Interindividual variation of the organism - as an indicator for assessing the severity of heat stroke in an experimental study of Wistar rats

doi:10.21203/rs.3.rs-2202186/v1

Introduction

Autopsies are often performed, but the evidence is insufficient and non-specific. The aim of the research was to determine the core temperature values of rats exposed to different water temperatures (37°C, 41°C, 44°C), before the start of the experiment (Tb), after immersion in water (Tu), after 20 minutes of exposure (Tu) and at death. (Ts) rats for the purpose of hyperthermia and heat stroke.

Material and Method

Forty rats were divided into five groups depending on the temperature and length of exposure to water: control group-CG37, G41-hyperthermia- group which exposure time was a 20 minutes on 41°C, G41-heat stroke- group exposed until death on 41°C, G44- hyperthermia- group which exposure time was a 20 minutes on 44°C, G44- heat stroke- group exposed until death on 44°C. A RET-4 probe was used to measure the core temperature of rats.

Results

Significant changes in the body temperature of rats were observed during the lethal outcome, p < 0.0005. A significant difference was also observed in postmortem temperature of groups G41 and G44, p = 0.01. a significant difference between body temperatures in groups CG37, G41-hyperthermia, G41- heat stroke, G44-hyperthermia and G44-heat stroke (p < 0.0005), and the significance of the differences in the CG37 group was p = 0.044.

Conclusion

Exposure of albino rats to different water temperatures also led to a change in the internal temperature; normothermia was established through thermoregulation in the control group, and in the other groups, hyperthermia and heat stress occurred.

temperature

variation

heat

rats

heat stroke

A large number of deaths both in Europe and in the United States of America have been attributed to exposure to high temperatures (1, 2). Central Europe experienced its hottest summer in 2003 since 1500, with average temperatures around 3.5˚C above the upper air temperature limits. The number of deaths was between 22,000 and 45,000, in a period of two weeks, according to data published by the Red Cross Organization (3). Common causes of death were dehydration and heat stroke in hospitalized patients in France(4). A 7% increase in mortality recorded during the same period in Switzerland was associated with heat stroke (5). The core temperature of internal organs is regulated at approximately 36.6°C (6), but due to exposure to high temperatures it can vary significantly, from the lowest recorded temperature of 13.7°C experienced by human survivors (7) to a maximum of 41.5°C without any complications for the body (8). Core body temperature in humans is the main regulated variable in thermoregulation (9). Body core temperature is the result of local thermal balance depending on the place of measurement (10). It has been proven that the influence of high air temperature triggered certain processes in the organism and that compensatory mechanisms failed. At autopsy in cases of sudden death after exertion, atherosclerosis of the coronary vessels was found in more than 60%, left ventricular hypertrophy of unknown etiology in 7.8%, and valvular heart disease in 7.1% (3–5). Sudden death during exertion can depend on the predisposing heart disease. In comparison with the control group, a statistically significant association was observed between the subjects' heat exhaustion and changes in the heart tissue. Establishing absolute evidence in support of the diagnosis of death caused by heatstroke is a major problem in forensic pathology. Primarily, it is not routine to always measure the temperature of the deceased. In addition, macroscopic findings are not site-specific, especially if the survival time was short.The diagnosis of hyperthermia is based on investigative actions, circumstances, and reasonable exclusion of other causes of death.

The aim of the research was to determine the core temperature values of rats exposed to different water temperatures before the start of the experiment (Tb), after immersion in water (Tu), after 20 minutes of exposure (Tu) and at death. (Ts) rats for the purpose of hyperthermia and heat stroke.

The study was processed as a prospective experimental, randomized study based on the albino Wistar rat model of hyperthermia. The research was conducted at the Medical faculty of the University of Sarajevo, in accordance with the Principles for the Care of Laboratory Animals (11). After the approval of the Ethics Committee of the Faculty of Medicine of the University of Sarajevo (registration number 02-3-4-1253/20, Bosnia and Herzegovina), the total number of adult Wistar rats, body weight from 200 to 300 g, were included in experiment.Target temperature points in the water bath were determined and one rat at a time was exposed to a total of 40 rats included in the experiment. Rats were divided into five groups depending on the temperature and length of exposure to water: control group exposed to water temperature of 37°C (CG), groups exposed to water temperature of 41°C and 44°C for 20 minutes (G41-hyperthermia; G44-hyperthermia), groups exposed to water temperature of 41°C and 44°C with exposure time until death (G41-heat stroke; G44-heat stroke). Heat stroke is defined as an increase in core temperature above 40.5°C (8, 9).

The rats were anesthetized with ketamine in a dose of 1.2 mL/kg tw + /- 10% (USP Rotexmedica-Germany) (12). The experimental protocol was made according to the previously described methodology of induction of hyperthermia and heat shock (13). A probe (RET-4 probe for mice and rats) was used to measure the core temperature of rats, and the core temperature was read on a thermometer (Physitemp Instruments Clifton, Physitemp Thermalert Model TH-8, USA).

Basal temperature was the lowest in group G44 and was 38.02 ± 0.55, and the highest in G37 was 38.25 ± 0.12°C. The immersion temperature ranged from 37.84°C in G41 to 38.67 ± 0.90° in G44. In group KG37, the lowest temperature of 37.75°C was observed after 20 minutes of exposure, and the highest in G44, 43.09 ± 0.79°C. The death temperature was lower in G41°C 38.34 ± 3.59 compared to 41.51 ± 2.73°C in G44 (Table 1).

Table 1

Mean values of measured body temperature of rats of experimental groups at four time points
T (°C)	Grupa	x̄	±SD	95% IP
T (°C)	Grupa	x̄	±SD	DG	GG
T-b (°C)	KG37	38.25	0.12	38.13	38.37
	G41	37.80	0.48	37.53	38.07
	G44	38.02	0.55	37.72	38.32
T-u (°C)	KG37	38.17	0.23	37.95	38.39
	G41	37.84	0.50	37.56	38.12
	G44	38.67	0.90	38.19	39.15
T-20 (°C)	KG37	37.75	0.38	37.40	38.11
	G41	40.04	0.43	39.79	40.28
	G44	43.09	0.70	42.71	43.46
T-s(°C)	G41	38.34	3.59	36.35	40.33
	G44	41.51	2.73	40.05	42.97
± X ̅- mean value; ±SD-standard deviation; IP-Confidence Interval;DG-Lower Bound; GG- upper limit; T-b- basal temperature of rats; T-u immersion temperature of rats; T-temperature in the 20th minute; T-s- temperature of death; p-probability KG37-control group of rats exposed to water temperature of 37˚C; G41-group exposed to water temperature 41˚C; G44-group of rats exposed to a water temperature of 44˚C;

By analyzing the mean values of body temperature measured at four time points, showed that a basal body temperature of rats of group KG37 was not significantly different from the basal temperature of rats G41 and G44, p = 0.125, so multiple comparisons between groups were not performed. The temperature after water immersion depending on the group was significantly different between groups G41 and G44, p = 0.005. After exposure to water temperature for a period of 20 minutes, depending on the group, it was observed that the body temperatures of rats differed significantly between G37 and G41, KG37 and G44, p < 0.0005 and G41 and G44, p < 0.0005. A significant difference was also observed in postmortem temperature of groups G41 and G44, p = 0.01 (Table 2).

Table 2. Difference in body temperature values of rats of experimental groups at three time points

T (°C)	Group	Group	p	95% IP
T (°C)	Group	Group	p	DG	GG
T-u (°C)	G44	G41	0.005	-1.42	-0.22
T-20 (°C) T-s (°C)	KG37	G41	<0.0005	-2.91	-1.65
	KG37	G44	<0.0005	-5.95	-4.71
	G44	G41	<0.0005	2.56	3.54
	G41	G44	0.01	-5.86	-0.46

T (°C) - Temperature in degrees Celsius; p-probability; IP-Confidence Interval;DG-Lower Bound; GG- upper limit; T-u immersion temperature of rats; T-temperature in the 20th minute; T-s- temperature of death; p-probability; KG37 (-control group of rats exposed to water temperature of 37˚C; G41-group exposed to water temperature 41˚C; G44-group of rats exposed to a water temperature of 44˚C;

Repeated measurements of body temperature at three time points (T-b, T-u, T-20) by group showed a significant difference between body temperatures in all three groups. In the control group (KG37) a significant difference was observed in three measurements (p = 0.044), and in groups G41 and G44 a significant difference in the body temperature of the rats was observed in three measurements (p < 0.0005) with a drop in the temperature of the KG37 group in 20 minutes and an increase in temperature in groups G41 and G44 (Fig. 1)

Repeated measurements of body temperature at three time points per group, examining not only the influence of temperature but also the influence of length of exposure to water temperature depending on the experimental group, showed a significant difference between body temperatures in groups CG37, G41-hyperthermia, G41- heat stroke, G44-hyperthermia and G44-heat stroke (p < 0.0005), and the significance of the differences in the CG37 group was p = 0.044 (Fig. 2).

The mean values of the measured body temperatures of the rats and the significance of the difference of the measured temperatures are presented in the table in 3 a and b.(Table 3)

Table 3. Multiple comparison of body temperature of rats of groups at three time points

a)

T (°C)	Grupa	Grupa	P	95% IP
T (°C)	Grupa	Grupa	P	DG	GG
T-u (°C)	G41-AM	G41-PM	0.04	-2.07	-0.03
	G41-PM	G44-AM	0.03	-2.03	-0.6
T-20 (°C)	KG37	G41-AM	<0.0005	-3.00	-1.25
		G41-PM	<0.0005	-3.2	-1.57
		G44-AM	<0.0005	-6.08	-4.39
		G44-PM	<0.0005	-6.27	-4.58
	G41-AM	G44-AM	<0.0005	-3.95	-2.26
	G41-AM	G44-PM	<0.0005	-4.14	-2.45
	G41-PM	G44-AM	<0.0005	-3.64	-2.00
	G41-PM	G44-PM	<0.0005	-3.82	-2.19

b)

T (°C)	Grupa	Grupa	p	95% IP
T (°C)	Grupa	Grupa	p	DG	GG
	KG37	G41-PM	<0.0005	-9.19	-4.29
		G44-AM	<0.0005	6.99	-409
		G44-PM	<0.0005	-12.01	-7.11
	G41-AM	G41-PM	<0.0005	-8.56	-3.66
T-s		G44-AM	0.001	-6.36	-1.46
		G44-PM	<0.0005	-11.38	-6.48
	G41-PM	G44-PM	0.013	-5.19	-4.58
	G44-AM	G44-PM	<0.0005	-7.39	-2.65
	G44-PM	G44-AM	<0.0005	2.65	7.39

T (°C) - Temperature in degrees Celsius; p-probability; IP-Confidence Interval;DG-Lower Bound; GG- upper limit; T-b- basal temperature; T-u- rat immersion temperature; T-s- temperature of death; KG37-control group of rats exposed to water temperature of 37˚C; G41-AM (G41-hyperthermia)- antemortem group exposed to water temperature 41˚C (length of exposure 20 minutes); G41-PM (G41-heat stroke) - postmortem group exposed to water temperature 41˚C (length of exposure until death); G44-AM (G44-hyperthermia)-antemortem group of rats exposed to water temperature of 44˚C (length of exposure 20 minutes); G44-PM (G41-heat stroke) -postmortem group of rats exposed to water temperature of 44˚C (length of exposure until death);

Large temperature fluctuations and climate changes affect the global temperature, with the period between 2011 and 2020 being the warmest in the last 140 years (14–17). Since 2015, the warmest years are 2016, 2019 and 2020. The mean global temperature was 1.2°C above the level in 2020, (17) and between 2030 and 2052, it is predicted to rise by another 1.5°C (18). These temperature changes of the environment directly affect the internal temperature of the organism because the basic temperatures increase and the loss of water in the body also increases the reserve consumption of the compensatory mechanisms of the organism (18).

Heatstroke leads to oxidative cellular stress and impaired thermocompensatory mechanisms, and can range from subtle heat exhaustion to death. The organism includes compensatory mechanisms of heat release through sweating, tachycardia, and ultimately the activation of pro-inflammatory and inflammatory cytokines. Heatstroke is defined as a state of elevated body temperature of 40.05°C and above this value, which is a consequence of overemphasized compensatory mechanisms and exhausted body reserves with interindividual variations (19, 20). Through heat stroke, a condition occurs that in the range of 10 to 50% leads to death through cardiac dysfunction with global hypokinesia (21). Given that the number of deaths caused by heatstroke is increasing, it is necessary to understand the behavioral response of the organism to increased environmental temperature, which is directly related to global warming (22). In the world, the number of deaths that occur in closed spaces with an increase in air temperature inside the room, for example during bathing, in saunas, Turkish baths, and among athletes due to strenuous training, especially in the summer months, is increasing. Two significant causes of sudden death in athletes are death due to arrhythmia and heat stroke. Death caused by arrhythmia is more in the center of medical attention while heatstroke is less considered (23). Autopsy rates remain low and vary between countries, and protocols for performing autopsies in cases of suspected sudden death also differ (24).

Determining heat stroke as the cause of death is very difficult in forensic medicine due to the non-specificity of biochemical analyses. In our rat model of thermal climate, a thermoregulatory response was included by exposing rats to different water temperatures (37, 41 and 44°C). An esophageal probe was used to measure core temperature. The proven physiological body temperature of rats is 37.5–37.7°C (25), and the aim of the research was to determine the core values of rats exposed to different water temperatures (37, 41 and 44°C), before the start of the experiment (Tb), after immersion in water (Tu), after 20 minutes of exposure (Tu) and at the moment of death. (Ts) rats for the purpose of hyperthermia and heat stroke. Basal temperature was the lowest in group G44 and the highest in control group, which is in correlations with interindividual variations. The death temperature was lower in G41 compared to G44, which presents of the physiological state and the absence of pathological processes in the rats before the experiment started. Given that the analysis of basal temperatures in the groups werw not signifficant, this points to the conclusion about the physiological state of the organism and normothermia in rats before the start of the experiment. A significant difference appears during immersion in water and after 20 minutes and at the moment of death, which points to the use of control mechanisms of the thermoregulation center. Heat stroke was detected in rats exposed to a highest temperature of 44 degrees.

Our results are in accordance with the literature data that indicate that the minimum time required for the onset of heatstroke, with the compensatory mechanisms of the organism involved, is 20 minutes from the moment of the target temperature (19). Basal temperature is the result of the organism's physiological thermoregulation itself, while increased ambient temperature and the length of exposure of the body affect the increase in internal temperature, and the inclusion of the compensatory mechanisms of the organism with the exchange of heat between the body and the environment. The temperature of rats at the time of death was 44.02 ± 0.38°C in group G44-heat stroke, and 41.20 ± 0.76°C in G41. The rats exposed to water temperatures of 41°C and 44°C had longer survival in group G41 175.50 minutes, compared to rats in group G44 4.14 minutes (p = 0.001), therefore, the short time period of exposure to high temperature caused that there were more rats of group 44 that did not reach the temperature threshold for heat stroke. In a study by Quinn et al. (26), a predetermined temperature is used as the temperature standard of heatstroke, which was also done in our research, and is considered an interindividual variation (27–30). In a study of healthy individuals, the internal temperature reached values of 40°C after 10 minutes of exposure. In addition, deterioration of brain and heart function was observed after immersion in water at a temperature of 44°C, but no brain and heart damage was observed after immersion in water heated to 40°C. The degree of damage depends on the temperature of the environment and the length of exposure of the body to that environment (30). Heat stress induces systemic inflammation through increased cytokine and white blood cell concentrations, which contributes to hyperthermia and reduced metabolic activities, as metabolic reservoirs are depleted to deploy homeostatic mechanisms. Excessive inflammation in heat stress induces the release of damage-associated molecular ligands from apoptotic cells in the final stage of pyroptosis (31).

The modern epoch of climate change also caused research on the topic of thermoregulation of the organism in accordance with adaptation to high temperatures. Thermoregulation of heat is described by the central integrator model of peripheral and central heat inputs that activate thermoeffector reactions when the core temperature moves above physiological. However, further research should consider more complex models that include several integrators and other afferent signals taking into account the stages of the heat flow mechanism, but also the interindividual variations of the response that cannot be excluded.

Exposure of albino rats to different water temperatures also led to a change in the internal core temperature; In the control group, normothermia was established through thermoregulation, and in the other groups, hyperthermia and heat stress occurred.

Ethics declarations

Ethics approval and consent to participate: Approved

Animal ethics

All the procedures were in accordance with the Guide for the Animal Care and Use of Laboratory Animals by the US National Institutes of Health (NIH Publication No. 85–23, Revised 1996), and has approved of the Ethics Committee of the Faculty of Medicine of the University of Sarajevo (registration number 02-3-4-1253/20, Bosnia and Herzegovina). The research was conducted at the Medical faculty of the University of Sarajevo, in accordance with the Principles for the Care of Laboratory Animals (11).

Consent for publication

All authors have seen and approved the manuscript.

Competing interests

The authors declare no competing interests.

Authors' contributions

E.D. was responsible for designing the study while A.S, L.D and Z.A were responsible for recruitment and sampling. M K. and E.M carried out the hematological part of the work and accomplishment of ethical approvals. E.M. L.D and E.D. performed estimation of temperatures. E.D and M.K contributed in statistical analysis and data collection. E. D, Z.A, L.D contributed in literature collection, manuscript writing, and thorough reviewing. All authors have read and approved the final manuscript.

Funding

None

Availability of data and materials

It can be requested from the corresponding author

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No competing interests reported.

Interindividual variation of the organism - as an indicator for assessing the severity of heat stroke in an experimental study of Wistar rats

Status:

Version 1

Abstract

Introduction

Material and Method

Results

Conclusion

Figures

1. Introduction

2. Material And Methods

3. Results

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

Status:

Version 1