Heat Stroke In Mice Research Articles

Rifaximin alleviates intestinal barrier disruption and systemic inflammation via the PXR/NFκB/MLCK pathway and modulates intestinal Lachnospiraceae abundance in heat-stroke mice

Heatstroke is a critical condition with a high mortality rate and intestinal barrier dysfunction is a key factor in its progression to sepsis in some patients. This study aimed to explore the protective effects of rifaximin on the intestinal barrier in heat-stroke mice and the underlying mechanisms. A mouse model of heat stroke was established, followed by rifaximin intervention. Rifaximin significantly improved survival rates, reduced core body temperature, and alleviated intestinal tissue damage. Further mechanistic studies revealed that rifaximin restored heat stroke-induced damage to intestinal barrier function by upregulating the expression of the tight junction proteins, ZO-1 and occludin. Additionally, 16S rRNA sequencing showed that rifaximin significantly increased the abundance of Lachnospiraceae in the gut and enhanced short-chain fatty acid butyrate levels. In vitro experiment results revealed that butyrate promotes the expression of the intestinal epithelial cell protein MUC2, thereby strengthening the intestinal barrier. Rifaximin also activated the pregnane X receptor (PXR) signaling pathway and inhibited the NF-κB/MLCK signaling pathway, reducing the permeability of intestinal epithelial cells. This study demonstrated that rifaximin protects the intestinal barrier in mice with heat stroke through multiple pathways by modulating the gut microbiota, increasing butyrate production, and activating the PXR signaling pathway. These findings provide a new theoretical basis for the clinical application of rifaximin in heat stroke treatment.

Hyperbaric oxygen therapy attenuates heatstroke-induced hippocampal injury by inhibiting microglial pyroptosis

Background: Central nervous system (CNS) injury is the most prominent feature of heatstroke and the hippocampus is prone to damage. However, the mechanisms underlying the heatstroke-induced hippocampal injury remain unclear. Hyperbaric oxygen (HBO) therapy prevents CNS injury in heatstroke mice. However, the underlying mechanisms of HBO in heatstroke-induced hippocampal injury remain unclear. This study aimed to elucidate the protective effects of HBO against hippocampal injury and its potential role in microglial pyroptosis in heatstroke rats.Methods: A rat heatstroke model and a heat stress model with a mouse microglial cell line (BV2) were, respectively, used to illustrate the effect of HBO on heat-induced microglial pyroptosis in vivo and in vitro. We used a combination of molecular and histological methods to assess microglial pyroptosis and neuroinflammation both in vivo and in vitro.Results: The results revealed that HBO improved heatstroke-induced survival outcomes, hippocampal injury, and neurological dysfunction in rats. In addition, HBO mitigates microglial pyroptosis and reduces the expression of pro-inflammatory cytokines in the hippocampus of heatstroke rats. In vitro experiments showed that HBO attenuated BV2 cell injury under heat stress. Furthermore, HBO prevented heat-induced pyroptosis of BV2 cells, and the expression of pro-inflammatory cytokines IL-18 and IL-1β was reduced. Mechanistically, HBO alleviates heatstroke-induced neuroinflammation and hippocampal injury by preventing microglial pyroptosis. Conclusions: In conclusion, HBO attenuates heatstroke-induced neuroinflammation and hippocampal injury by inhibiting microglial pyroptosis.

Metabolic dysfunction in the myocardium three months after exertional heat stroke in mice

In humans, exposure to heat stroke has proven to be a risk factor for development of cardiovascular disease later in life. We hypothesized that exposure to exertional heat stroke (EHS) would result in the development of similar long-term cardiac disease in mice. In this study we followed mice for three months after EHS, using ultrasound imaging of the heart, metabolomics and histological analysis of ventricular tissue and biomarker analysis after 4 weeks of recovery. Methods: Mice were instrumented with temperature telemetry devices and were trained to run on forced running wheels using an incremental exercise protocol. EHS mice ran in an temperature elevated environmental chamber; females 37.5°C; males 34°C; whereas, matched exercise control mice (EXC) ran in 22-23°C. Running stopped when EHS mice reached symptom limitation (unconsciousness). The mice were followed for 12 wks post EHS, with two ultrasound measures at 2 and 10 wks of recovery. At the end of the study, mice were anesthetized, a blood sample drawn, and ventricular tissue collected for metabolomics. RESULTS: Male but not female EHS mice gained ~10% greater body mass compared to EXC over the course of recovery ( P<0.01). Hearts from male but not female EHS mice exhibited elevations in heart mass per tibia length ( P<0.01). At three months, metabolomics in male EHS hearts revealed significant elevations in glucose and glucose metabolites and significant decreases in acylcarnitines consistent with a metabolic shift toward glucose and away from lipid metabolism. In contrast, female hearts exhibited fewer abnormalities but with significant elevations in lipid and arachidonate metabolites. At two weeks post, there were no differences in male ultrasound measures between EHS and EXC mice but in females there was a reduced posterior wall velocity ( P=0.012) and a trend towards narrower posterior wall thickness ( P= 0.06) . Between 2 and 12 wks post EHS, there were significant elevations in fractional shortening in males ( P<0.05) and elevations in posterior wall velocity ( P<0.01). In contrast, females exhibited significant reductions posterior wall velocity. From blood samples, male EHS mice had increased HDL and females exhibited decreased HDL (p<0.01 in both). LDL increased only in males P<0.05. Vascular adhesion molecules sICAM ( P<0.01) and Pecam1 ( P<0.01) were significantly decreased in EHS males and females and e-Selectin was decreased in EHS males only ( P<0.001). Total serum protein was significantly reduced in both male and females ( P<0.01). Male EHS mice also exhibited significant elevations in white blood cell count and total lymphocytes ( P=0.01) suggesting ongoing activation of the immune system . CONCLUSIONS: In this controlled preclinical model of EHS, both male and female mice exhibited long-lasting cardiovascular and systemic disorders that could account for the lingering incidence of cardiovascular disease and poor overall health in heat stroke victims. Supported by US Army Med. Research and Devel. Command BA180078 and the BK and Betty Stevens Endowment. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Intestinal alkaline phosphatase improves intestinal permeability and alleviates multiple organ dysfunction caused by heatstroke

ObjectiveHeatstroke (HS) is a severe acute disease related to gastrointestinal barrier dysfunction, systemic inflammation and multiple organ injury. Many of the functions of Intestinal alkaline phosphatase (IAP) have been linked to gut homeostasis, gut barrier function and inflammation. However, the protective effect of IAP on heatstroke is not fully elucidated. This study aims to explore the protective effect of IAP on heatstroke by maintaining intestinal barrier and improving permeability. MethodsMale C57BL/6 mice were placed in a controlled climate chamber (ambient temperature: 40.0 ± 0.5 °C; humidity: 60 ± 5 %) until the maximum core temperature (Tc, max) reached 42.7 °C (the received criterion of HS). Then heat exposed mice (n = 195) were divided into three groups: 0.2 mL of 0.9 % physiological saline (HS) or vehicle (HS + Vehicle) or 300 IU IAP (HS + IAP) by gavage at 0, 24, and 48 h after onset. Control group mice (Con) (n = 65) were not exposed to heat and were gavaged with 0.9 % physiological saline of the same volume at the same time. ResultsIAP treatment significantly reduced the levels of endotoxin, FD4, and D-lactate in the blood of heatstroke mice, reduced intestinal permeability and maintained the integrity of the intestinal barrier by increasing the expression of tight junction proteins. Meanwhile, IAP treatment alleviated liver and kidney damage caused by heatstroke, reduced serum levels of inflammatory cytokines, and thus improved survival rate of mice after heatstroke. ConclusionThis study indicates that IAP can improve the intestinal barrier function and intestinal permeability by increasing intestinal tight junctions, reduce systemic inflammation and multiple organ injury and improving the survival rate of heatstroke. Therefore, we consider IAP may be added to enteral nutrition formulas as a potential means for diseases characterized by intestinal permeability disorders, including heatstroke.

Vancomycin modestly attenuates symptom severity during onset of and recovery from exertional heat stroke in mice.

Increased intestinal permeability during exertion and subsequent leakage of bacteria into circulation is hypothesized to accelerate exertional heat stroke (EHS) onset and/or exacerbate EHS severity. To provide proof of concept for this theory, we targeted intestinal microbiota via antibiotic prophylaxis and determined whether vancomycin would delay EHS onset and/or mitigate EHS severity and mortality rates using a mouse model of EHS. Mice were 1) designated as EHS or Exercise Control (ExC) and 2) given 7 days of vancomycin (VEHS, VExC) or untreated water (EHS, ExC) before EHS/Exercise. Following EHS/ExC, mice were euthanized immediately (0 h) or returned to their home cage (25°C) and euthanized after 3 h or 24 h. VEHS mice exhibited reduced abundance and altered composition of fecal bacteria (with notable decreases in genera within orders Clostridiales and Bacteroidales); increased water consumption, lower core temperature (TC) before and during heating (TCMax), lower circulating markers of organ damage and inflammation at 24 h; and reduced hepatic activation of stress pathways at 0 and 3 h compared with EHS mice. Vancomycin-induced alterations to the intestinal microbiota likely influenced EHS outcomes, but it is unconfirmed whether this is due to attenuated bacterial leakage into circulation or other (in)direct effects on physiology and behavior (e.g., decreased TC, increased water consumption). To our knowledge, this is the first study quantitating antibiotic effects in conscious/unanesthetized, exertional HS animals.NEW & NOTEWORTHY Vancomycin prophylaxis lowered core temperature before and during EHS, mitigated EHS-associated rise of hepatic biomarkers and cytokines/chemokines in circulation (particularly at 24 h), and corresponded to inhibited phosphorylation of hepatic c-Jun NH2-terminal kinase on Threonine 183/Tyrosine 185 at 0 and 3 h in conscious, unanesthetized mice. However, vancomycin also induced cecal enlargement suggesting its off-target effects could limit its utility against EHS.

Holistic view of heat acclimation alleviated intestinal lesion in mice with heat stroke based on microbiome-metabolomics analysis.

The severity of heat stroke (HS) is associated with intestinal injury, which is generally considered an essential issue for HS. Heat acclimation (HA) is considered the best strategy to protect against HS. In addition, HA has a protective effect on intestinal injuries caused by HS. Considering the essential role of gut microbes in intestinal structure and function, we decided to investigate the potential protective mechanism of HA in reducing intestinal injury caused by HS. HA model was established by male C57BL/6J mice (5-6 weeks old, 17-19 g) were exposed at (34 ± 0.7)°C for 4 weeks to establish an animal HA model. The protective effect of HA on intestinal barrier injury in HS was investigated by 16S rRNA gene sequencing and nontargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics. According to the experimental results, HA can change the composition of the gut microbiota, which increases the proportion of lactobacilli, faecal bacteria, and urinobacteria but decreases the proportion of deoxycholic acid. Moreover, HA can reduce liver and kidney injury and systemic inflammation caused by HS and reduce intestinal injury by enhancing the integrity of the intestinal barrier. In addition, HA regulates inflammation by inhibiting NF-κB signalling and increasing tight junction protein expression in HS mice. HA induces changes in the gut microbiota, which may enhance tight junction protein expression, thereby reducing intestinal inflammation, promoting bile acid metabolism, and ultimately maintaining the integrity of the intestinal barrier. In conclusion, HA induced changes in the gut microbiota. Among the gut microbiota, lactobacilli may play a key role in the potential protective mechanism of HA.

Mesenchymal stem cell-derived exosomal miR-548x-3p inhibits pyroptosis of vascular endothelial cells through HMGB1 in heat stroke

Heat stroke (HS) is an acute physical illness associated with a higher risk of organ dysfunction. This study is the first to explore exosomal miR-548x-3p derived from human bone marrow mesenchymal stem cells (BMSCs) in the pyroptosis of vascular endothelial cells (VECs) associated with HS. Human BMSCs-derived exosome alleviated the injury of the heart, liver, kidney and ileum tissues, the increase of IL-1β, IL-18 and TNF-α levels, pyroptosis of endothelial cells and the increase of HGMB1, NLRP3, ASC, caspase1 and GSDMD-N protein expression in HS mice and HS-induced human umbilical vein endothelial cells (HUVECs). miR-548x-3p was down-expressed in HS patients, while up-expressed in BMSCs-derived exosome. BMSCs-ExomiR-548x-3p mimics to inhibit pyroptosis, inflammation and HGMB1/NLRP3 activation in HS-induced HUVECs and HS mice, which were blocked by overexpression of HMGB1. In conclusion, human BMSCs-derived exosomes carried miR-548x-3p mimics to inhibit pyroptosis of VECs through HMGB1 in HS mice.

Ovariectomy Increases Intestinal Damage Post Exertional Heat Stroke In Mice

Ovariectomy Increases Intestinal Damage Post Exertional Heat Stroke In Mice

Mechanism of regulatory T cells in heat stroke-induced acute kidney injury

To investigate the mechanism of regulatory T cells (Treg) in heat stroke (HS)-induced acute kidney injury (AKI). Male SPF Balb/c mice were randomly divided into control group, HS group (HS+Rat IgG), HS+PC61 group, and HS+Treg group (n = 6). The HS mice model was established by making the body temperature of the mice reach 42.7 centigrade at room temperature 39.5 centigrade with relative humidity 60% for 1 hour. In HS+PC61 group, 100 μg PC61 antibody (anti-CD25) was injected through the tail vein in consecutive 2 days before the model was established to eliminate Tregs. Mice in HS+Treg group was injected with 1×106 Treg via tail vein immediately after successful modeling. The proportion of Treg infiltrated in the kidney, serum creatinine (SCr) and histopathology, levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) both in the serum and kidney tissue, as well as proportion of neutrophils and macrophages located in the kidney were observed at 24 hours after HS. HS dampened renal function and exaggerated kidney injury, up-regulated levels of inflammatory cytokines both in local kidney and circulation, and increased infiltration of neutrophils and macrophages to the injured kidneys. The proportion of Treg (Treg/CD4+) infiltrated in kidney was significantly decreased in HS group, compared with control group [(3.40±0.46)% vs. (7.67±0.82)%, P < 0.01]. Compared with HS group, local Tregs in kidney were almost completely depleted via PC61 antibody [(0.77±0.12)% vs. (3.40±0.46)%, P < 0.01]. Depletion of Tregs could exacerbate HS-AKI, indicating by increased serum creatinine [SCr (mmol/L): 348.22±35.36 vs. 254.42±27.40, P < 0.01] and pathological injury (Paller score: 4.70±0.20 vs. 3.60±0.20, P < 0.01), incremental levels of IFN-γand TNF-α both in injured kidney and serum [serum IFN-γ (ng/L): 747.70±64.52 vs. 508.46±44.79, serum TNF-α (ng/L): 647.41±26.62 vs. 464.53±41.80, both P < 0.01], and more infiltrated neutrophils and macrophages in the injured kidney [neutrophil proportion: (6.63±0.67)% vs. (4.37±0.43)%, macrophage proportion: (38.70±1.66)% vs. (33.19±1.55)%, both P < 0.01]. On the contrast, adoptive transfer of Tregs could reverse the aforementioned effects of Treg depletion, indicating by incremental proportion of Tregs in the injured kidney [(10.58±1.19)% vs. (3.40±0.46)%, P < 0.01], decreased serum creatinine [SCr (mmol/L): 168.24±40.56 vs. 254.42±27.40, P < 0.01] and pathological injury (Paller score: 2.73±0.11 vs. 3.60±0.20, P < 0.01), reduced levels of IFN-γ and TNF-α both in injured kidney and serum [serum IFN-γ (ng/L): 262.62±22.68 vs. 508.46±44.79, serum TNF-α (ng/L): 206.41±22.58 vs. 464.53±41.80, both P < 0.01], and less infiltrated neutrophils and macrophages in the injured kidney [neutrophil proportion: (3.04±0.33)% vs. (4.37±0.43)%, macrophage proportion: (25.68±1.93)% vs. (33.19±1.55)%, both P < 0.01]. Treg might be involved in HS-AKI, possibly via down-regulation of pro-inflammatory cytokines and infiltration of inflammatory cells.

Ovariectomy aggravates the pathophysiological response to exertional heat stroke in mice.

Female mice have a greater capacity for exercising in the heat than male mice, reaching greater power output and longer times of heat exposure before succumbing to exertional heat stroke (EHS). Differences in body mass, size, or testosterone do not explain these distinct sex responses. Whether the ovaries could account for the superior exercise capacity in the heat in females remains unknown. Here, we determined the influence of ovariectomy (OVX) on exercise capacity in the heat, thermoregulation, intestinal damage, and heat shock response in a mouse EHS model. We performed bilateral OVX (n = 10) or sham (n = 8) surgeries in young adult (4 mo) female C57/BL6J mice. Upon recovery from surgeries, mice exercised on a forced wheel placed inside an environmental chamber set at 37.5 °C and 40% relative humidity until experiencing loss of consciousness (LOC). Terminal experiments were performed 3 h after LOC. OVX increased body mass by the time of EHS (sham = 3.8 ± 1.1, OVX = 8.3 ± 3.2 g, P < 0.05), resulted in shorter running distance (sham = 753 ± 189, OVX = 490 ± 87 m, P < 0.05), and shorter time to LOC (sham = 126.3 ± 21, OVX = 99.1 ± 19.8 min, P < 0.05). Histopathological assessment of the intestines revealed damage in the jejunum (sham = 0.2 ± 0.7, OVX = 2.1 ± 1.7 AU, P < 0.05) and ileum (sham = 0.3 ± 0.5, OVX = 1.8 ± 1.4 AU, P < 0.05). OVX increased mesenteric microvascular density (sham = 101 ± 25, OVX = 156 ± 66 10-2 mm/mm2, P < 0.05) and decreased concentration of circulatory heat shock protein 72 (HSP72) (sham = 26.7 ± 15.8, OVX = 10.3 ± 4.6 ng/mL, P < 0.05). No differences were observed in cytokines or chemokines between groups. Our findings indicate that OVX aggravates the pathophysiological response to EHS in mice.NEW & NOTEWORTHY Females outperform males in a mouse model of exertional heat stroke (EHS). Here, we show for the first time the impact of ovariectomy (OVX) on EHS pathophysiology. OVX resulted in a shorter exercise capacity in the heat, greater intestinal damage, and lower heat shock response following EHS.

The impact of castration on physiological responses to exertional heat stroke in mice.

The capability of male mice to exercise in hot environments without succumbing to exertional heat stroke (EHS) is markedly blunted compared to females. Epidemiological evidence in humans and other mammals also suggests some degree of greater vulnerability to heat stroke in males compared to females. The origins of these differences are unknown, but testosterone has previously been shown to induce faster elevations in core temperature during acute, passive heat exposure. In this study, we tested the hypothesis that loss of testosterone and related sex hormones through castration would improve the performance and heat tolerance of male mice during EHS exposure. Twenty-four male mice were randomly divided into 3 groups, untreated EHS mice (SHAM-EHS), castrated EHS mice (CAS+EHS) and naïve exercise controls (NAIVE). Exercise performance and physiological responses in the heat were monitored during EHS and early recovery. Two weeks later, blood and tissues were collected and analyzed for biomarkers of cardiac damage and testosterone. Core temperature in CAS+EHS rose faster to 39.5°C in the early stages of the EHS trial (P<0.0001). However, both EHS groups ran similar distances, exhibited similar peak core temperatures and achieved similar exercise times in the heat, prior to symptom limitation (unconsciousness). CAS+EHS mice had ~10.5% lower body mass at the time of EHS, but this provided no apparent advantage in performance. There was no evidence of myocardial damage in any group, and testosterone levels were undetectable in CAS+EHS after gonadectomy. The results of these experiments exclude the hypothesis that reduced performance of male mice during EHS trials is due to the effects of male sex hormones or intact gonads. However, the results are consistent with a role of male sex hormones or intact gonads in suppressing the early and rapid rise in core temperature during the early stages of exercise in the heat.

A Potential Driver of Disseminated Intravascular Coagulation in Heat Stroke Mice: Neutrophil Extracellular Traps.

Highlights What are the main findings? NETs contribute to the activation of the coagulation cascade and have been successfully proved to be a potential driver of DIC in HS mice. What is the implication of the main finding? This work provides a novel alternative treatment strategy for the treatment of DIC in HS patients.Aims: Disseminated intravascular coagulation (DIC) is a common complication of heat stroke (HS) patients, and it is one of the important reasons leading to multiple organ failure and even death. The association between neutrophil extracellular traps (NETs) and DIC is unclear in HS mice. Methods and results: Here, HS was induced by the combination of hyperthermia (HT) and lipopolysaccharide (LPS). The DIC was evaluated by measuring prothrombin time (PT), D-dimer, thrombomodulin (TM), fibrinogen (FIB), and platelet (PLT). The expression of citrullinated-histone (CitH3) was analyzed by Western blotting. The formation of NETs was observed by immunofluorescence microscopy. The risk of HS-induced DIC was increased when HT was combined with LPS. The markers of NETs were significantly higher than those in the control group, and the NETs derived from HS promoted the development of DIC. DNase I improved coagulation dysfunction via the clearance of NETs caused by neutrophil aggregation. Conclusions: Degradation of NETs reduced the risk of developing DIC, and thus the survival rate of mice was improved. These results indicate that NETs may hold potential alternative therapeutic strategies for the treatment of DIC in HS patients.

Outer membrane vesicles derived from heatstroke-associated intestinal microbiota promote multiple organ injury in mice.

Multiple organ injury is a common issue in heatstroke (HS); however, the underlying pathogenesis remains unclear. As an early event in HS, intestinal injury is an active participant that drives organ injury. Outer membrane vesicles (OMVs), a group of vesicles shed by unbalanced intestinal microbiota as "danger signals," mediate different functional cargo transport in cells and modulate varying biological events in distant target cells. However, the role of OMVs in HS-mediated organ damage remains unclear. Therefore, this study examined OMV production in HS and explored the effect of regulating multiple organ injury. To construct a mouse model, animals were exposed to hyperthermia. OMVs from the intestinal microbiota of HS and control mice were extracted by standardized differential ultracentrifugation. Thereafter, OMVs were characterized and infused into recipient mice via the tail vein. Cl-amidine (a pan-peptidylarginine deiminase inhibitor and OMV production inhibitor) was injected intraperitoneally (2mg/kg) 2h before HS treatment, and the absorption of HS OMVs by different organs was tracked. The effect of OMVs on inducing organ pathological changes, inflammatory infiltration, inflammatory cytokine expression, and serum organ injury biomarkers was demonstrated. HS increased OMV production by intestinal microbiota; OMVs were absorbed by different organs in vivo, and were especially enriched in the liver and lung. Compared to control OMVs, infusion with HS OMVs induced significant organ pathological changes, elevated inflammatory cell (macrophages and neutrophil) infiltration, inflammatory cytokine (TNF-ɑ, IL-1β, IL-6) expression, as well as serum biomarkers of organ injury. Similarly, inhibition of endogenous OMVs alleviated these organ injury indicators induced by HS. To our knowledge, the present study is the first to illustrate that OMVs induce acute organ impairment during severe HS, offering a foundation for subsequent studies and providing novel therapeutic targets.

High-Fat Diet Impairs Muscle Function and Increases the Risk of Environmental Heatstroke in Mice.

Environmental heat-stroke (HS) is a life-threatening response often triggered by hot and humid weather. Several lines of evidence indicate that HS is caused by excessive heat production in skeletal muscle, which in turn is the result of abnormal Ca2+ leak from the sarcoplasmic reticulum (SR) and excessive production of oxidative species of oxygen and nitrogen. As a high fat diet is known to increase oxidative stress, the objective of the present study was to investigate the effects of 3 months of high-fat diet (HFD) on the HS susceptibility of wild type (WT) mice. HS susceptibility was tested in an environmental chamber where 4 months old WT mice were exposed to heat stress (41 °C for 1 h). In comparison with mice fed with a regular diet, mice fed with HFD showed: (a) increased body weight and accumulation of adipose tissue; (b) elevated oxidative stress in skeletal muscles; (c) increased heat generation and oxygen consumption during exposure to heat stress; and finally, (d) enhanced sensitivity to both temperature and caffeine of isolated muscles during in-vitro contracture test. These data (a) suggest that HFD predisposes WT mice to heat stress and (b) could have implications for guidelines regarding food intake during periods of intense environmental heat.

The Influence of Ovariectomy on Performance and Thermoregulation During Exertional Heat Stroke in Mice

BackgroundExertional heat stroke (EHS) is a condition characterized by central nervous system dysfunction and multi‐organ injury in hyperthermic victims. Most studies addressing EHS pathophysiology are performed in males. This is a concern since there is an increasing participation of females in sports, active military service, and sex‐dependent susceptibilities to EHS. Preclinical data suggest that female mice outperform male mice regarding distance ran, speed, and time to collapse in a model of EHS. Importantly, differences in body mass between males and females do not explain these contrasting heat tolerances. Whether the female sex hormones could account for the superior performance in the heat remains unknown. Our goal was to determine the influence of female sex hormones on performance and thermoregulatory parameters in a preclinical model of EHS.MethodsWe performed ovariectomy (OVX, n=8) or sham (n=8) surgeries in young adult (18 weeks old) female C57/BL6 mice. Ovariectomy was performed by the bilateral removal of the ovaries. To allow for real time monitoring of core temperature during the entire experiment, all mice were implanted with telemetry emitters at the time of ovariectomy/sham surgeries. Upon recovery from surgeries and training (~6 weeks), all mice underwent the EHS protocol in an environmental chamber set at 37.5℃ and 40% relative humidity. Mice exercised on a running wheel until experiencing loss of consciousness.ResultsOvariectomy increased body mass by 10% at the time of EHS (Sham = 25 ± 1 vs OVX = 28 ± 2 g, P&lt;0.05), resulted in 35% shorter distance (Sham = 753 ± 189 vs. 490 ± 87 m, P&lt;0.05), 25% shorter time to collapse (Sham = 126 ± 21 vs. OVX = 95 ± 12 min, P&lt;0.05), and 15% lower final speed (Sham = 9 ± 1 vs. OVX = 7.5 ± 1 m/min, P &lt; 0.05).ConclusionOvariectomy negatively impacts tolerance to EHS. Ovariectomized mice not only displayed a marked decrease in performance during the EHS trial but also gained more mass than their controls. This suggests that female sex hormones are required for a greater tolerance against EHS.

Long‐term myocardial DNA methylation and metabolomic responses to exertional heat stroke in mice

Epidemiological studies indicate that exposure to exertional heat stroke (EHS) can increase the risk of all‐cause cardiovascular and systemic disease and promote early mortality. Using a preclinical mouse model of EHS, we previously characterized profound DNA methylation changes in immune cells at 30 d of recovery and severe cardiometabolic disturbances in the ventricular myocardium (VM) at 9‐14 d. These observations indicate that a single exposure to EHS can result in long‐term epigenetic and metabolic alterations. However, whether a long‐term epigenetic responses occur in the VM and or whether cardiometabolic perturbations persist is unknown.PURPOSETo determine if long‐term alterations to (1) DNA methylation and (2) the metabolome occur within the VM after 30 d of recovery from EHS and (3) whether epigenetic alterations predict imbalances within the metabolome.METHODSSixteen C57BL6 female mice underwent a standardized EHS protocol using a forced running wheel (environmental temp: 37.5°C, 40% humidity) or a matched exercise control trial (EXC) (22.5°C). The EHS mice achieved peak core temps of ~42.4°C, accompanied by transient loss of consciousness. Mice were sacrificed after 30 d of recovery; left ventricles were isolated and frozen in liquid nitrogen for DNA methylation sequencing and untargeted metabolomics.RESULTSFollowing 30 d of recovery from EHS, more than 6,000 differentially methylated cytosines (DMCs) were observed within promoter regions; 900 differentially methylated regions (DMRs) were also observed (i.e ≥ 5 differentially DMCs within 300 base pairs) at (Q &lt; 0.05) compared to EXC. Pathway analysis of promoter DMRs indicated changes in genes that modulate basic cell functions, DNA binding, transcription, and metabolism. Cardiometabolic perturbations were found to largely resolve compared to observations at 9‐14 d. Some metabolic disturbances remained, e.g. reductions in α‐ketoglutarate and increases in several lipid intermediates. These remnant alterations are known to be common in early onset heart failure and chronic kidney disease. Repressive DMCs (P &lt; 0.05) were observed within transcriptional regulatory regions of enzymes that regulate α‐ketoglutarate, e.g. isocitrate dehydrogenase 1 &amp; 2 (IDH1 &amp; IDH2), suggesting a potential link with metabolomic findings.CONCLUSIONSA single EHS episode results in a robust epigenetic response in VM when observed at 30 d of recovery, whereas all but a subset of metabolites within the VM had fully recovered. These long‐term molecular changes may induce altered or maladaptive physiological outcomes in response to future environmental or medical challenges.

A Preclinical Model of Exertional Heat Stroke in Mice.

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.

A Preclinical Model of Exertional Heat Stroke in Mice

A Preclinical Model of Exertional Heat Stroke in Mice

Metabolomic profiling identifies a novel mechanism for heat stroke‑related acute kidney injury.

Heat stroke can induce a systemic inflammatory response, which may lead to multi-organ dysfunction including acute kidney injury (AKI) and electrolyte disturbances. To investigate the pathogenesis of heat stroke (HS)-related AKI, a mouse model of HS was induced by increasing the animal's core temperature to 41°C. Blood samples obtained from the tail vein were used to measure plasma glucose and creatinine levels. Micro-positron emission tomography-computed tomography (micro-PET/CT), H&E staining and transmission electron microscopy were conducted to examine metabolic and morphological changes in the mouse kidneys. Immunohistochemistry (IHC) and western blot analyses were performed to investigate the expression of apoptosis-inducing factor mitochondria-associated 2 (Aifm2), high-mobility group box 1 (HMGB1) and receptor for advanced glycosylation end products (RAGE). Liquid chromatography-mass spectrometry analysis was conducted to find differential metabolites and signaling pathways. The HS mouse model was built successfully, with significantly increased creatinine levels detected in the serum of HS mice compared with controls, whereas micro-PET/CT revealed active metabolism in the whole body of HS mice. H&E and TUNEL staining revealed that the kidneys of HS mice exhibited signs of hemorrhage and apoptosis. IHC and western blotting demonstrated significant upregulation of Aifm2, HMGB1 and RAGE in response to HS. Finally, 136 differential metabolites were screened out, and enrichment of the ‘biosynthesis of unsaturated fatty acids’ pathway was detected. HS-associated AKI is the renal manifestation of systemic inflammatory response syndrome, and may be triggered by the HMGB1/RAGE pathway. Metabolomics indicated increased adrenic acid, docosahexaenoic acid and eicosapentaenoic acid may serve as metabolic biomarkers for AKI in HS. The findings suggested that a correlation between the HMGB1/RAGE pathway and biosynthesis of unsaturated fatty acids may contribute to the progression of HS-related AKI.

Role of the Receptor for Advanced Glycation End Products in Heat Stress-Induced Endothelial Hyperpermeability in Acute Lung Injury.

ObjectiveTo study the role of the receptor for advanced glycation end products (RAGE) in endothelial barrier dysfunction induced by heat stress, to further explore the signal pathway by which RAGE contributes to heat-induced endothelia response, and thereby find a novel target for the clinical treatment of ALI (acute lung injury) induced by heatstroke.MethodsThis study established the animal model of heatstroke using RAGE knockout mice. We observed the role of RAGE in acute lung injury induced by heatstroke in mice by evaluating the leukocytes, neutrophils, and protein concentration in BALF (Bronchoalveolar lavage fluids), lung wet/dry ratio, histopathological changes, and the morphological ultrastructure of lung tissue and arterial blood gas analysis. To further study the mechanism, we established a heat stress model of HUVEC and concentrated on the role of RAGE and its signal pathway in the endothelial barrier dysfunction induced by heat stress, measuring Transendothelial electrical resistance (TEER) and western blot.ResultsRAGE played a key role in acute lung injury induced by heatstroke in mice. The mechanism C-Jun is located in the promoter region of the RAGE gene. C-Jun increased the RAGE protein expression while HSF1 suppressed RAGE protein expression. The overexpressed RAGE protein then increased HUVEC monolayer permeability by activating ERK and P38 MAPK under heat stress.ConclusionThis study indicates the critical role of RAGE in heat stress-induced endothelial hyperpermeability in acute lung injury and suggests that RAGE could be a potential therapeutic target in protecting patients against acute lung injury induced by heatstroke.