Heatstroke (HS) is a complex clinical event defined as a core temperature (Tc) greater than 40°C accompanied by central nervous system (CNS) disturbances such as confusion, ataxia, or coma (1). Heatstroke can be categorized into passive HS (PHS) or exertional HS (EHS) owing to the absence or presence of a physical effort component during heat exposure (2–4). Both conditions can be fatal, with EHS being the third most frequent cause of death in athletes (4).
Heatstroke results from thermoregulatory failure coupled with an exaggerated acute-phase response (1). From mouse models, the thermoregulatory profile during passive heat stress has been characterized as a triphasic pattern, with an initial linear core temperature (Tc) increase, a subsequent equilibrium plateau, and a final rapid progression to HS (5). Prolonged physical activity in hot and humid environmental conditions increases the risk of HS (6, 7). During exercise, a significant amount of extra heat is released from active skeletal muscles. The dissipation of extra heat depends on a temperature gradient from the core through the skin and then to the environment. However, during heat exposure, this gradient is diminished. In addition, cutaneous blood flow decreases drastically as exhaustion appears during physical activity in a hot environment (8). The diminution of cutaneous blood flow with exhaustive exercise in the heat jeopardizes the ability to dissipate heat from the skin. A spiraling increase in Tc during prolonged physical effort has been observed in humans with both high (9) and low physical performance characteristics (10). Therefore, EHS may have a distinct thermoregulatory profile compared with PHS. However, the Tc progression profile during heat stress in the presence of physical effort has not yet been reported. Besides, in comparison with PHS, EHS is more complex to study, since it is difficult to dissociate the direct effects of physical activity from those from the hot environment. If hyperthermia from thermoregulatory failure is the primary factor of death in EHS, then EHS should not be more lethal than an equivalent or even higher heat load in the absence of a physical effort. However, the influence of exercise during heat exposure on HS mortality has never been tested.
The aim of the current study was to test the hypothesis that physical effort during heat exposure may affect thermoregulatory profile and mortality of animals that underwent HS. The theory was tested by measuring HS-related thermoregulatory profiles and mortalities of conscious, unrestrained rats exposed to heat in the presence or absence of physical effort.
MATERIALS AND METHODS
Adult male Sprague-Dawley rats (10–12 weeks; weighing 325.7 ± 15.9 g) were purchased from the Guangdong Medical Laboratory Animal Center (Guangzhou, China). The animals were housed in the animal center of the General Hospital of Guangzhou Military Command, according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 86–23, 1985 revision). The rats (4/cage) were housed under controlled environmental condition (12-h light/dark cycle, 55% ± 5% humidity, 23°C ambient temperature) with free access to standard chow and water. All experimental protocols involving animals were approved by the Animal Care and Use Committee of the General Hospital of Guangzhou Military Command.
Tail-cuff and femoral arterial blood pressure measurements
Systolic blood pressure (SBP) was determined in conscious rats using a noninvasive tail-cuff blood pressure measurement (BPM) system (Biowill, Shanghai, China), as previously described (11). The tail-cuff BPM system was previously validated by femoral artery BPM system (12), and the present study further validated this approach. Briefly, 8 rats were anesthetized using an intraperitoneal injection of 5% chloral hydrate solution (300 mg/kg). The right femoral artery was cannulated with a trocar (24G) for blood pressure monitoring. After 3 days of recovery, each rat underwent the passive heating protocol as described below under Heating Protocol, and tail-cuff and femoral pressure measurement values were recorded simultaneously at 10-min intervals until SBP started to drop from the peak level. Measurements were correlated between the 2 methods using the Pearson test.
Core temperature was monitored by rectal temperature using a thermocouple (BW-TH1101, Biowill, ShangHai, China) inserted 6.5 cm into the rectum.
Before starting the heating and exercise protocols, the rats were acclimatized to the artificial climate chamber (length × width × height = 4 m × 3 m × 2 m; provided by the Department of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, China), motor-driven treadmill (BW-ZHPT-6, 8-channel rat treadmill; Biowill), tail-cuff SBP monitoring system, and rectal thermocouple. The rats remained in their home cages and were then placed in the chamber with an ambient temperature (Ta) of 27°C ± 0.5°C and humidity of 55% ± 5% for 8 h each day for 2 weeks and for 24 h the day before the experiment, with ad libitum food and water. Exercise acclimation was performed by making the rats run at a speed of 15 m/min (without incline) for 10 min each day for 2 weeks and rest for 3 days before the experiment. All the rats used in this study underwent rectal temperature, tail-cuff SBP and heart rate (HR) measurement twice each day for 2 weeks before the experiment.
Approximately 24 h before heat stress, the rats were randomly assigned to the rest-heated or run-heated group (n = 64 per group), or their respective control groups (n = 60 per group). All animals were weighed 1 h before the experiment, and food and water were removed from the cages. To induce HS, the animals were placed in the artificial climate chamber, set at an ambient temperature (Ta) of 39.5°C ± 0.2°C, with a relative humidity of 60% ± 5%. Then, the rats underwent or not the exercise protocol. During this time, the rats in the control groups were exposed to a Ta of 27°C ± 0.5°C, with a relative humidity of 55% ± 5%, and underwent exercise or not. Core temperature, SBP, and HR were measured at 10-min intervals. The time point at which the SBP started drop down from the peak level was taken as a reference point of HS onset (13–19). At the onset of HS, heat exposure was terminated, and the rats were immediately removed from the chamber, weighed, and returned to a Ta of 25°C ± 0.5°C with food and water ad libitum. Core temperature was further monitored at 10-min intervals for 4 h.
The run-heated group ran on a treadmill that was placed in the artificial climate chamber. The control group ran without heating. Rats ran at 15 m/min without incline but were allowed a 2-min rest period every 8 min of work for the measurement of SBP and Tc. Heat tolerance and exercise ability vary between individual rats (6). During preliminary experiments, we observed that the shortest time span beyond which rats would exhibit exhaustion signs (defined as rats not keeping pace and unable of righting themselves when placed on their back) under run-heated condition was 55 min. Thus, the total running time was limited to 50 min (including the rest periods) to make sure that each rat within a group had an equal amount of exercise. After the 50-min run, the treadmill was stopped, but heating was continued until HS onset.
Thermal stress determination
Thermal stress was quantified according to previously reported methods, with some modifications (5). Supplementary Figure 1 (Supplemental Digital Content 1, at https://links.lww.com/SHK/A295) provides a graphical representation of the calculations that were performed to describe the thermoregulatory responses of heat-stress rats. The severity of hyperthermia was calculated as Tc,HS (5). Tc,HS is the Tc at HS onset defined as SBP drop from the peak level. Total thermal area (in °C·min) was used as an indication of thermal load and was calculated when Tc exceeded 39.5°C. Core temperature of 39.5°C was used because it equals the heat stress temperature (39.5°C) and represents the temperature above which rats radiate excess body heat in the environment (5). The ascending (39.5°C to Tc,HS) and descending (Tc,HS to 39.5°C) aspects were segregated to further describe thermal area. Gross heating rate (°C/min) is the average Tc elevation rate during the whole heat stress period and was calculated using the following formula: Gross Heating Rate (°C/min) = (Tc,HS − Tc at the Start of Heating) / Heat Exposure Time (min). Hypothermia was defined as Tc less than 35°C (7). Tc,min (°C) is the lowest Tc value during the 4-h recovery period.
Cooling rate determination
Cooling rate (CR) (°C/min) is the Tc descending rate at Ta of 25°C ambient during the first 30 min after HS onset and was calculated using the following formula: CR = (Tc,HS − Tc after 30-min recovery) / 30 (min) (5).
Dehydration was determined using the difference between preheat and postheat body weight (BW): Percent Dehydration = [(Preheat BW − Postheat BW) / Preheat BW] × 100%.
Statistical analysis was performed using SPSS version 17.0 (IBM, Armonk, NY, USA). Survival was expressed as percentage, whereas other data are expressed as mean values ± standard deviation (SD). One-way analysis of variance (ANOVA) with Bonferroni post hoc test was used to compare groups for BW, dehydration, heat exposure time, Tc end of run, thermal area, Tc,HS, heating rate, SBP at HS onset, and Tc,min. Cox regression was used to estimate hazard ratios and 95% confidence intervals (CIs) of HS mortality. Survival was analyzed by the Kaplan-Meier method and compared by the log rank test. The chi-square test was used to compare survival rate between the run-heated and rest-heated groups. Inverse model curve estimation was used to analyze the correlations between HS mortality and Tc,HS or thermal area. A 2-tailed P ≤ 0.05 was considered statistically significant.
Validation of the tail-cuff SBP measurement
The validity and reliability between tail-cuff and femoral SBP measurements are presented in Figure 1. Strong correlation between the 2 measurements were detected (Pearson r = 0.955, P < 0.001).
HS thermoregulatory profiles in Sprague-Dawley rats
Figure 2 shows the Tc, SBP, and HR regulatory profiles of the representative individual rats of the rest-heated group (Fig. 2A) and run-heated group (Fig. 2B) as well as their respective control groups. The heat exposure time ranged from 160 to 200 min in the rest-heated group and from 70 to 100 min in the run-heated group.
The Tc of the rest-heated group exhibited an initial steep rise (from zero to 20 min; heating rate = 10.1% ± 1.1%) owing to uncompensated environmental heat load because Ta was higher than Tc. Once Tc became higher than Ta, rats could effectively dissipated extra body heat to the environment and showed a decreased Tc slope (from 20 to approximately 140–170 min; heating rate, 1.8% ± 0.2%). A second increase in Tc slope subsequently developed (from approximately 140–170 min to HS onset; heating rate, 6.1% ± 0.8%), indicating a breakdown of thermoregulatory control (Fig. 2A).
Core temperature progression in the run-heated group also exhibited an initial steep rise in Tc (from zero to 20 min; heating rate, 10.1% ± 1.1%) owing to heat exposure and exercise (from zero to 50 min; heating rate, 8.8% ± 0.5%), a relatively slower rise after exercise during heat exposure (from 50 to approximately 60–80 min; heating rate, 2.0% ± 0.5%), and a final rapid progression until HS onset (from approximately 60 to 80 min to HS onset; heating rate, 4.9% ± 0.5%) (Fig. 2B).
There were significant correlations between Tc and SBP (r = 0.955 and r = 0.922; P < 0.001), and HR (r = 0.988 and r = 0.998; P < 0.001) during heat exposure in the rest-heated and run-heated groups.
Table 1 presents each measured and calculated parameters for the 2 groups of HS animals and their respective controls. Compared with the rest-heated group, physical effort during heat stress significantly shortened the latency of HS onset (ANOVA, P < 0.001). By the time of HS onset, the 2 HS groups had virtually identical Tc,HS (ANOVA, P > 0.05), whereas the run-heated group had a significantly smaller ascending thermal area than the rest-heated group (ANOVA, P < 0.001), indicating that physical effort during heat exposure may not increase the severity of heat-induced hyperthermia, but it decreased the thermal load for HS. In addition, the run-heated group had a significantly larger descending thermal area than the rest-heated group (ANOVA, P < 0.001), indicating that the animals maintained longer in heat stress situation after occurrence of HS. The total thermal area of the run-heated group was still significantly smaller than the rest-heated group (ANOVA, P < 0.001).
In all cases, heat stress induced significantly higher SBP and HR than the control conditions (ANOVA, P < 0.001), but there were no differences in SBP and HR between the rest-heated and run-heated groups at HS onset (ANOVA, P > 0.05) (Table 1).
Dehydration was presented as the percentage of BW loss. As shown in Table 1, there was no difference in BW loss between the 2 control groups (ANOVA, P > 0.05). In all cases, HS induced significantly greater dehydration than the control conditions (ANOVA, P < 0.001), but differences were not detected between the rest-heated and run-heated HS rats (ANOVA, P > 0.05).
Cooling rate and hypothermia after HS onset
As shown in Table 1, the run-heated rats had significantly slower CRs than the rest-heated rats (ANOVA, P < 0.05). Despite the differences in CR, the rest-heated and run-heated rats developed hypothermia within 1 h after heat exposure. There were no significant differences between the rest-heated and run-heated groups with regard to the Tc,min (ANOVA, P > 0.05). The Tc,min was inversely correlated with Tc,HS in both the rest-heated (r = −0.926, P < 0.001) and run-heated (r = −0.910, P < 0.001) groups.
Table 1 shows the survival rates of the rest-heated and run-heated groups and their respective control groups. All control rats survived the 72-h observation period. Although physical effort alone could cause mild thermal load (total thermal area, 38.3 ± 12.9°C·min), no death was observed in the control groups during the 72-h observation period. The rest-heated and run-heated groups had a 46.9% and 31.3% survival rate at 72 h, respectively (chi-square, P < 0.01). As shown in Figure 3, the survival time was significantly shorter in the run-heated group than in the rest-heated group (Kaplan-Meier, P < 0.001). The main factors related to poorer survival were Tc,HS (P = 0.029) and total thermal area (P = 0.012) in the rest-heated group and Tc,HS (P = 0.007) in the run-heated group (Table 2).
Risk factors of HS mortality
The relationships between HS responses and HS mortality are shown in Table 2. Among the listed HS responses, the main factors correlated with poorer survival were Tc,HS (Cox regression, P = 0.019 or P = 0.002, respectively), total thermal area (Cox regression, P = 0.000 or P = 0.037, respectively), ascending thermal area (Cox regression, P = 0.000 or P = 0.041 respectively), and descending thermal area (Cox regression, P = 0.000 or P = 0.002, respectively) in both of the rest-heated and run-heated groups. To further demonstrate the results of multivariate Cox regression analysis, all survivors and fatalities in the rest-heated or run-heated group are separately pooled into 2 large groups, respectively, for the comparison of potential risk factors for the mortality of HS. In both of the rest-heated and run-heated groups, rats that subsequently died had higher Tc,HS and total thermal area than the surviving rats (ANOVA, P < 0.001). In addition to higher ascending thermal area, the fatalities had higher descending thermal area than the survivors (ANOVA, P < 0.001), indicating that significant heat injury can occur after withdrawal from the heat.
To further investigate the relationship between Tc,HS or total thermal area with HS mortality, we applied curve estimate analysis. The results have shown that there was continuum of increasing incidence of HS death with increasing Tc,HS or thermal area in both the run-heated and rest-heated HS rats (curve estimate, P < 0.001). Interestingly, in comparison with the rest-heated rats, the run-heated animals had a similar Lethal Dose, 50% (LD50) of Tc,HS (43.3°C) and far lower LD50 of thermal area (322.9 vs. 159.9).
The aforecited dose-response curves provide an objective method of classifying the severity of hyperthermia and thermal load based on the incidence of mortality within the total population. Thus, each animal in the rest-heated or run-heated group was retrospectively assigned into 1 of 4 groups based on intervals of Tc,HS or total thermal area: (1) LD0–25, (2) LD25–50, (3) LD50–75, and (4) LD75–100. This classification allows comparing run-heated and rest-heated rats whose reaction to experimental treatment resulted in a similar probability of death and comparing survivors versus dead rats over a wide range of thermal load and Tc.
As previously mentioned, rats that subsequently died had a higher Tc,HS and total thermal area than survivors. However, when comparing the total thermal area or Tc,HS of both survivors and fatalities within a narrow range of Tc,HS (Fig. 4A) or total thermal area (Fig. 4B), respectively, these distinctions disappear when the mortality is less than 50%. In the present study, the distinction of total thermal area between rats that subsequently died and surviving rats exist only when mortality is more than 75%. A possible explanation is that the means presented in Figure 4 are isolated according to narrow ranges in total thermal area or Tc,HS, and this eliminates the comparison of all survivors with all rats that subsequently died, which effectively masks the existence of both heat-sensitive and heat-resistant individuals. Results from Figure 4 indicate that Tc,HS is more suitable to predict mortality than thermal area.
The relationship between Tc,HS and survival is depicted in Figure 5. This histogram represents the results from 128 rats that either ran or rested at Ta of 39.5°C until HS onset. Death occurred when Tc,HS was more than 43°C in both groups. All rats that endured a Tc,HS higher than 43.5°C died.
We hypothesized that physical effort during heat exposure may affect thermoregulatory profile and mortality of HS. Results from the present study supported our hypothesis, suggesting that (1) the thermoregulatory profile of EHS rats was different from PHS rats in certain aspects; (2) working component during heat exposure contributed to an increased mortality rate of HS; and (3) in both EHS and PHS rats, the incidences of mortality are correlated with the severity of hyperthermia.
A hallmark symptom of HS is CNS dysfunction (1). Previous studies demonstrated that animals would develop CNS symptoms just after the mean arterial blood pressure peaks and begins to decrease (20). Therefore, the drop of mean arterial blood pressure from its peak has been recognized as a sign of HS onset in animal models of HS (13–19). So far, BPM using femoral artery catheter is the most accurate BPM method. However, owing to exercise component, artery catheter is not suitable for the EHS animal model. Some authors suggested to use preset Tc to define HS (5, 21, 22), whereas others suggested to make animals run to exhaustion at normal ambient temperature as the sign of EHS onset (6). However, EHS occurs predominantly in hot and humid environment. Furthermore, heat tolerance capability as well as exercise capability varies between individual animals. Thus, HS symptoms may or may not develop at the same Tc level or exhaustion. Our rat EHS model overcomes these experimental limitations by applying the tail-cuff BPM system. The accuracy of this method has been validated by direct BPM methods in previous studies (11, 12, 23) and in the present study. The tail-cuff method is noninvasive and does not require surgical intervention, making it suitable for the EHS model. According to our data, by the time SBP peaks and starts to decrease during heat exposure (representing HS onset), both PHS (rest-heated) and EHS (run-heated) rats exhibited a wide variability in Tc,HS (ranging from 43°C to 43.8°C in PHS rats and from 43°C to 43.7°C in EHS rats; Table 1). These results indicated the limitation of the use of a predetermined Tc as an indicator of HS onset.
Although the thermoregulatory responses to passive heat exposure have been documented in previous mouse models of HS (5), this is the first study to demonstrate that physical effort could significantly affect thermoregulatory profile of HS rats during heat exposure and HS recovery Table 3). During heat exposure, either rest-heated or run-heated rats exhibited a characteristic triphasic Tc progression pattern. However, compared with the rest-heated rats, the run-heated rats had a significantly shorter latency of HS onset and thus had a significantly smaller ascending and total thermal area. Despite the significant difference in HS latency and thermal area, no significant difference in Tc,HS was observed between EHS and PHS rats, indicating that physical effort during heat stress did not significantly influence Tc,HS (Table 1). It seemed that the exercise component during heat stress did not increase the severity of hyperthermia but decreased the thermal load requirement to develop HS. A possible explanation for the thermoregulatory profile of HS rats is that there might be a threshold of thermal control in animals, and Tc progression elicited by heat exposure in the presence or absence of physical effort surpasses this threshold, causing the breakdown of thermal control and inducing HS. However, a previous study failed to detect thermal injury to the hypothalamus at autopsy of fatal cases of HS (24). Therefore, the exact mechanisms for the heat stress thermoregulatory profile still need further exploration.
When moved back to the environment with Ta of 25°C ± 0.5°C after HS onset, all animals exhibited rapid Tc decrease. In comparison with the rest-heated rats, the run-heated rats had a significantly slower CR and therefore had a larger descending thermal area (Table 1), indicating that physical effort could affect heat dissipation after heat exposure. Animal passive heat stress was followed by a period of hypothermia, which has been previously reported (5). However, in the present study, we first observed that hypothermia also occurred after EHS, and the exercise component had no influence on hypothermia level. The pathophysiological mechanism for postheat hypothermia remains unknown. We noticed that hypothermia level was associated with Tc,HS in HS rats, and previous studies have reported that preventing the postheat hypothermia could significantly increase mortality of HS animals (5, 25). This suggests that hypothermia level might be related to hyperthermia severity, and it may be a protective thermoregulatory event.
Previous studies have reported that an elevation in thermal area correlates with decreased survival rate (5, 6). According to our data, the rest-heated rats had significantly larger total thermal area than the run-heated rats, and therefore, we expected the run-heated rats to experience decreased heat-induced mortality in our model. Surprisingly, this combination of heat and exercise was more fatal than acute exposure to excessive heat at rest. Although exercise per se is a stressor, we did not observe death in the exercise control group during the 72-h observation period. These data suggested that heat plus exercise are more lethal than heat alone. Analyzed by multivariate Cox proportional hazard regression, Tc,HS, total thermal area, ascending thermal area, and descending thermal area were all found to be independent prognostic factors for the overall survival of HS rats. Higher value of those parameters correlated with higher mortality in the rest-heated and run-heated rats, respectively. Compared to the rest-heated rats, the run-heated ones had relatively lower Tc,HS, total thermal area, and ascending thermal area values. Thus, we preclude those parameters as the reasons for the relative higher mortality of the run-heated rats. The descending thermal area represents thermal area during HS recovery. In comparison with the rest-heated rats, the run-heated rats had relative slower CR and so had larger descending thermal area, indicating that physical effort during heat exposure could jeopardize the ability of heat dissipation after HS onset. Although CR did not emerge out of multivariate analysis, the significant correlation between descending thermal area with survival suggested that the impaired heat loss capacity during recovery may be a possible mechanism of relative poor survival of EHS rats.
Regarding thermal area and Tc,HS, some previous studies suggested thermal area as the more accurate predictor of HS mortality, whereas others indicated Tc,HS as the more accurate one. Applying curve estimate analysis, we found that the LD50 of Tc,HS was almost identical between the rest-heated and run-heated groups, whereas the LD50 of thermal area in the rest-heated group was more than 1-fold higher than in the run-heated group. Furthermore, in both HS groups, dead rats had significantly higher Tc,HS than survivors when mortality was more than 50%, whereas in the run-heated group, the distinction in thermal area only existed when mortality was more than 75%. Taken together, these data suggest that Tc,HS is a more accurate predictor of HS mortality than thermal area, especially in the presence of exercise.
Although there was a good agreement between Tc,HS and mortality rates, some HS animals still exhibited individual variability in susceptibility to heat-induced mortality. Indeed, some animals were able to survive Tc,HS reaching 43.5°C, whereas others succumbed to Tc,HS reaching 43°C. Under these circumstances, the point of collapse does not ensure similar chances of survival among different individuals. However, none of the animals survived at Tc,HS higher than 43.5°C, indicating that with increasing hyperthermia severity, animals seem to have similar response to heat-induced mortality.
Both exercise or heat exposure per se can cause dehydration, and dehydration is a common phenomenon in patients with HS (4). However, the influence of dehydration on EHS mortality has never been experimentally defined. Although we observed significant dehydration in rats at HS onset in our run-heated and rest-heated models, there were no differences in dehydration between the 2 heated groups, which may be attributed to the significantly longer heat exposure time of the rest-heated rats compared with the run-heated rats (Table 1). Although dehydration can significantly impair cardiovascular and thermoregulatory responses to HS (5), the presence of similar dehydration levels despite differences in survival and severity suggests that this variable may not be a causative factor for the relatively higher mortality of EHS rats.
The present study suffers from some limitations. First, because of the working component and electrical stimulation, we could not apply biotelemetry to monitor Tc progression as reported by Leon et al (5). Although we performed acclimation training for the rats before the experiment, the present measurement method still might affect normal behavior and physiological mechanisms in rats and therefore methodological confounders might still be present. In addition, it remains an animal model, and these results might not be directly applied to humans.
In conclusion, thermoregulatory profile of the run-heated animals was different from that of the rest-heated animals. Although the exercise component had no influence on heat-induced hyperthermia severity at low and comparable thermal loads, mortality was higher in the run-heated group compared with the rest-heated group. A possible mechanism for the relatively poor survival of EHS rats is the impaired heat dissipation during recovery. The present model can serve as an experimental paradigm to support further studies to determine the physiological mechanisms behind the morbidity and mortality of EHS.
The authors thank Professor Jin-qiang Guo, Department of Heat Environmental Medicine, for his assistance in the use of the artificial climate chamber. Expert statistical assistance from M.D. Danli Kong, Department of Public Health and Statistic of Guangdong Medical College, is appreciated.
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