The protocol describes the development of a standardized, repeatable, preclinical model of exertional heat stroke (EHS) in mice free from adverse external stimuli such as electric shock. The model provides a platform for mechanistic, preventative, and therapeutic studies.
Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, whereas exertional heat stroke (EHS) occurs during physical activity. EHS differs from CHS in etiology, clinical presentation, and sequelae of multi-organ dysfunction. Until recently, only models of CHS have been well established. This protocol aims to provide guidelines for a refined preclinical mouse model of EHS that is free from major limiting factors such as the use of anesthesia, restraint, rectal probes, or electric shock. Male and female C57Bl/6 mice, instrumented with core temperature (Tc) telemetric probes were utilized in this model. For familiarization with the running mode, mice undergo 3 weeks of training using both voluntary and forced running wheels. Thereafter, mice run on a forced wheel inside a climatic chamber set at 37.5 °C and 40%-50% relative humidity (RH) until displaying symptom limitation (e.g., loss of consciousness) at Tc of 42.1-42.5 °C, although suitable results can be obtained at chamber temperatures between 34.5-39.5 °C and humidity between 30%-90%. Depending on the desired severity, mice are removed from the chamber immediately for recovery in ambient temperature or remain in the heated chamber for a longer duration, inducing a more severe exposure and a higher incidence of mortality. Results are compared with sham-matched exercise controls (EXC) and/or naïve controls (NC). The model mirrors many of the pathophysiological outcomes observed in human EHS, including loss of consciousness, severe hyperthermia, multi-organ damage as well as inflammatory cytokine release, and acute phase responses of the immune system. This model is ideal for hypothesis-driven research to test preventative and therapeutic strategies that may delay the onset of EHS or reduce the multi-organ damage that characterizes this manifestation.
Heat stroke is characterized by central nervous system dysfunction and subsequent organ damage in hyperthermic subjects1. There are two manifestations of heatstroke. Classic heat stroke (CHS) affects mostly elderly populations during heat waves or children left in sun-exposed vehicles during hot summer days1. Exertional heat stroke (EHS) occurs when there is an inability to thermoregulate adequately during physical exertion, typically, but not always, under high ambient temperatures resulting in neurological symptoms, hyperthermia, and subsequent multi-organ dysfunction and damage2. EHS occurs in recreational and elite athletes as well as military personnel and in laborers with and without concomitant dehydration3,4. Indeed, EHS is the third leading cause of mortality in athletes during physical activity5. It is extremely challenging to study EHS in humans as the episode can be lethal or lead to long-term negative health outcomes6,7. Therefore, a reliable preclinical model of EHS could serve as a valuable tool to overcome the limitations of retrospective and associative clinical observations in human EHS victims. Preclinical models of CHS in rodents and pigs have been well characterized8,9,10. However, preclinical models of CHS do not directly translate into EHS pathophysiology due to the unique effects of physical exercise on the thermoregulatory profile and innate immune response11. In addition, previous attempts to develop preclinical EHS models in rodents posed significant restrictions, including superimposed stress stimuli induced by electric shock, insertion of a rectal probe, and predefined maximum core body temperatures with high mortality rates12,13,14,15,16 that do not match current epidemiological data. These represent significant limitations that may confound data interpretation and provide unreliable biomarker indexes. Therefore, the protocol aims to characterize and describe the steps of a standardized, highly repeatable, and translatable preclinical model of EHS in mice that is largely free from the limitations mentioned above. Adjustments to the model that can result in graded physiological outcomes from moderate to fatal heat stroke are described. To the authors' knowledge, this is the only preclinical model of EHS with such characteristics, making it possible to pursue relevant EHS research in a hypothesis-driven manner11,17,18.
All procedures have been reviewed and approved by the University of Florida IACUC. C57BL/6J male or female mice, ~4 months old, weighing within a range of 27-34 g and 20-25 g, respectively, are used for the study.
1. Surgical implantation of the telemetric temperature monitoring system
2. Familiarization: Voluntary and forced wheel running
3. EHS protocol
The typical thermoregulatory profiles during the entirety of the EHS protocol and early recovery of a mouse is illustrated in Figure 1A. This profile comprises four distinct phases that can be defined as the chamber heating stage, incremental exercise stage, steady-state exercise stage, and a recovery stage by either a rapid cooling (R) or severe (S) method17. The main thermoregulatory outcomes include maximum Tc achieved (Tc,max) and the time required to reach Tc,max. Ascending thermal area allows for determining the effective exposure to temperature >39.5 °C21 and hypothermia depth (Tc,min). Typical values for these variables summarized from several studies are shown in Table 1. Other outcome variables routinely measured include the total distance run, the maximum speed achieved, and the percentage weight lost during the EHS protocol (a surrogate measure for dehydration). Again, typical values can be observed in Table 1. Female mice are more resistant to heat stroke in this model and run nearly 2-fold longer distances than male mice17, as illustrated schematically in Figure 1B and summarized numerically in Table 1.
Terminal experiments have been performed at different time points post-EHS, ranging from immediately before and after collapse19 to 30 days11,17,22. This model consistently demonstrates histological damage to the intestines, kidney, and liver19. Other expected results include common biomarkers of stress or immune responsiveness11,17, (Table 2), as well as end organ dysfunction including indicators of liver (alanine transaminase), muscle (creatine kinase), intestinal (fatty acid binding protein 2), and kidney (creatinine: blood urea nitrogen ratio) as shown in Table 319. Future investigations may consider measuring other markers of tissue damage or oxidative stress.
In the R preclinical model, >99% of the animals survive until sample collection. However, in the S model, as described above, the mortality increases to >30% (N = 32, P < 0.003). A typical recovery temperature profile for the S model is illustrated in Figure 1A (dashed red line), where Tc stays above 37 °C throughout the 2 h recovery period. The partition of EHS recovery periods during each stage of the EHS protocol and recovery is compared in Figure 2 between the classic and the S models. Interestingly, there is no difference in the time required to recover to 39.5 °C in the two models. However, the time to cool to the environmental temperature (37.5 °C, above normal body temperature) was greatly prolonged (P < 0.0001).
Figure 1: Thermoregulatory profiles during the entirety of the EHS protocol and early recovery of a mouse. (A) The typical core temperature profile of a C57Bl6 mouse undergoing the protocol on the vertical axis. On the horizontal axis, as time progresses from chamber heating (-50) to the beginning of the incremental portion of the protocol. As the mouse reaches 41 °C, speed is kept constant during the steady-state phase until it reaches symptom-limitation. During recovery, core temperature drops at different rates for severe (red dashed line) and rapid cooling (solid line) models. (B) Schematic representation of the sex differences observed in core temperature and duration. The dashed line is male, and the solid line is female. Please click here to view a larger version of this figure.
Figure 2: Duration in which mouse's core temperature remained >39.5 °C for rapid cooling (R) and slow cooling (S) protocols. Note that significant differences exist in the Tc,max to 37.5 °C and Tc,max to Tc,min segments. Data are mean ± standard deviation. Please click here to view a larger version of this figure.
Males | Females | EXC | |
Tc,max (°C) | 42.1 ± 0.2 | 42.3 (42.2–42.4) | 38.5 ± 0.2 |
Time to Tc (min) | 123 ± 11 | 208 (152–252) | 113 ± 10 |
%Weight Loss in EHS | 8.1 ± 2.1 | 6.0 (5.1–7.6 | 4.5% ± 1.0% |
Hypothermia depth (°C) | 33.0 ± 1.1 | 31.7 (30.7–33.1) | n/a |
Ascending thermal area (°C >39.5 • S) | 96.5 ± 14.7 | 240 (202–285) | n/a |
Total Distance (m) | 444.9 ± 89.3 | 623 (424–797) | Matched |
Maximum speed (m/min) | 5.3 ± 0.6 | 8.1 (7.1–9.2) | 5.2 |
Table 1: Expected temperature and exercise responses using the rapid cooling model of exertional heat stroke. All data from environmental temperature = 37.5°C, 30%-40% relative humidity. Means ± SD summarized from King et al. 201519, Garcia et al. 201817, Garcia et al. 202018.
Tc,max = maximum core temperature achieved at or near symptom limitation during exertional heat stroke (EHS).
% Weight loss = %weight difference from immediately before and after EHS. Ascending thermal area = an indicator of thermal load. It is the product of time x temperature > 39.5 °C during the EHS protocol.
Male | Female | |||||||
Males | EXC | 30 min | 3 h | 24 h | EXC | 30 min | 3 h | 24 h |
Corticosterone (ng/mL) | 50 ± 10 | 175 ± 42 | 152 ± 28 | 46 ± 26 | 72 ± 11 | 219 ± 78 | 259 ± 36 | 95 ± 24 |
IL-6 (pg/mL) | 3.8 ± 0 | 58.0 ± 50.0 | 37.0 ± 43 | 5.1 ± 4.0 | 3.7 ± 0.3 | 97.0 ± 48 | 10.4 ± 16.0 | 5.0 ± 4.2 |
GCS-F (pg/mL) | 34.2 ± 16.4 | 573 ± 462 | 1080 ± 52 | 87.8 ± 40.5 | 44.2 ± 20.0 | 238 ± 194 | 1712 ± 1700 | 208.4 ± 193 |
Table 2: Biomarker of stress hormone/cytokine responses in a rapid cooling model of the exertional heat stroke.
Data are means ± SD, All data from environmental temperature = 37.5 °C, 30%-40% relative
humidity. Summarized from Garcia et al. 201817.
Time point | EXC | 30 min | 3 h | 24 h |
Creatine Kinase (IU/L) | 215 ± 108 | 309 ± 145 | 1392 ± 1797 | 344 ± 196 |
Blood Urea Nitrogen (mg/dL) | 23 ± 2.7 | 66 ± 2.6 | 34 ± 8.5 | 17.2 ± 0.4 |
Creatinine:BUN ratio | 131 ± 70.0 | 210.7 ± 22.8 | 268.6 ± 118 | 52.3 ± 14 |
Alanine transaminase | 25 ± 3.7 | 367 ± 744 | 123 ± 167 | 207 ± 236 |
FABP-2 (ng/mL) | 2.3 ± 1.0 | 10.2 ± 1.0 | 2.6 ± 3.1 | 1.2 ± 0.5 |
Table 3: Biomarkers of organ Injury in male mice during recovery from rapid cooling model of exertional heat stroke.
Data are means ± SD. All data from environmental temperature = 37.5 °C. King et al. 201519.
This technical review aims to provide guidelines for the performance of a preclinical model of EHS in mice. Detailed steps and materials required for the execution of a reproducible EHS episode of variable severities are provided. Importantly, the model largely mimics the signs, symptoms, and multi-organ dysfunction observed in human EHS victims11,19. Furthermore, this model allows for the examination of the mechanism underlying short- and long-term EHS recovery19,20,22,23 and the effect of interventions on thermoregulation, performance measurements in the heat, rate of temperature reductions after stroke, and indicators of multi-organ dysfunction and functional tests of recovery. This model allows investigators to draw comparisons between other models that may be relevant for comparisons such as those describing malignant hyperthermia or rhabdomyolysis24,25,26.
This preclinical model eliminates unnecessary stressors, such as the use of electrical stimulation, rectal probes, anesthesia, or predetermined Tc cut-offs. Further, it highlights sex differences and innate tolerance to EHS. There are, however, some critical steps that must be adhered to. For instance, minor elevations in relative humidity may prolong the duration of the protocol because mice are able to use condensation of water vapor to cool themselves (opposite of the effects of humidity in humans)19. Also, it is important to note that when using the S mode, the empty cage must be kept inside the chamber during the entire duration of the test. If the cage is left outside the chamber, exposed to room temperature, it creates a sufficient gradient to cool the mouse even if quickly returned to the heated chamber20. A unique but not necessarily required feature of the protocol is using a small, forced running wheel (17.1 cm diameter). This diameter requires the mice to lift their upper torsos to meet the wheel as speed increases and undergo considerable coordination to keep up with the speed of the wheel and step on the widely spaced rungs of the wheel. Therefore, the efficiency, speed, and performance using such a wheel are much different from when mice run on a flat surface such as a treadmill or much larger diameter wheels available. If different diameter wheels are used, the example data shown here are unlikely to be representative. Given that the running activity is more complex in the smaller wheel, its use may appropriately simulate complex motor activities in the heat typical of diverse activities rather than simply running on flat surfaces.
The ability to select the severity by adjusting the cooling rate is another advantage of this model. The main therapeutic intervention known to be effective in counteracting negative outcomes of EHS is immediate cooling below 40 °C27. Therefore, the rapid cooling approach described in the R model is recommended for those trying to reverse-translate an EHS episode into exercise settings where cooling stations are readily available. However, in many other instances, such as in military scenarios or sports events held in remote settings, victims are often left in the heat, post-collapse, often for hours until medical support is available. This makes the slow cooling (S) approach a valid model for more severe outcomes. Presumably, this approach could be further modified to provide a wide range of severity of outcomes and to test cooling protocols.
Perhaps the most critical step in this procedure is ensuring proper implantation of the telemetric temperature device and allowing for ample recovery post-surgery. The ensuing inflammation process involved in the recovery can greatly alter the ability of the mouse to respond favorably to the EHS protocol, as infections and inflammation have been shown to impact thermoregulatory responses during EHS negatively3,27. Proper suturing is imperative for the success of the surgery and for promoting proper wound healing. It is critical to ensure that the muscle layer has been sutured separately from the skin layer. The muscle layer should also be cut only along the linea alba to ensure unnecessary blood loss and damage to the muscle. It is imperative to administer analgesics at proper times and provide sufficient time for the animals to recover fully from surgery before introducing the in-cage running wheels. The mouse must be monitored during recovery for signs and symptoms of distress and weight loss.
Throughout the development of this protocol, a variety of successful modifications were tested. The first modification included the pace at which the training was conducted and the elimination of the free-wheeling portion during acclimation. Because of equipment limitations, training was carried out utilizing the same protocol but with incremental increases in the speed of 0.5 m/min every 10 min for 60 min; free-wheeling was not utilized in the initial training session. These small changes did not affect the overall outcome or training status of the mouse. A second modification that was tested was the placement of the mouse during the increase in environmental chamber temperature. The protocol states that the mouse must rest in the home cage until the target environmental temperature is reached. However, to eliminate the opening of the chamber door at the target temperature, the mouse was placed in the forced running wheel to rest while the chamber was reaching the target temperature. The Tc and activity of the mice did not significantly differ whether the mouse was resting in the wheel or the home cage during this time period. Lastly, a variety of environmental conditions were tested ranging from 37.5-39.5 °C with 30%-90% RH19. The overall pattern remained similar while Tc,max, and exercise duration did differ. Manipulation of the target temperature and humidity can therefore be tailored to individual research goals.
There are a few additional limitations to bear in mind for this protocol. For example, because the protocol is symptom-limited, the mouse will not run beyond the point of collapse, this makes it difficult to make a more severe model based on exercise intensity. However, the modified cooling protocol rectifies this limitation. Another limitation is that any future therapeutic or intervention must be administered remotely, before or after the EHS protocol. If the animal had to be stopped for therapeutic administration, the Tc would immediately drop, and the thermoregulatory profile would be altered.
Although these limitations present a few logistical issues, this model displays advantageous features compared to other models that have employed stressful stimuli or invasive equipment. In the future, this model can be used to uncover the mechanisms underlying EHS and test novel interventions that may delay the onset of EHS or prevent the multi-organ dysfunction that ensues. In summary, this protocol establishes guidelines for the execution of a reliable preclinical model of EHS in mice and hopefully identifies the potential pitfalls to avoid when recreating this approach in other environments and future investigations.
The authors have nothing to disclose.
This work was funded by the Department of Defense W81XWH-15-2-0038 (TLC) and BA180078 (TLC) and the BK and Betty Stevens Endowment (TLC). JMA was supported by financial aid from the Kingdom of Saudi Arabia. Michelle King was with the University of Florida at the time this study was conducted. She is currently employed by the Gatorade Sports Science Institute, a division of PepsiCo R&D.
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