = no/little cold stress, 3–4 = low cold stress, 5–6 = moderate cold stress, 7–8 = high cold stress, and 9–10 = very high cold stress. The CSI was calculated [21] as: CSI = 6.67 (TREt TRE0) (35 TRE0)1 + 3.33 (TSKt TSK0) (14 TSK0)1. The CSI was calculated before (TRE0, TSK0) and at the end of the cold stress procedure (TREt, TSKt); 14 – water temperature; 35 – TRE threshold. TRE and TSK were assigned a weighting using the constants 6.67 and 3.33, respectively. Heart rate measurement HR was recorded using a HR monitor (S-625X, Polar Electro, Kempele, Finland) throughout the experiment. Resting HR was calculated as the average from the last 20 min of the baseline recording, and the stress HR was calculated as the average during the cold stress procedure. Measurements of metabolic thermoregulation A mobile spirometry system (Oxycon Mobile, Jaeger/VIASYS Healthcare, Hoechberg, Germany) was used to measure pulmonary gas exchange at rest and during cold-water immersion. This system uses a tightly fitting face mask covering the nose and mouth with a lightweight integrated flow meter (Triple V volume sensor) with a dead space of 30 ml. It monitors oxygen consumption (VO2) and carbon dioxide production (VCO2) every 5 s on a breathby-breath basis. The processing, recording, and battery system comprises two units attached to a belt, which was kept as close as possible to the subject’s nose and mouth during immersion. The data were stored on a memory card and PC hardware. Calibration of this instrument was performed before recording according to the manufacturer’s instructions. As recommended [36], the data collected during the first 5 min of each 20 min immersion were not used in any calculations because of reflex hyperventilation caused by cold-water immersion. Resting VO2 (in l min1) and VCO2 (in l min1) were calculated as average from the last 20 min of the baseline recording, and stress VO2 and VCO2 were calculated as the average during the cold-water immersion sessions. MHP (in W) was calculated from the respiratory gas exchange measure-
Tissue insulation (It, in °C m2 W1) was calculated using the equation similar to that reported previously [39]: It = ((TREt TSKt) BSA)/((0.92 MHP) + (DTRE) 0.965 0.6 Wt), where TREt and TSKt are the final rectal and skin measurements, respectively, and DTRE is the mean difference of TRE between baseline and the end of cold stress. The TRE and TSK gradient (TRE TSK), as a measure of insulation, was also calculated. Blood samples The concentrations of stress markers and proinflammatory mediators were measured in blood before and after cold (within 5 min) exposure. The stress hormones were cortisol (in nmol/l), norepinephrine (in nmol/l), epinephrine (in ng/ml), and dopamine (in ng/ml). The proinflammatory mediators were tumor necrosis factor-alpha (TNF-a, in pg/ml), interleukin-6 (IL-6, in pg/ml), and neopterin (in ng/ml). Blood samples for measurement of norepinephrine, epinephrine, and dopamine were collected in vacuum tubes with EDTA as an anticoagulant (EDTA-K3, 3 ml), mixed gently by inverting 8–10 times, and kept at 2–8 °C until centrifugation. The blood was centrifuged at 1200g for 15 min within 30 min of blood collection. Plasma samples were separated from the red cells as soon as possible (maximum 10–15 min) after centrifugation. Blood samples for measurement of cortisol, TNF-a, IL-6, and neopterin were collected by venipuncture into vacuum tubes for serum with gel separator (5 ml). Samples were allowed to clot and the serum was separated by centrifugation at 1200g for 15 min. All samples were stored at 70 °C until analysis. Norepinephrine and epinephrine concentrations were measured using a CatCombi enzyme-linked immunosorbent assay (ELISA) kit, dopamine concentration was measured using a dopamine ELISA kit, and neopterin concentration was measured using a neopterin ELISA kit (IBL, Hamburg, Germany). The solid-phase ELISA was based on the sandwich principle. The antigen in the samples was incubated in the coated wells with a goat anti-rabbit antibody and with an enzyme-conjugated secondary antibody directed toward a different region of the antigen molecule. After the substrate reaction, the intensity of the developed color is proportional to the amount of antigen. Results of samples were determined directly using the standard curve.
Please cite this article in press as: R. Solianik et al., Gender-specific cold responses induce a similar body-cooling rate but different neuroendocrine and immune responses, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.04.015
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R. Solianik et al. / Cryobiology xxx (2014) xxx–xxx
IL-6 concentration was measured using a IL-6-EASIA kit, and TNF-a concentration was measured using a TNF-a-EASIA kit (DIAsource, Nivelles, Belgium). The assay uses monoclonal antibodies directed against distinct epitopes of IL-6 or against distinct epitopes of TNF-a. A calibration curve was plotted and IL-6 or TNF-a concentration in samples was determined by interpolation from the calibration curve. The use of the Gemini analyzer reader and a sophisticated data reduction method result in high sensitivity in the low range and in an extended calibration range. Cortisol concentration was measured using an AIA-2000 automated enzyme immunoassay analyzer (Tosoh Corp., Tokyo, Japan). Cortisol in the test sample competes with enzyme-labeled cortisol for a limited number of binding sites on a cortisol-specific antibody immobilized on magnetic beads. The amount of enzyme-labeled cortisol that binds to the beads was inversely proportional to the cortisol concentration in the test sample. A standard curve using a range of known standard concentrations was constructed and unknown cortisol concentrations were calculated using this curve. Statistical analysis To compare differences between men and women, t tests for independent samples were used for data with a normal distribution (height, Wt, BMI, FFM, %BF, skinfold thickness, BSA, %BSA/Wt), and the Mann–Whitney U test was used for age, which was not normally distributed. A mixed Analysis of variance (ANOVA) design was used to analyze the changes in temperature, insulation, physiological stress, metabolic thermoregulatory, and immune system variables, and to compare these between men and women. Repeated-measures ANOVA was used to analyze the effects of cold stress on all variables, and the means were compared between men and women using a univariate ANOVA. The data are reported as mean ð xÞ, standard deviation (SD), 95% confidence interval (95% CI), and mean differences between the baseline and cold stress directly after or during cold-water immersion (D). The partial eta squared ðg2p Þ was estimated as a measure of cold stress effect size. The level of significance was set at P < 0.05. If a significant effect was found, the statistical power (%SP, in percent) was calculated. Pearson correlation coefficients were used to identify relationships between variables. All statistical analysis was performed using SPSS v.21.0 (IBM Corp., Armonk, NY, USA).
Results Participants’ characteristics The baseline characteristics of the men and women are described in Table 1. Gender differences were found in all subject characteristics except age and BMI. Height, Wt, FFM, and BSA were higher in men (P < 0.001 for each), and %BSA/Wt and mean skinfold thickness (P < 0.001, P < 0.05, respectively) were lower in men. Body temperatures and thermal insulation Table 2 summarizes the body temperatures and thermal insulation for men and women during the experiment. Men and women had the same duration of cold-water immersion: men, x 2.28 h (SD 0.76, 95% CI 1.90–2.65); women, x 2.38 h (SD 0.68, 95% CI 1.99– 2.77). TRE, TMU, and TSK decreased (P < 0.001, %SP 100) after cold exposure. There were no differences between men and women in any body temperature variable or in the rate of TRE cooling. It was higher (P < 0.05, %SP 64.1) and there was a greater rate of decrease in TRE (r = 0.41, P < 0.05) in women. The temperature gradient between TRE and TSK was significantly higher (P < 0.001,
%SP 100) at the end of cold exposure than at baseline, but the gradient did not differ significantly between men and women. Physiological stress response Table 3 presents the values for the cold stress response. The CSI was similar in men and women, and indicated moderate cold strain. HR increased (P < 0.001, %SP 100) during cold exposure in men and women but did not differ at baseline and during cooling between men and women. All stress markers were similar between men and women at baseline and after cold exposure except for epinephrine concentration, which was significantly higher (P < 0.05, %SP 84.1) in men compared with women after cold exposure. Cold stress induced a significant increase in cortisol (P < 0.05, %SP 51.9) and epinephrine (P < 0.001, %SP 99.6) concentrations only in men, whereas these hormone levels did not change in women. The concentrations of norepinephrine (P < 0.05, %SP 87.0, and P < 0.01, %SP 97.6 for men and women, respectively) and dopamine (P < 0.05, %SP 86.8 and %SP 80.7 for men and women, respectively) increased after cold exposure. At the end of the cold stress experiment, CSI was significantly related to norepinephrine concentration (r = 0.55, P < 0.05) but not to the concentrations of cortisol, epinephrine, or dopamine (r = 0.21, r = 0.20, and r = 0.47, respectively). Physiological heat production Table 4 summarizes the thermoregulatory metabolic response in men and women. MHP was similar in men and women at baseline. Cold-water immersion increased the total MHP and normalized MHP (P < 0.001, %SP 100) in both groups. During cold exposure, significant gender differences were observed in the total MHP (P < 0.001, %SP 99.9), MHP normalized to total Wt (P < 0.05, %SP 60.3), and MHP normalized to BSA (P < 0.001, %SP 93.5). The DMHP was significantly negatively related to BMI and %BF in men (r = 0.61, P < 0.05 and r = 0.72, P < 0.001, respectively) but not in women (r = 0.33 and r = 0.36, P > 0.05, respectively). Cold-water immersion increased the Mshiv rate (men, x 121.29 W m2, SD 29.31, 95% CI 106.71–135.87; women, x 82.84 W m2, SD 22.42, 95% CI 69.90–95.78) and the perception of shivering (men, x 2.30, SD 0.46, 95% CI 2.07–2.53; women, x 2.75, SD 0.39, 95% CI 2.52–2.97). A significantly greater increase in both variables was observed in men during cold exposure (P < 0.001, %SP 97.6 and P < 0.05, %SP 79.7, respectively). Mshiv was positively related to FFM (r = 0.51, P < 0.05) and negatively related to %BF (r = 0.76, P < 0.001). Mshiv was significantly negatively related to BMI and %BF in women (r = 0.81, P < 0.001 for each) but not in men (r = 0.36 and r = 0.45, P > 0.05, respectively). Immune system response The data on the response of proinflammatory mediators to cold stress for men and women are presented in Table 5. IL-6 and TNF-a concentrations did not differ between men and women at baseline and after cold exposure. However, the DTNF-a was greater in men (P < 0.05, %SP 55.5) than in women. The neopterin level decreased in both men and women (P < 0.05, %SP 86.3 and %SP 53.5, respectively), but the TNF-a level decreased only in men (P < 0.001, %SP 99.4). The CSI was not significantly related to the concentration of TNF-a, IL-6 (r = 0.24, P > 0.05 for each), or neopterin (r = 0.24, P > 0.05) at the end of cold stress. There were no significant correlations between DIL-6 and Dnorepinephrine (r = 0.30, P > 0.05), DIL-6 and Dcortisol (r = 0.14, P > 0.05), DIL-6 and DTNF-a (r = 0.12, P > 0.05), or Depinephrine and DTNF-a (r = 0.34, P > 0.05).
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R. Solianik et al. / Cryobiology xxx (2014) xxx–xxx Table 2 Thermal insulation and body temperature variables at baseline and at the end of cold stress. Men
Women
x
SD
95% CI
x
SD
95% CI
0.24 0.58
36.87–37.11 35.65–36.23
36.89 35.97⁄,a 0.75
0.15 0.57
36.81–36.98 35.64–36.30
g2p
36.99 35.94⁄,a 0.74
D
1.05
0.64
0.73–1.37
0.92
0.56
0.60–1.24
0.61
0.55
0.34–0.88
0.48
0.39
0.25–0.71
0.43 2.46
36.09–36.51 29.39–31.84
35.97 30.58⁄,a 0.85
0.58 2.30
35.64–36.31 29.25–31.91
g2p
36.30 30.62⁄,a 0.84
D
5.68
2.52
4.43–6.94
5.39
2.34
4.04–6.75
1.05 1.24
31.84–32.89 18.22–19.45
32.20 20.29⁄,a 0.93
0.45 3.37
31.94–32.46 18.35–22.24
g2p
32.37 18.84⁄,a 0.99
D
13.53
1.55
12.75–14.30
11.91
0.11
0.03
0.09–0.12
1.10 1.37
4.07–5.17 16.43–17.79
g2p
4.62 17.11⁄,a 0.99
D
12.47
1.58
11.68–13.54
Rectal temperature, °C At rest After cooling
Rectal temperature cooling rate, °C h1 Muscle temperature, °C At rest After cooling
Skin temperature, °C At rest After cooling
Insulation, °C m2 W1 Rectal-skin gradient, °C At rest After cooling
3.33
9.99–13.83
0.14§,b
0.05
0.11–0.17
4.69 15.67⁄,a 0.92
0.39 3.47
4.46–4.91 13.67–17.67
10.99
3.42
9.02–12.96
2 pÞ
Data are presented as mean ( x), standard deviation (SD), 95% confidence interval (95% CI), partial eta squared ðg and mean difference between baseline and the end of cold stress (D). a,b Denote significant differences of P < 0.001 and P < 0.05, respectively between baseline and the end of cold exposure (⁄) or between men and women (§).
Table 3 Physiological stress response. Men x Cold strain index
Women SD
5.93
1.92 8.69 10.435
g2p
62.94 83.36⁄,a 0.75
D
20.41
95% CI
SD
95% CI
5.44
1.97
4.31–6.58
58.62–67.26 78.21–88.50
68.57 85.55⁄,a 0.86
6.60 7.93
64.76–72.38 80.97–90.13
12.16
14.36–26.46
16.98
7.19
12.83–21.13
96.11 73.45
489.54–585.12 541.89–614.94
514.48 550.30 0.05
132.35 96.28
430.39–598.57 489.13–611.48
g2p
537.33 578.41⁄,b 0.21
D
41.00
81.73
0.36–81.64
35.83
172.65
73.84 to 145.53
6.73 41.08⁄,b 0.75
3.54 19.33
3.02–10.44 20.79–61.36
7.69 34.89⁄,a 0.66
3.71 20.82
5.20–10.18 20.91–48.88
34.34
21.65
11.62–57.07
27.20
29.56
13.39–41.02
Heart rate, b min1 At rest During cooling
Cortisol, nmol/l At rest After cooling
Norepinephrine, ng/ml At rest After cooling
g
2 p
D Epinephrine, ng/ml At rest After cooling
4.97–6.89
x
1.25 1.72
1.25–2.5 3.50–5.21
2.07 2.40§,b 0.02
1.84 1.32
0.83–3.30 1.32–3.44
g2p
1.87 4.36⁄,a 0.58
D
2.48
2.16
1.41–3.56
0.33
2.80
1.55 to 2.21
Dopamine, ng/ml At rest After cooling
21.59 55.70
0.60–45.93 57.87–174.78
22.28 87.38⁄,b 0.50
14.20 54.94
12.13–32.44 51.01–123.75
g2p
23.27 116.33⁄,b 0.75
D
93.17
58.87
31.38–154.95
65.10
57.62
23.88–106.32
Data are presented as mean ð xÞ, standard deviation (SD), 95% confidence interval (95% CI), partial eta squared (g2p ) and mean difference between baseline and cold exposure directly after or during the cold stress (D). a,bDenote significant differences of P < 0.001 and P < 0.05, respectively between baseline and the end of cold exposure (⁄) or between men and women (§).
Please cite this article in press as: R. Solianik et al., Gender-specific cold responses induce a similar body-cooling rate but different neuroendocrine and immune responses, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.04.015
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R. Solianik et al. / Cryobiology xxx (2014) xxx–xxx
Table 4 Total metabolic heat production (MHP) and MHP normalized to body mass and surface area at baseline and during cold-water immersion. Men
MHP, W At rest During cooling
g2p D
Women
x
SD
95% CI
x
SD
95% CI
62.89 303.83⁄,a 0.92
22.90 69.87
51.41–74.19 269.08–338.57
55.05 186.75⁄,§,a 0.90
14.31 50.00
46.79–63.32 157.88–215.62
241.02
73.07
204.69–277.36
131.70§,a
44.56
105.97–157.43
1
MHP, W kg At rest During cooling
0.80 3.97⁄,a 0.89
0.25 1.12
0.68–0.92 3.41–4.53
0.89 3.05⁄,a,§,b 0.85
0.27 1.12
0.73–1.04 2.40–370
3.17
1.14
2.60–3.74
2.16§,b
0.95
1.62–2.71
10.50 37.77
26.42–36.87 135.95–173.51
31.72 108.38⁄,§,a 0.90
8.58 34.17
26.76–36.67 88.65–128.11
g2p
31.65 154.73⁄,a 0.91
D
123.08
39.68
103.35–142.81
76.67§,a
29.66
59.54–93.79
g2p D 2
MHP, W m At rest During cooling
Data are presented as mean ð xÞ, standard deviation (SD), 95% confidence interval (95% CI), partial eta squared (g2p ) and mean difference between baseline and cold exposure during the cold stress (D). a,bDenote significant differences of P < 0.001 and P < 0.05, respectively between baseline and the end of cold exposure (⁄) or between men and women (§).
Table 5 Proinflammatory mediator concentrations at baseline and after cold stress. Tumor necrosis factor
Men x
Alpha, pg/ml At rest After cooling
Women SD 1.09 1.17
4.73–5.81 3.75–4.91
–0.94
0.83 26.70 33.69
g2p
14.17 17.21 0.13
D
3.04 5.30 4.14⁄,b 0.38
g2p D Interleukin 6, pg/ml At rest After cooling
Neopterin, nmol/l At rest After cooling
g2p D
5.27 4.33⁄,a 0.57
x
95% CI
–1.16
SD
95% CI
5.07 4.79 0.13
1.27 1.37
4.22–5.91 3.87–5.71
1.35 to 0.52
0.28§,b
0.73
0.53 to 0.53
0.90–27.45 0.46–33.97
19.32 17.82 0.04
46.27 39.24
8.04
0.96 to 7.03
1.51
8.18
7.00 to 3.99
1.43 1.04
4.59–6.01 3.62–4.66
2.10 1.97
3.77–6.60 3.20–5.86
1.52
1.91 to 0.40
0.97
1.17 to 0.30
5.19 4.53⁄,b 0.34 0.66
11.76 to 50.41 8.55 to 44.18
Data are presented as mean ð xÞ, standard deviation (SD), 95% confidence interval (95% CI), partial eta squared (g2p ) and mean difference between baseline and end of cold stress (D). a,bDenote significant differences of P < 0.001 and P < 0.05, respectively between baseline and the end of cold exposure (⁄) or between men and women (§).
Discussion The aim of this study was to test the hypothesis that in response to cold stress: (i) men are more reliant on metabolic strategies, whereas women are more reliant on insulative strategies; (ii) men are more reliant on shivering thermogenesis; and (iii) women experience greater cold-induced stress, which will result in greater changes in endocrine and immune responses during whole-body immersion that decreases TRE to 35.5 °C. As expected, men showed a greater metabolic response and women – a greater insulative response. In addition, more shivering thermogenesis was observed in men than in women. Contrary to our expectations, the CSI was similar in men and women. Most of the endocrine and immune responses after cold stress did not differ between men and women, although the Depinephrine and DTNF-a were greater in men. As expected, all temperature variables decreased during cold stress. Body temperature variables did not differ between men and women before and after cold exposure. Contrary to our expectations, TRE decreased but the TRE cooling rate was the same in men and women. We hypothesized that these differences were related to different body-heating strategies. During cold exposure, the tis-
sue temperature decreases as the body attempts to maintain a normal body temperature by increasing MHP and by minimizing heat loss, primarily through shivering and peripheral vasoconstriction [35]. On exposure to cold, both men and women exhibited an insulative response (a reduction in TSK relative to TRE) and a metabolic response (an increase in heat production and shivering). However, MHP and shivering were significantly greater in men than in women. When exposed to cold stress, the unacclimatized animal acutely defends body temperature by shivering thermogenesis, which increases heat production [40]. Mshiv was positively related to FFM, indicating that the greater muscle mass contributed more to metabolic heat produced by involuntary shivering. Mshiv was negatively related to %BF, suggesting a reason for the lower insulation capacity in men. We do not know whether the women in this study displayed greater nonshivering thermogenesis during cold exposure; however, we can speculate that nonshivering thermogenesis may have been greater in women because they exhibited a similar decrease in TSK and TRE, and less shivering and MHP compared with men. The similar rate of decrease in TRE can be explained by the greater insulation (It), which would have decreased heat loss.
Please cite this article in press as: R. Solianik et al., Gender-specific cold responses induce a similar body-cooling rate but different neuroendocrine and immune responses, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.04.015
R. Solianik et al. / Cryobiology xxx (2014) xxx–xxx
Recent evidence suggests that nonshivering thermogenesis by sympathetic, norepinephrine-induced mitochondrial heat production in BAT is a component of this metabolic response in healthy men and that body fat content and BMI correlate negatively with BAT content [42]. Our finding of a significant negative correlation between BMI and MHP, %BF, and MHP in men but not in women is consistent with this inverse relationship between body fat level and BAT content. In women, Mshiv was negatively related to %BF and BMI, suggesting a lower shivering rate and greater nonshivering thermogenesis. According to Moran et al. [21], larger changes in TRE and TSK during cold strain indicate greater physiological stress. We expected that women would experience a higher stress level (CSI) and exhibit greater activation of the sympathetic–adrenomedullary and hypothalamic–pituitary–adrenocortical systems, as generally occurs in response to stress [10]. However, we found a significant relationship only between CSI and norepinephrine level, and no relationships between CSI and other blood markers. Contrary to our expectation, the CSI and most stress indicators (cortisol, norepinephrine, and dopamine concentrations) did not differ between men and women. As observed in a previous study [37], cold-water immersion did not change the epinephrine level in women. Proinflammatory cytokines are signaling molecules of the inflammatory immune system that initiate and coordinate the cascade of immune events [9]. The most studied hormones with regard to stress are the proinflammatory cytokines IL-6 and TNFa, which are involved in the acute-phase response to inflammatory stressors [14]. However, we found no significant relationship between CSI and proinflammatory markers. We had expected that a higher CSI would increase the immune system response more in women than in men. In contrast to Brenner et al. [4], we did not observe an increase in plasma IL-6 concentration in response to cold exposure. They reported positive relationships between changes in the concentrations of IL-6, norepinephrine, and cortisol, and that the innate immune system was not adversely affected by brief cold exposure. Although we found changes in norepinephrine and cortisol levels, we found no relationship between these hormones and IL-6 level. Charmandari et al. [6] have concluded that IL-6 plays a major role in the overall control of inflammation by stimulating glucocorticoid secretion and by suppressing the secretion of TNF-a. In contrast, we did not observe any relationships between these variables or changes in IL-6 level, although TNF-a concentration decreased significantly in men. Recent evidence indicates that glucocorticoids and catecholamines, the major stress hormones, inhibit the production of proinflammatory cytokines such as TNF-a production [9]. Interestingly, the concentration of proinflammatory TNF-a decreased and epinephrine concentration increased after cold exposure only in men. Van der Poll et al. [41] observed that endogenous epinephrine or that administered as a component of sepsis treatment can inhibit TNF-a. However, we found no significant negative relationships between these variables after cold exposure. Neopterin is another marker of the immune system and is involved in activation of cellular immunity [22]. After cold exposure, neopterin concentration decreased in both men and women. We believe that further studies are needed to explain fully whether and to what extent cold exposure stimulates or suppresses the immune response. The limitation of the present study is that we used two sampling time points to assess the neuroendocrine and immune responses in men and women and that the single time point at the end of cold stress does not cover the whole response curve. It is known, that in vitro mild hypothermia can suppress inflammatory reactions, and moderate hypothermia can delay the induction of pro-inflammatory cytokines [13]. We have induced significant
7
response of blood stress markers (Table 3) and proinflammatory mediators (Table 5). By contrast we did not observe any IL-6 response. However previous studies [4,17] have shown that IL-6 response is already evoked after 1 h cooling, thus we expected that our cold stress duration would be sufficient to evoke IL-6 response. It is of great importance to assess kinetics of stress and proinflammatory markers, and further research is warranted according to gender-differences in response to cold. Conclusions In this study, we observed differences in the cold response between men and women. Specifically, men relied more on metabolic strategies and women – on insulative strategies. Men used shivering thermogenesis more than women. Despite the lack of difference in the CSI between men and women, the neuroendocrine and immune responses differed. Men exhibited a higher epinephrine level and a lower TNF-a level during cold stress. Contrary to our expectations, the cooling rate was the same in men and women. Conflict of interest Authors declare that there is no conflict of interest. References [1] H. Anttonen, A. Pekkarinen, J. Niskanen, Safety at work in cold environments and prevention of cold stress, Ind. Health 47 (2009) 254–261. te˙, The effect of two [2] M. Brazaitis, S. Kamandulis, A. Skurvydas, L. Daniusevicˇiu kinds of T-shirts on physiological and psychological thermal responses during exercise and recovery, Appl. Ergon. 42 (2010) 46–51. te˙, Z. Senikiene˙, The [3] M. Brazaitis, A. Skurvydas, K. Vadopalas, L. Daniusevicˇiu effect of heating and cooling on time course of voluntary and electrically induced muscle force variation, Medicina 47 (2011) 39–45. [4] I.K. Brenner, J.W. Castellani, C. Gabaree, A.J. Young, J. Zamecnik, R.J. Shephard, P.N. Shek, Immune changes in humans during cold exposure: effects of prior heating and exercise, J. Appl. Physiol. 87 (1999) 699–710. [5] A.C. Burton, Human calorimetry: II. The average temperature of the tissues of the body, J. Nutr. 9 (1935) 261–280. [6] E. Charmandari, C. Tsigos, G. Chrousos, Endocrinology of the stress response, Annu. Rev. Physiol. 67 (2005) 259–284. [7] C.F. Chi, Y.C. Shih, W.L. Chen, Effect of cold immersion on grip force, EMG, and thermal discomfort, Int. J. Ind. Ergon. 42 (2012) 113–121. [8] A.M. Cypess, S. Lehman, G. Williams, I. Tal, D. Rodman, A.B. Goldfine, F.C. Kuo, E.L. Palmer, Y.H. Tseng, A. Doria, G.M. Kolodny, C.R. Kahn, Identification and importance of brown adipose tissue in adult humans, N. Engl. J. Med. 360 (2009) 1509–1517. [9] I.J. Elenkov, G.P. Chrousos, Stress hormones, proinflammatory and antiinflammatory cytokines, and autoimmunity, Ann. N. Y. Acad. Sci. 966 (2002) 290–303. [10] M. Gunnar, K. Quevedo, The neurobiology of stress and development, Annu. Rev. Psychol. 58 (2007) 145–173. [11] M. Hadi, C.C. Chen, M. Whatley, K. Pacak, J.A. Carrasquillo, Brown fat imaging with (18)F-6-fluorodopamine PET/CT, (18)F-FDG PET/CT, and (123)I-MIBG SPECT: a study of patients being evaluated for pheochromocytoma, J. Nucl. Med. 48 (2007) 1077–1083. [12] I. Janssen, S.B. Heymsfield, Z.M. Wang, R. Ross, Skeletal muscle mass and distribution in 468 men and women aged 18–88 yr, J. Appl. Physiol. 89 (2000) 81–88. [13] A. Kimura, S. Sakurada, H. Ohkuni, Y. Todome, K. Kurata, Moderate hypothermia delays proinflammatory cytokine production of human peripheral blood mononuclear cells, Crit. Care Med. 30 (2002) 1499–1502. [14] A. Koj, Initiation of acute phase response and synthesis of cytokines, Biochim. Biophys. Acta 1317 (1996) 84–94. [15] J.C. Launay, G. Savourey, Cold adaptations, Ind. Health 47 (2009) 221–227. [16] E.C. LaVoy, B.K. McFarlin, R.J. Simpson, Immune responses to exercising in a cold environment, Wilderness Environ. Med. 22 (2011) 343–351. [17] E.C. Lee, G. Watson, D. Casa, L.E. Armstrong, W. Kraemer, J.L. Vingren, B.A. Spiering, C.M. Maresh, Interleukin-6 responses to water immersion therapy after acute exercise heat stress: a pilot investigation, J. Athl. Train. 47 (2012) 655–663. [18] B.B. Lemire, D. Gagnon, O. Jay, G.P. Kenny, Differences between sexes in rectal cooling rates after exercise-induced hyperthermia, Med. Sci. Sports Exerc. 41 (2009) 1633–1639. [19] T.M. Makinen, Different types of cold adaptation in humans, Front. Biosci. (Schol. Ed.) 2 (2010) 1047–1067.
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Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Gender-specific cold responses induce a similar body-cooling rate but different neuroendocrine and immune responses q Solianik Rima a,⇑, Albertas Skurvydas a, Astra Vitkauskiene˙ b, Marius Brazaitis a a b
Sports Research and Innovation Institute, Lithuanian Sports University, Sporto str. 6, LT-44221 Kaunas, Lithuania Department of Laboratory Medicine, Medical Academy, Lithuanian University of Health Sciences, Eiveniu str. 2, LT-50028 Kaunas, Lithuania
a r t i c l e
i n f o
Article history: Received 23 December 2013 Accepted 24 April 2014 Available online xxxx Keywords: Acute cold stress Cold response strategies Thermoregulation
a b s t r a c t This study investigated whether there are any gender differences in body-heating strategies during cold stress and whether the immune and neuroendocrine responses to physiological stress differ between men and women. Thirty-two participants (18 men and 14 women) were exposed to acute cold stress by immersion to the manubrium level in 14 °C water. The cold stress continued until rectal temperature (TRE) reached 35.5 °C or for a maximum of 170 min. The responses to cold stress of various indicators of body temperature, insulation, metabolism, shivering, stress, and endocrine and immune function were compared between men and women. During cold stress, TRE and muscle and mean skin temperatures decreased in all subjects (P < 0.001). These variables and the TRE cooling rate did not differ between men and women. The insulative response was greater in women (P < 0.05), whereas metabolic heat production and shivering were greater (P < 0.05) in men. Indicators of cold strain did not differ between men and women, but men exhibited larger changes in the indicators of neuroendocrine (epinephrine level) and in immune (tumor necrosis factor-a level) responses (both P < 0.05). The results of the present study indicated that men exhibited a greater metabolic response and shivering thermogenesis during acute cold stress, whereas women exhibited a greater insulative response. Despite the similar experience of cold strain in men and women, the neuroendocrine and immune responses were larger in men. Contrary to our expectations, the cooling rate was similar in men and women. Ó 2014 Elsevier Inc. All rights reserved.
Introduction The thermal stress encountered in many cold work environments may negatively affect various aspects of human performance and behavior [1]. Both laboratory and field studies can be found on this subject. It is well established that acute cold exposure increases the levels of catecholamine [16,31] and other stress hormones [16,27], and impairs motor [3,30] and cognitive performance [24]. There is no current consensus about the responses of the immune system to cold, in particular whether these changes lead to suppression [16,32] or activation [4,28,44] of the immune system. Despite the common opinion that gender affects the body’s response to cold [15,19], most thermal research has been conducted in men, and thus our general understanding of the responses to cold stress is based on the men. Women exhibit a faster body-cooling rate during cold stress [18,20,34,37], and this may increase the immune and physiological responses in women.
q Statement of funding: This research received no grant from any funding agency in the public, commercial or not-for-profit sectors. ⇑ Corresponding author. Fax: +370 37 204 515. E-mail address: [email protected] (R. Solianik).
The main cold responses can be insulative (a reduction in skin temperature relative to core temperature) or metabolic (increase in energy expenditure by shivering or nonshivering thermogenesis) [19,42]. The evidence suggests that differences in thermoregulation between men and women are related mainly to anthropometric differences. Adipose tissue and vasoconstricted skeletal muscle are responsible for thermal insulation [35]. Women have more fat mass [34], whereas men have more skeletal muscle mass [12]. During shivering, insulative muscle function is lost. According to van der Lans et al. [40], the greater reliance of shivering in men should increase metabolic heat production (MHP). We hypothesized that women would exhibit a greater insulative response (more tissue insulation) and men would exhibit a greater metabolic response (more MHP and shivering). The body surface area to mass ratio has an important influence on heat loss and body-cooling rate during exposure to the cold [35]. This ratio is larger for women, thereby facilitating core temperature cooling [7]. Current evidence suggests the importance of the increased contribution of brown adipose tissue (BAT) to nonshivering thermogenesis during cold exposure [29,40,42]. BAT in humans is
http://dx.doi.org/10.1016/j.cryobiol.2014.04.015 0011-2240/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: R. Solianik et al., Gender-specific cold responses induce a similar body-cooling rate but different neuroendocrine and immune responses, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.04.015
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activated by elevated plasma catecholamine level [33,45]. The probability of detecting BAT increases with norepinephrine level [11] and is higher for women than men [8,26]. It has been thought that BAT is unnecessary in adults, who have a higher basal metabolic rate and greater muscle mass for shivering [8]. Thus, it is not surprising that Cypess et al. [8] observed a greater capacity to activate and increase BAT mass in women. BAT plays an important role in cold-induced nonshivering thermogenesis, and there is an inverse relationship between BAT activity and shivering [23]. The greater shivering ability in men is a function of their larger muscle mass [12] and thus their BAT activity during cold exposure is lower than in women. The main aim of this study was to compare the physiological and immune response to cold stress between men and women. No studies have reported gender-related differences in cold-response strategies, in particular the responses of the neuroendocrine and immune systems. Considering the anthropometric differences mentioned above, we hypothesized that, in response to cold stress, men will use metabolic strategies, including shivering thermogenesis, and that women would use insulative strategies. Considering the findings of Moran et al. [21] showing greater cold strain with faster cooling of core temperature in response to cold stress, we hypothesized that, during controlled cold stress, women will experience a greater stress level and thus larger changes in markers of the neuroendocrine and immune systems.
Methods Participants Fifty participants were assessed for eligibility. Subjects with Raynaud’s syndrome, asthma, neurological pathology, or conditions that could be worsened by exposure to cold water were excluded from this study. The criteria for inclusion were: (1) age 18–25 years; (2) no excessive sport activities, i.e., 8 h the night before the experiment and to refrain from alcohol, heavy exercise and caffeine for at least 24 h before the experiment. To avoid an effect of diet-induced thermogenesis, the subjects fasted from 10 h before the start of the experimental trial until it ended [43]. The experimental acute cold stress procedure was performed in the morning (07:00–11:00). The subject dressed in a swimsuit and self-inserted the rectal probe, and then the strap for recording heart rate (HR) was attached to the chest. The subject was then asked to lie down in a semirecumbent position for 30 min at an ambient temperature of 22 °C and 60% relative humidity. Resting pulmonary gas exchange and HR were recorded during the last 20 min. The resting (control) skin, muscle (TMU), and rectal (TRE) temperatures were measured, and blood samples were taken and stored for later analysis. The subject then underwent the cold stress procedure. The participant was immersed in a semirecumbent position up to the level of the manubrium in a 14 °C water bath. After 20 min of immersion, the subject exited from the bath and rested for 10 min by sitting in the room environment, and then the same procedure was repeated. The cold stress continued until TRE decreased to 35.5 °C or until 170 min in total (120 min maximum total immersion time), at which time the immersion ended regardless of the TRE. The exposure time until the TRE was achieved was recorded. TRE, HR, and subjective rating of shivering were recorded every 5 min throughout the cooling experiment. Pulmonary gas exchange was recorded only during each 20 min water immersion. Immediately after the cold stress experiment, the subject was towel dried, the skin, TRE and TMU were measured, and blood samples were taken.
Experimental measurements Anthropometric measurements The subject’s weight (Wt, in kg), fat-free mass (FFM, in kg), body fat (%BF, in percent) (TBF-300 body composition scale; Tanita, UK Ltd., West Drayton, UK), and height (in cm) were estimated during the familiarization. The subject’s body surface area (BSA, in m2) was estimated using the following best-fit equations: BSA = 0.01281 weight0.44 height0.60 for men and BSA = 0.01474 weight0.47 height0.55 for women [38]. Body mass index (BMI, in kg m2) and BSA/Wt ratio (%BSA/Wt, in percent) were calculated. Skinfold thickness (in mm) was measured using a skinfold caliper (SH5020, Saehan, Masan, Korea) at 10 sites: chin,
Table 1 Baseline characteristics of study participants. Men (n = 18)
Age, year Height, cm Mass, kg Body mass index, kg m2 Fat free mass, kg Body fat, % Mean skinfold thickness, mm Body surface area, m2 Surface to mass ratio, %
Women (n = 14)
x
SD
95% CI
x
SD
95% CI
20.72 181.72 77.98 23.61 66.14 14.92 12.06 1.97 2.55
0.96 5.96 8.61 2.47 5.65 3.43 4.14 0.12 0.15
20.25–21.20 178.76–184.69 73.70–82.27 22.38–24.83 63.33–68.95 13.21–16.62 9.94–14.19 1.92–2.03 2.47–2.62
21.36 170.64§,a 63.34§,a 21.74 45.65§,a 27.11§,a 17.89§,b 1.75§,a 2.78§,a
2.47 6.94 9.87 3.06 3.86 5.93 5.30 0.15 0.21
19.93–22.78 166.64–174.65 57.64–69.03 19.98–23.51 43.42–47.88 23.69–30.54 14.83–20.96 1.66–1.83 2.66–2.90
Data are presented as mean ð xÞ, standard deviation (SD) and 95% confidence interval (95% CI). a,bDenote significant differences of P < 0.001 and P < 0.05, respectively between men and women (§).
Please cite this article in press as: R. Solianik et al., Gender-specific cold responses induce a similar body-cooling rate but different neuroendocrine and immune responses, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.04.015
R. Solianik et al. / Cryobiology xxx (2014) xxx–xxx
subscapular, pectoral, suprailium, midaxillary, abdomen, triceps, anterior thigh, medial collateral ligament, and medial calf, and the mean skinfold thickness was calculated [20].
3
ments of VO2 and the respiratory exchange ratio (RER = VCO2/VO2) according to Péronnet and Massicotte [25] as follows: MHP = (281.65 + 80.65 RER) VO2. For comparative purposes, MHP was normalized against Wt and BSA.
Temperature recording Measurement of shivering
TRE was monitored throughout the experimental trial with a thermocouple (Rectal Probe, Ellab, Hvidovre, Denmark; accuracy ± 0.01 °C) inserted to a minimum of 12 cm past the anal sphincter. The data for the TRE baseline and the end point of cold stress were used for analysis. The TRE cooling rate per hour was calculated. The TMU and skin temperature were measured before and at the end of the cold stress procedure. The TMU was measured with a needle microprobe (MKA, Ellab; accuracy ± 0.01 °C) inserted at a depth of 3 cm under the skin covering the gastrocnemius lateralis muscle in the right leg. No local anesthesia was administered before needle insertion. After the first measurement, the insertion area was marked with a sterile skin marker to ensure repeatability of needle insertion before and after the cooling exposure. Back, thigh, and forearm skin temperatures were monitored by surface thermistors (DM852, Ellab; accuracy ± 0.01 °C). The mean skin temperature (TSK, in °C) was calculated by the Burton [5] equation as: TSK = 0.5Back + 0.36Thigh + 0.14Forearm.
The method to measure subjective shivering for the whole body has been described elsewhere [2]. Briefly, the shivering sensation ranged from 1 (vigorously shivering) to 4 (not at all). The subject was instructed to relate his or her sensations at the time of reporting every 5 min throughout the cold stress procedure. A mean rating score was calculated for each session. As the sites of body temperature measurements reflect the core and skin temperatures, predictions of shivering (Mshiv, in W m2) are based largely on the data from these sites. A predictive formula to estimate metabolic rate due to shivering metabolism has been proposed [36] as: Mshiv = (155.5 (37 TRE) + 47.9 (33 TSK) 1.57 (33 TSK)2)/(%BF)0.5, where 37 °C – TRE set point; 33 °C – TSK set point. The value of Mshiv was determined from the difference between the resting and the end of the cold stress procedure, when TRE and TSK were the lowest.
Cold strain index calculation
Thermal insulation calculation
The cold strain index (CSI) was based on TRE and TSK as a rating of cold stress on a universal scale of 0–10 with 1–2 = no/little cold stress, 3–4 = low cold stress, 5–6 = moderate cold stress, 7–8 = high cold stress, and 9–10 = very high cold stress. The CSI was calculated [21] as: CSI = 6.67 (TREt TRE0) (35 TRE0)1 + 3.33 (TSKt TSK0) (14 TSK0)1. The CSI was calculated before (TRE0, TSK0) and at the end of the cold stress procedure (TREt, TSKt); 14 – water temperature; 35 – TRE threshold. TRE and TSK were assigned a weighting using the constants 6.67 and 3.33, respectively. Heart rate measurement HR was recorded using a HR monitor (S-625X, Polar Electro, Kempele, Finland) throughout the experiment. Resting HR was calculated as the average from the last 20 min of the baseline recording, and the stress HR was calculated as the average during the cold stress procedure. Measurements of metabolic thermoregulation A mobile spirometry system (Oxycon Mobile, Jaeger/VIASYS Healthcare, Hoechberg, Germany) was used to measure pulmonary gas exchange at rest and during cold-water immersion. This system uses a tightly fitting face mask covering the nose and mouth with a lightweight integrated flow meter (Triple V volume sensor) with a dead space of 30 ml. It monitors oxygen consumption (VO2) and carbon dioxide production (VCO2) every 5 s on a breathby-breath basis. The processing, recording, and battery system comprises two units attached to a belt, which was kept as close as possible to the subject’s nose and mouth during immersion. The data were stored on a memory card and PC hardware. Calibration of this instrument was performed before recording according to the manufacturer’s instructions. As recommended [36], the data collected during the first 5 min of each 20 min immersion were not used in any calculations because of reflex hyperventilation caused by cold-water immersion. Resting VO2 (in l min1) and VCO2 (in l min1) were calculated as average from the last 20 min of the baseline recording, and stress VO2 and VCO2 were calculated as the average during the cold-water immersion sessions. MHP (in W) was calculated from the respiratory gas exchange measure-
Tissue insulation (It, in °C m2 W1) was calculated using the equation similar to that reported previously [39]: It = ((TREt TSKt) BSA)/((0.92 MHP) + (DTRE) 0.965 0.6 Wt), where TREt and TSKt are the final rectal and skin measurements, respectively, and DTRE is the mean difference of TRE between baseline and the end of cold stress. The TRE and TSK gradient (TRE TSK), as a measure of insulation, was also calculated. Blood samples The concentrations of stress markers and proinflammatory mediators were measured in blood before and after cold (within 5 min) exposure. The stress hormones were cortisol (in nmol/l), norepinephrine (in nmol/l), epinephrine (in ng/ml), and dopamine (in ng/ml). The proinflammatory mediators were tumor necrosis factor-alpha (TNF-a, in pg/ml), interleukin-6 (IL-6, in pg/ml), and neopterin (in ng/ml). Blood samples for measurement of norepinephrine, epinephrine, and dopamine were collected in vacuum tubes with EDTA as an anticoagulant (EDTA-K3, 3 ml), mixed gently by inverting 8–10 times, and kept at 2–8 °C until centrifugation. The blood was centrifuged at 1200g for 15 min within 30 min of blood collection. Plasma samples were separated from the red cells as soon as possible (maximum 10–15 min) after centrifugation. Blood samples for measurement of cortisol, TNF-a, IL-6, and neopterin were collected by venipuncture into vacuum tubes for serum with gel separator (5 ml). Samples were allowed to clot and the serum was separated by centrifugation at 1200g for 15 min. All samples were stored at 70 °C until analysis. Norepinephrine and epinephrine concentrations were measured using a CatCombi enzyme-linked immunosorbent assay (ELISA) kit, dopamine concentration was measured using a dopamine ELISA kit, and neopterin concentration was measured using a neopterin ELISA kit (IBL, Hamburg, Germany). The solid-phase ELISA was based on the sandwich principle. The antigen in the samples was incubated in the coated wells with a goat anti-rabbit antibody and with an enzyme-conjugated secondary antibody directed toward a different region of the antigen molecule. After the substrate reaction, the intensity of the developed color is proportional to the amount of antigen. Results of samples were determined directly using the standard curve.
Please cite this article in press as: R. Solianik et al., Gender-specific cold responses induce a similar body-cooling rate but different neuroendocrine and immune responses, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.04.015
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R. Solianik et al. / Cryobiology xxx (2014) xxx–xxx
IL-6 concentration was measured using a IL-6-EASIA kit, and TNF-a concentration was measured using a TNF-a-EASIA kit (DIAsource, Nivelles, Belgium). The assay uses monoclonal antibodies directed against distinct epitopes of IL-6 or against distinct epitopes of TNF-a. A calibration curve was plotted and IL-6 or TNF-a concentration in samples was determined by interpolation from the calibration curve. The use of the Gemini analyzer reader and a sophisticated data reduction method result in high sensitivity in the low range and in an extended calibration range. Cortisol concentration was measured using an AIA-2000 automated enzyme immunoassay analyzer (Tosoh Corp., Tokyo, Japan). Cortisol in the test sample competes with enzyme-labeled cortisol for a limited number of binding sites on a cortisol-specific antibody immobilized on magnetic beads. The amount of enzyme-labeled cortisol that binds to the beads was inversely proportional to the cortisol concentration in the test sample. A standard curve using a range of known standard concentrations was constructed and unknown cortisol concentrations were calculated using this curve. Statistical analysis To compare differences between men and women, t tests for independent samples were used for data with a normal distribution (height, Wt, BMI, FFM, %BF, skinfold thickness, BSA, %BSA/Wt), and the Mann–Whitney U test was used for age, which was not normally distributed. A mixed Analysis of variance (ANOVA) design was used to analyze the changes in temperature, insulation, physiological stress, metabolic thermoregulatory, and immune system variables, and to compare these between men and women. Repeated-measures ANOVA was used to analyze the effects of cold stress on all variables, and the means were compared between men and women using a univariate ANOVA. The data are reported as mean ð xÞ, standard deviation (SD), 95% confidence interval (95% CI), and mean differences between the baseline and cold stress directly after or during cold-water immersion (D). The partial eta squared ðg2p Þ was estimated as a measure of cold stress effect size. The level of significance was set at P < 0.05. If a significant effect was found, the statistical power (%SP, in percent) was calculated. Pearson correlation coefficients were used to identify relationships between variables. All statistical analysis was performed using SPSS v.21.0 (IBM Corp., Armonk, NY, USA).
Results Participants’ characteristics The baseline characteristics of the men and women are described in Table 1. Gender differences were found in all subject characteristics except age and BMI. Height, Wt, FFM, and BSA were higher in men (P < 0.001 for each), and %BSA/Wt and mean skinfold thickness (P < 0.001, P < 0.05, respectively) were lower in men. Body temperatures and thermal insulation Table 2 summarizes the body temperatures and thermal insulation for men and women during the experiment. Men and women had the same duration of cold-water immersion: men, x 2.28 h (SD 0.76, 95% CI 1.90–2.65); women, x 2.38 h (SD 0.68, 95% CI 1.99– 2.77). TRE, TMU, and TSK decreased (P < 0.001, %SP 100) after cold exposure. There were no differences between men and women in any body temperature variable or in the rate of TRE cooling. It was higher (P < 0.05, %SP 64.1) and there was a greater rate of decrease in TRE (r = 0.41, P < 0.05) in women. The temperature gradient between TRE and TSK was significantly higher (P < 0.001,
%SP 100) at the end of cold exposure than at baseline, but the gradient did not differ significantly between men and women. Physiological stress response Table 3 presents the values for the cold stress response. The CSI was similar in men and women, and indicated moderate cold strain. HR increased (P < 0.001, %SP 100) during cold exposure in men and women but did not differ at baseline and during cooling between men and women. All stress markers were similar between men and women at baseline and after cold exposure except for epinephrine concentration, which was significantly higher (P < 0.05, %SP 84.1) in men compared with women after cold exposure. Cold stress induced a significant increase in cortisol (P < 0.05, %SP 51.9) and epinephrine (P < 0.001, %SP 99.6) concentrations only in men, whereas these hormone levels did not change in women. The concentrations of norepinephrine (P < 0.05, %SP 87.0, and P < 0.01, %SP 97.6 for men and women, respectively) and dopamine (P < 0.05, %SP 86.8 and %SP 80.7 for men and women, respectively) increased after cold exposure. At the end of the cold stress experiment, CSI was significantly related to norepinephrine concentration (r = 0.55, P < 0.05) but not to the concentrations of cortisol, epinephrine, or dopamine (r = 0.21, r = 0.20, and r = 0.47, respectively). Physiological heat production Table 4 summarizes the thermoregulatory metabolic response in men and women. MHP was similar in men and women at baseline. Cold-water immersion increased the total MHP and normalized MHP (P < 0.001, %SP 100) in both groups. During cold exposure, significant gender differences were observed in the total MHP (P < 0.001, %SP 99.9), MHP normalized to total Wt (P < 0.05, %SP 60.3), and MHP normalized to BSA (P < 0.001, %SP 93.5). The DMHP was significantly negatively related to BMI and %BF in men (r = 0.61, P < 0.05 and r = 0.72, P < 0.001, respectively) but not in women (r = 0.33 and r = 0.36, P > 0.05, respectively). Cold-water immersion increased the Mshiv rate (men, x 121.29 W m2, SD 29.31, 95% CI 106.71–135.87; women, x 82.84 W m2, SD 22.42, 95% CI 69.90–95.78) and the perception of shivering (men, x 2.30, SD 0.46, 95% CI 2.07–2.53; women, x 2.75, SD 0.39, 95% CI 2.52–2.97). A significantly greater increase in both variables was observed in men during cold exposure (P < 0.001, %SP 97.6 and P < 0.05, %SP 79.7, respectively). Mshiv was positively related to FFM (r = 0.51, P < 0.05) and negatively related to %BF (r = 0.76, P < 0.001). Mshiv was significantly negatively related to BMI and %BF in women (r = 0.81, P < 0.001 for each) but not in men (r = 0.36 and r = 0.45, P > 0.05, respectively). Immune system response The data on the response of proinflammatory mediators to cold stress for men and women are presented in Table 5. IL-6 and TNF-a concentrations did not differ between men and women at baseline and after cold exposure. However, the DTNF-a was greater in men (P < 0.05, %SP 55.5) than in women. The neopterin level decreased in both men and women (P < 0.05, %SP 86.3 and %SP 53.5, respectively), but the TNF-a level decreased only in men (P < 0.001, %SP 99.4). The CSI was not significantly related to the concentration of TNF-a, IL-6 (r = 0.24, P > 0.05 for each), or neopterin (r = 0.24, P > 0.05) at the end of cold stress. There were no significant correlations between DIL-6 and Dnorepinephrine (r = 0.30, P > 0.05), DIL-6 and Dcortisol (r = 0.14, P > 0.05), DIL-6 and DTNF-a (r = 0.12, P > 0.05), or Depinephrine and DTNF-a (r = 0.34, P > 0.05).
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R. Solianik et al. / Cryobiology xxx (2014) xxx–xxx Table 2 Thermal insulation and body temperature variables at baseline and at the end of cold stress. Men
Women
x
SD
95% CI
x
SD
95% CI
0.24 0.58
36.87–37.11 35.65–36.23
36.89 35.97⁄,a 0.75
0.15 0.57
36.81–36.98 35.64–36.30
g2p
36.99 35.94⁄,a 0.74
D
1.05
0.64
0.73–1.37
0.92
0.56
0.60–1.24
0.61
0.55
0.34–0.88
0.48
0.39
0.25–0.71
0.43 2.46
36.09–36.51 29.39–31.84
35.97 30.58⁄,a 0.85
0.58 2.30
35.64–36.31 29.25–31.91
g2p
36.30 30.62⁄,a 0.84
D
5.68
2.52
4.43–6.94
5.39
2.34
4.04–6.75
1.05 1.24
31.84–32.89 18.22–19.45
32.20 20.29⁄,a 0.93
0.45 3.37
31.94–32.46 18.35–22.24
g2p
32.37 18.84⁄,a 0.99
D
13.53
1.55
12.75–14.30
11.91
0.11
0.03
0.09–0.12
1.10 1.37
4.07–5.17 16.43–17.79
g2p
4.62 17.11⁄,a 0.99
D
12.47
1.58
11.68–13.54
Rectal temperature, °C At rest After cooling
Rectal temperature cooling rate, °C h1 Muscle temperature, °C At rest After cooling
Skin temperature, °C At rest After cooling
Insulation, °C m2 W1 Rectal-skin gradient, °C At rest After cooling
3.33
9.99–13.83
0.14§,b
0.05
0.11–0.17
4.69 15.67⁄,a 0.92
0.39 3.47
4.46–4.91 13.67–17.67
10.99
3.42
9.02–12.96
2 pÞ
Data are presented as mean ( x), standard deviation (SD), 95% confidence interval (95% CI), partial eta squared ðg and mean difference between baseline and the end of cold stress (D). a,b Denote significant differences of P < 0.001 and P < 0.05, respectively between baseline and the end of cold exposure (⁄) or between men and women (§).
Table 3 Physiological stress response. Men x Cold strain index
Women SD
5.93
1.92 8.69 10.435
g2p
62.94 83.36⁄,a 0.75
D
20.41
95% CI
SD
95% CI
5.44
1.97
4.31–6.58
58.62–67.26 78.21–88.50
68.57 85.55⁄,a 0.86
6.60 7.93
64.76–72.38 80.97–90.13
12.16
14.36–26.46
16.98
7.19
12.83–21.13
96.11 73.45
489.54–585.12 541.89–614.94
514.48 550.30 0.05
132.35 96.28
430.39–598.57 489.13–611.48
g2p
537.33 578.41⁄,b 0.21
D
41.00
81.73
0.36–81.64
35.83
172.65
73.84 to 145.53
6.73 41.08⁄,b 0.75
3.54 19.33
3.02–10.44 20.79–61.36
7.69 34.89⁄,a 0.66
3.71 20.82
5.20–10.18 20.91–48.88
34.34
21.65
11.62–57.07
27.20
29.56
13.39–41.02
Heart rate, b min1 At rest During cooling
Cortisol, nmol/l At rest After cooling
Norepinephrine, ng/ml At rest After cooling
g
2 p
D Epinephrine, ng/ml At rest After cooling
4.97–6.89
x
1.25 1.72
1.25–2.5 3.50–5.21
2.07 2.40§,b 0.02
1.84 1.32
0.83–3.30 1.32–3.44
g2p
1.87 4.36⁄,a 0.58
D
2.48
2.16
1.41–3.56
0.33
2.80
1.55 to 2.21
Dopamine, ng/ml At rest After cooling
21.59 55.70
0.60–45.93 57.87–174.78
22.28 87.38⁄,b 0.50
14.20 54.94
12.13–32.44 51.01–123.75
g2p
23.27 116.33⁄,b 0.75
D
93.17
58.87
31.38–154.95
65.10
57.62
23.88–106.32
Data are presented as mean ð xÞ, standard deviation (SD), 95% confidence interval (95% CI), partial eta squared (g2p ) and mean difference between baseline and cold exposure directly after or during the cold stress (D). a,bDenote significant differences of P < 0.001 and P < 0.05, respectively between baseline and the end of cold exposure (⁄) or between men and women (§).
Please cite this article in press as: R. Solianik et al., Gender-specific cold responses induce a similar body-cooling rate but different neuroendocrine and immune responses, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.04.015
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R. Solianik et al. / Cryobiology xxx (2014) xxx–xxx
Table 4 Total metabolic heat production (MHP) and MHP normalized to body mass and surface area at baseline and during cold-water immersion. Men
MHP, W At rest During cooling
g2p D
Women
x
SD
95% CI
x
SD
95% CI
62.89 303.83⁄,a 0.92
22.90 69.87
51.41–74.19 269.08–338.57
55.05 186.75⁄,§,a 0.90
14.31 50.00
46.79–63.32 157.88–215.62
241.02
73.07
204.69–277.36
131.70§,a
44.56
105.97–157.43
1
MHP, W kg At rest During cooling
0.80 3.97⁄,a 0.89
0.25 1.12
0.68–0.92 3.41–4.53
0.89 3.05⁄,a,§,b 0.85
0.27 1.12
0.73–1.04 2.40–370
3.17
1.14
2.60–3.74
2.16§,b
0.95
1.62–2.71
10.50 37.77
26.42–36.87 135.95–173.51
31.72 108.38⁄,§,a 0.90
8.58 34.17
26.76–36.67 88.65–128.11
g2p
31.65 154.73⁄,a 0.91
D
123.08
39.68
103.35–142.81
76.67§,a
29.66
59.54–93.79
g2p D 2
MHP, W m At rest During cooling
Data are presented as mean ð xÞ, standard deviation (SD), 95% confidence interval (95% CI), partial eta squared (g2p ) and mean difference between baseline and cold exposure during the cold stress (D). a,bDenote significant differences of P < 0.001 and P < 0.05, respectively between baseline and the end of cold exposure (⁄) or between men and women (§).
Table 5 Proinflammatory mediator concentrations at baseline and after cold stress. Tumor necrosis factor
Men x
Alpha, pg/ml At rest After cooling
Women SD 1.09 1.17
4.73–5.81 3.75–4.91
–0.94
0.83 26.70 33.69
g2p
14.17 17.21 0.13
D
3.04 5.30 4.14⁄,b 0.38
g2p D Interleukin 6, pg/ml At rest After cooling
Neopterin, nmol/l At rest After cooling
g2p D
5.27 4.33⁄,a 0.57
x
95% CI
–1.16
SD
95% CI
5.07 4.79 0.13
1.27 1.37
4.22–5.91 3.87–5.71
1.35 to 0.52
0.28§,b
0.73
0.53 to 0.53
0.90–27.45 0.46–33.97
19.32 17.82 0.04
46.27 39.24
8.04
0.96 to 7.03
1.51
8.18
7.00 to 3.99
1.43 1.04
4.59–6.01 3.62–4.66
2.10 1.97
3.77–6.60 3.20–5.86
1.52
1.91 to 0.40
0.97
1.17 to 0.30
5.19 4.53⁄,b 0.34 0.66
11.76 to 50.41 8.55 to 44.18
Data are presented as mean ð xÞ, standard deviation (SD), 95% confidence interval (95% CI), partial eta squared (g2p ) and mean difference between baseline and end of cold stress (D). a,bDenote significant differences of P < 0.001 and P < 0.05, respectively between baseline and the end of cold exposure (⁄) or between men and women (§).
Discussion The aim of this study was to test the hypothesis that in response to cold stress: (i) men are more reliant on metabolic strategies, whereas women are more reliant on insulative strategies; (ii) men are more reliant on shivering thermogenesis; and (iii) women experience greater cold-induced stress, which will result in greater changes in endocrine and immune responses during whole-body immersion that decreases TRE to 35.5 °C. As expected, men showed a greater metabolic response and women – a greater insulative response. In addition, more shivering thermogenesis was observed in men than in women. Contrary to our expectations, the CSI was similar in men and women. Most of the endocrine and immune responses after cold stress did not differ between men and women, although the Depinephrine and DTNF-a were greater in men. As expected, all temperature variables decreased during cold stress. Body temperature variables did not differ between men and women before and after cold exposure. Contrary to our expectations, TRE decreased but the TRE cooling rate was the same in men and women. We hypothesized that these differences were related to different body-heating strategies. During cold exposure, the tis-
sue temperature decreases as the body attempts to maintain a normal body temperature by increasing MHP and by minimizing heat loss, primarily through shivering and peripheral vasoconstriction [35]. On exposure to cold, both men and women exhibited an insulative response (a reduction in TSK relative to TRE) and a metabolic response (an increase in heat production and shivering). However, MHP and shivering were significantly greater in men than in women. When exposed to cold stress, the unacclimatized animal acutely defends body temperature by shivering thermogenesis, which increases heat production [40]. Mshiv was positively related to FFM, indicating that the greater muscle mass contributed more to metabolic heat produced by involuntary shivering. Mshiv was negatively related to %BF, suggesting a reason for the lower insulation capacity in men. We do not know whether the women in this study displayed greater nonshivering thermogenesis during cold exposure; however, we can speculate that nonshivering thermogenesis may have been greater in women because they exhibited a similar decrease in TSK and TRE, and less shivering and MHP compared with men. The similar rate of decrease in TRE can be explained by the greater insulation (It), which would have decreased heat loss.
Please cite this article in press as: R. Solianik et al., Gender-specific cold responses induce a similar body-cooling rate but different neuroendocrine and immune responses, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.04.015
R. Solianik et al. / Cryobiology xxx (2014) xxx–xxx
Recent evidence suggests that nonshivering thermogenesis by sympathetic, norepinephrine-induced mitochondrial heat production in BAT is a component of this metabolic response in healthy men and that body fat content and BMI correlate negatively with BAT content [42]. Our finding of a significant negative correlation between BMI and MHP, %BF, and MHP in men but not in women is consistent with this inverse relationship between body fat level and BAT content. In women, Mshiv was negatively related to %BF and BMI, suggesting a lower shivering rate and greater nonshivering thermogenesis. According to Moran et al. [21], larger changes in TRE and TSK during cold strain indicate greater physiological stress. We expected that women would experience a higher stress level (CSI) and exhibit greater activation of the sympathetic–adrenomedullary and hypothalamic–pituitary–adrenocortical systems, as generally occurs in response to stress [10]. However, we found a significant relationship only between CSI and norepinephrine level, and no relationships between CSI and other blood markers. Contrary to our expectation, the CSI and most stress indicators (cortisol, norepinephrine, and dopamine concentrations) did not differ between men and women. As observed in a previous study [37], cold-water immersion did not change the epinephrine level in women. Proinflammatory cytokines are signaling molecules of the inflammatory immune system that initiate and coordinate the cascade of immune events [9]. The most studied hormones with regard to stress are the proinflammatory cytokines IL-6 and TNFa, which are involved in the acute-phase response to inflammatory stressors [14]. However, we found no significant relationship between CSI and proinflammatory markers. We had expected that a higher CSI would increase the immune system response more in women than in men. In contrast to Brenner et al. [4], we did not observe an increase in plasma IL-6 concentration in response to cold exposure. They reported positive relationships between changes in the concentrations of IL-6, norepinephrine, and cortisol, and that the innate immune system was not adversely affected by brief cold exposure. Although we found changes in norepinephrine and cortisol levels, we found no relationship between these hormones and IL-6 level. Charmandari et al. [6] have concluded that IL-6 plays a major role in the overall control of inflammation by stimulating glucocorticoid secretion and by suppressing the secretion of TNF-a. In contrast, we did not observe any relationships between these variables or changes in IL-6 level, although TNF-a concentration decreased significantly in men. Recent evidence indicates that glucocorticoids and catecholamines, the major stress hormones, inhibit the production of proinflammatory cytokines such as TNF-a production [9]. Interestingly, the concentration of proinflammatory TNF-a decreased and epinephrine concentration increased after cold exposure only in men. Van der Poll et al. [41] observed that endogenous epinephrine or that administered as a component of sepsis treatment can inhibit TNF-a. However, we found no significant negative relationships between these variables after cold exposure. Neopterin is another marker of the immune system and is involved in activation of cellular immunity [22]. After cold exposure, neopterin concentration decreased in both men and women. We believe that further studies are needed to explain fully whether and to what extent cold exposure stimulates or suppresses the immune response. The limitation of the present study is that we used two sampling time points to assess the neuroendocrine and immune responses in men and women and that the single time point at the end of cold stress does not cover the whole response curve. It is known, that in vitro mild hypothermia can suppress inflammatory reactions, and moderate hypothermia can delay the induction of pro-inflammatory cytokines [13]. We have induced significant
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response of blood stress markers (Table 3) and proinflammatory mediators (Table 5). By contrast we did not observe any IL-6 response. However previous studies [4,17] have shown that IL-6 response is already evoked after 1 h cooling, thus we expected that our cold stress duration would be sufficient to evoke IL-6 response. It is of great importance to assess kinetics of stress and proinflammatory markers, and further research is warranted according to gender-differences in response to cold. Conclusions In this study, we observed differences in the cold response between men and women. Specifically, men relied more on metabolic strategies and women – on insulative strategies. Men used shivering thermogenesis more than women. Despite the lack of difference in the CSI between men and women, the neuroendocrine and immune responses differed. Men exhibited a higher epinephrine level and a lower TNF-a level during cold stress. Contrary to our expectations, the cooling rate was the same in men and women. Conflict of interest Authors declare that there is no conflict of interest. References [1] H. Anttonen, A. Pekkarinen, J. Niskanen, Safety at work in cold environments and prevention of cold stress, Ind. Health 47 (2009) 254–261. te˙, The effect of two [2] M. Brazaitis, S. Kamandulis, A. Skurvydas, L. Daniusevicˇiu kinds of T-shirts on physiological and psychological thermal responses during exercise and recovery, Appl. Ergon. 42 (2010) 46–51. te˙, Z. Senikiene˙, The [3] M. Brazaitis, A. Skurvydas, K. Vadopalas, L. Daniusevicˇiu effect of heating and cooling on time course of voluntary and electrically induced muscle force variation, Medicina 47 (2011) 39–45. [4] I.K. Brenner, J.W. Castellani, C. Gabaree, A.J. Young, J. Zamecnik, R.J. Shephard, P.N. Shek, Immune changes in humans during cold exposure: effects of prior heating and exercise, J. Appl. Physiol. 87 (1999) 699–710. [5] A.C. Burton, Human calorimetry: II. The average temperature of the tissues of the body, J. Nutr. 9 (1935) 261–280. [6] E. Charmandari, C. Tsigos, G. Chrousos, Endocrinology of the stress response, Annu. Rev. Physiol. 67 (2005) 259–284. [7] C.F. Chi, Y.C. Shih, W.L. Chen, Effect of cold immersion on grip force, EMG, and thermal discomfort, Int. J. Ind. Ergon. 42 (2012) 113–121. [8] A.M. Cypess, S. Lehman, G. Williams, I. Tal, D. Rodman, A.B. Goldfine, F.C. Kuo, E.L. Palmer, Y.H. Tseng, A. Doria, G.M. Kolodny, C.R. Kahn, Identification and importance of brown adipose tissue in adult humans, N. Engl. J. Med. 360 (2009) 1509–1517. [9] I.J. Elenkov, G.P. Chrousos, Stress hormones, proinflammatory and antiinflammatory cytokines, and autoimmunity, Ann. N. Y. Acad. Sci. 966 (2002) 290–303. [10] M. Gunnar, K. Quevedo, The neurobiology of stress and development, Annu. Rev. Psychol. 58 (2007) 145–173. [11] M. Hadi, C.C. Chen, M. Whatley, K. Pacak, J.A. Carrasquillo, Brown fat imaging with (18)F-6-fluorodopamine PET/CT, (18)F-FDG PET/CT, and (123)I-MIBG SPECT: a study of patients being evaluated for pheochromocytoma, J. Nucl. Med. 48 (2007) 1077–1083. [12] I. Janssen, S.B. Heymsfield, Z.M. Wang, R. Ross, Skeletal muscle mass and distribution in 468 men and women aged 18–88 yr, J. Appl. Physiol. 89 (2000) 81–88. [13] A. Kimura, S. Sakurada, H. Ohkuni, Y. Todome, K. Kurata, Moderate hypothermia delays proinflammatory cytokine production of human peripheral blood mononuclear cells, Crit. Care Med. 30 (2002) 1499–1502. [14] A. Koj, Initiation of acute phase response and synthesis of cytokines, Biochim. Biophys. Acta 1317 (1996) 84–94. [15] J.C. Launay, G. Savourey, Cold adaptations, Ind. Health 47 (2009) 221–227. [16] E.C. LaVoy, B.K. McFarlin, R.J. Simpson, Immune responses to exercising in a cold environment, Wilderness Environ. Med. 22 (2011) 343–351. [17] E.C. Lee, G. Watson, D. Casa, L.E. Armstrong, W. Kraemer, J.L. Vingren, B.A. Spiering, C.M. Maresh, Interleukin-6 responses to water immersion therapy after acute exercise heat stress: a pilot investigation, J. Athl. Train. 47 (2012) 655–663. [18] B.B. Lemire, D. Gagnon, O. Jay, G.P. Kenny, Differences between sexes in rectal cooling rates after exercise-induced hyperthermia, Med. Sci. Sports Exerc. 41 (2009) 1633–1639. [19] T.M. Makinen, Different types of cold adaptation in humans, Front. Biosci. (Schol. Ed.) 2 (2010) 1047–1067.
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