Animal preparation and housing

The timeline of the study is presented in Fig. 1A. Adult male SD rats (350–400 g) were obtained from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. (Licence No: MA28BDKP-5). The rats were individually housed at 20–25 ℃ and 45–50% humidity under a 12-h light/dark cycle. The rats had free access to standard rodent feed and water for 1 week of acclimatization prior to the experiments.

Fig. 1

Experimental procedures and physiological variables. A Overview of the experimental process. B, C, D Trends in vital signs over time from heat exposure to 2 h post-heatstroke onset, including Tco, MAP, and HR. Following heatstroke onset, the MAP decreased sharply in both heatstroke rat groups, with a more pronounced and severe decrease observed in the HS+NS group. Significant differences in MAP were noted at 5, 10, and 20 min post-event (C). NC + NS, n = 13; NC+LR, HS+NS, and HS+LR, n = 14. Tco core body temperature, MAP mean arterial pressure, HR heart rate

Ethical approval and euthanasia procedure

All experimental protocols, including animal care and handling, were approved by the Ethics and Welfare Committee of Animal Experimentation at the Second Affiliated Hospital, Zhejiang University School of Medicine (Approval ID: AIRB-2023-1677). This approval confirms adherence to the ethical standards and guidelines for animal research. Postexperiment, the surviving rats were euthanized via an overdose of sodium pentobarbital, which was administered to ensure painless death, which was consistent with the ethical guidelines for animal euthanasia.

Surgical procedures and physiological monitoringAnaesthesia and surgical preparation

Anaesthesia was induced in the rats via intraperitoneal injection of sodium pentobarbital (40 mg/kg), which was carefully monitored to maintain an appropriate depth of anaesthesia throughout the procedures. Sodium pentobarbital is known to affect cardiovascular parameters temporarily; hence, additional doses were administered judiciously, particularly to avoid critical periods 10 min before and 30 min after heat stroke onset, to prevent data interference.

Vascular access and monitoring

Following anaesthesia, a 22 G cannula was inserted into the right common carotid artery for continuous physiological monitoring and blood sampling. Another cannula was inserted into the tail vein for fluid administration. Physiological variables such as the mean arterial pressure (MAP) and heart rate (HR) were continuously recorded via a bioinformation acquisition system (Chengdu Taimeng, BL-420 N). Core body temperature (Tco) was recorded every 10 min via a veterinary anal thermometer.

Heatstroke induction

The rats were placed on a heating pad set to 43 ℃ to simulate heatstroke conditions. Current studies use two criteria for heatstroke modeling in rats: a Tco exceeding 42 ℃ or 43 ℃, and a decrease in MAP. However, the standards for MAP reduction are inconsistent, varying from a decrease from the peak, a drop of 25 mmHg, or a reduction to below 30 mmHg [19,20,21]. In our research, we observed that after heat exposure, the MAP of rats gradually increased and fluctuated after reaching its peak, making the “start of decline” criterion challenging to apply. To ensure model consistency and minimize the impact of hypotension on organ damage, we defined heatstroke onset as an increase in Tco to 42 ℃, accompanied by a decrease in MAP of at least 10 mmHg, typically occurring within 75–80 min, as shown in Fig. 1B. After heatstroke induction, body weight was measured again, and weight loss due to heat exposure was calculated based on the difference from the baseline weight.

Sample size calculation

This study is a randomized controlled trial in which rat inflammatory markers were used as the basis for sample size calculations. According to previous studies [22], when the effects of HCL and LR on inflammatory markers in septic rats were compared, 6–7 rats per group achieved good statistical power. As this study divided each group into two subgroups on the basis of blood collection time, the final sample size for each group was determined to be 12–14 rats. During the experiment, if carotid artery cannulation failed or a rat died before the completion of infusion, it was removed from the study. After removal, the number of rats in each group remained no less than 12, or if the data available for analysis in each group were more than six, no additional animals were supplemented.

Experimental design and group allocation

The rats were randomized into the following four groups via a random number table method:

Normothermic control with NS (NC+NS, n = 13): These rats were maintained under normothermic conditions with NS infusion.

Normothermic control with LR (NC+LR, n = 14): These rats were maintained under normothermic conditions with LR infusion.

Heatstroke with NS resuscitation (HS+NS, n = 14): These rats were subjected to heatstroke and treated with NS for cooling and fluid resuscitation.

Heatstroke with LR resuscitation (HS+LR, n = 14): These rats were subjected to heatstroke and treated with LR for cooling and fluid resuscitation.

Resuscitation protocols

The NC+NS and NC+LR groups were maintained at a steady Tco of 36–36.5 ℃ using a water circulation blanket for 80 min, followed by NS or LR infusion at room temperature. This protocol was designed to simulate the administration of fluid without the confounding effects of heat stress, serving as a control to assess the baseline impact of NS under normothermic conditions.

The HS+NS and HS+LR groups were exposed to 43 ℃ conditions until heatstroke onset. Immediately following heat stroke induction, the rats were removed from the heat source and placed in an ambient environment of 20–25 ℃. Resuscitation fluids at 4 ℃ (NS for the HS+NS group and LR for the HS+LR group) were administered.

Fluid composition and administration

NS contained 0.9% saline with 154 mmol/L chloride (Shuanghe Pharmaceutical).

LR contains sodium chloride, sodium lactate, potassium chloride, and calcium chloride (chloride at 108 mmol/L, lactate at 27 mmol/L) (Weigao Pharmaceutical).

Administration Protocol Despite the existence of well-established methods for modelling heatstroke in rats, no literature has defined the standards for the volume and rate of cooling and volume resuscitation fluids in heatstroke rats. Excessive infusion volumes can result in death due to volume overload, whereas insufficient volumes do not allow for the observation of the pathophysiological effects of different fluid types on heatstroke rats.

We based the infusion volume calculation in this study on the chloride ion input. On the basis of the conversion rules of body surface area between humans and rats [23], following established recommendations of cooling and volume resuscitation in heatstroke patients [2, 5], and considering the blood volume of the rats, our research team conducted a thorough investigation to determine the appropriate infusion volume. We ultimately established the following infusion protocol for this study: 4 mL per 100 g body weight over 10 min using a constant rate infusion pump (KellyMed, ZNB-XD).

Postinfusion monitoring and analysis

Blood samples for biochemical and inflammatory marker analyses were collected at 30 and 120 min postresuscitation. Blood inulin and p-aminohippuric acid concentrations were measured at 15, 45, and 75 min after infusion. Half of the rats underwent immediate euthanasia for organ and histological examination to assess renal and cardiac integrity and apoptosis. The levels of inflammasome-related proteins in kidney tissue were quantified via Western blot analysis. The remaining rats were observed for a 24-h period to assess survival.

Adaptation of methodology for renal function assessment

Initially, our strategy aimed to assess renal blood flow (RBF) and the glomerular filtration rate (GFR) via traditional clearance methods for p-aminohippuric acid and inulin. However, owing to insufficient urine output from heat-stroked rats, which compromised the reliability of these measurements, we adapted our approach. We opted to estimate the RBF and GFR on the basis of the serum concentrations of these markers, providing a viable alternative to reflect changes in renal function more consistently.

For the revised approach, we administered infusion fluids containing inulin (1.0 g/L) and p-aminohippuric acid (0.2 g/L). Blood samples were collected at 15, 45, and 75 min postinfusion, from the onset of heat stroke, and stored at − 80 ℃ until analysis. The serum levels of these compounds were quantified via ELISA kits (Shanghai Fankew Industrial Co.) following the manufacturer’s guidelines.

Blood sample analysis for organ function and inflammatory markersCollection and storage

Blood samples were drawn at 30 and 120 min post-heatstroke induction. Immediate analyses for arterial blood gases and electrolytes were conducted (EDAN, Shenzhen Libang Precision Instrument Co., Ltd.), whereas samples for prothrombin time (PT) and complete blood count (CBC) were analysed within 4 h of collection. The remaining blood samples were centrifuged at 3000 rpm for 15 min to separate the serum, which was then stored at − 80 ℃ until further analysis.

Biochemical analysis and inflammatory markers

Biochemical parameters, including PT, blood urea nitrogen (BUN), serum creatinine (Scr), and alanine aminotransferase (ALT), were measured via an automatic biochemical analyser (LWC400, Shenzhen Lanyun Medical Device Technology Co., Ltd.). The CBC was determined with a fully automatic blood analyser (Jiangxi Tekang Technology Co., Ltd.). Serum levels of IL-6, TNF-α, neutrophil gelatinase-associated lipocalin (NGAL), cardiac troponin I (cTnI), and catecholamines were quantified via specific ELISA kits (Wuhan Kelu Biotechnology Co., Ltd. and Shanghai Fankew Industrial Co.), following the manufacturers' protocols.

Tissue sample analysis for organ function and inflammation

The tissues were rinsed, weighed, and homogenized in PBS to ensure complete disruption. The homogenates were centrifuged, and the supernatants were used for further analysis of inflammatory and damage markers.

Histological and ultrastructural evaluationHistology

Both kidney and heart tissues were fixed, embedded in paraffin, and sectioned into 4-μm slices for histological examination. These sections were stained with haematoxylin and eosin (H&E) and evaluated microscopically via a blinded semiquantitative scoring method to assess organ damage. Renal damage was quantified via a scale designed for acute renal failure [24], with a focus on 100 cortical tubules across at least 10 different areas per kidney. The scoring criteria included tubular epithelial flattening, cell membrane bleb formation, brush border loss, cytoplasmic vacuolation, cell necrosis, tubular lumen obstruction, and interstitial oedema, with a maximum possible score of 10 points per tubule. Cardiac tissue damage was assessed by examining cellular features such as eosinophilic cytoplasm and nucleus, cytoplasmic vacuolation, inflammatory cell infiltration, and interstitial vessel congestion and haemorrhage via a standardized scoring system [25].

Transmission electron microscopy (TEM)

Ultrastructural analysis of kidney tissues was performed via TEM after fixation with 2.5% glutaraldehyde and 1% OsO4. The tissues were subjected to a series of dehydration and embedding processes, sectioned, double-stained with uranium acetate and lead citrate, and examined under a HITACHI HT7650 microscope.

Evaluation of apoptosis in renal cortex tissue sections

Apoptosis was assessed via a one-step TUNEL assay kit (HKI0011, Haoke). The tissues were fixed, embedded, and sectioned. After being deparaffinized in xylene, the tissue sections were rehydrated through a graded ethanol series. Following deparaffinization and rehydration, the sections underwent antigen retrieval via an EDTA solution and were allowed to cool. A TUNEL reaction mixture was subsequently applied, and the sections were incubated at 37 ℃. After incubation, the sections were washed and stained with DAPI for nuclear visualization. Finally, the prepared sections were mounted and digitally scanned via a fluorescence microscope (Eclipse Ci-L, Nikon, Japan). Renal cortex tissue images were captured at 20 × magnification via SlideViewer microscope software. We performed TUNEL staining on kidney specimens from four rats in each group. Apoptotic cells, indicated by red fluorescence, were quantified in 10 randomly selected high-power fields and averaged by an observer blinded to the samples.

Western blot analysis of protein expression in kidney tissue

For Western blot analysis, kidney tissues were first rinsed, minced, and homogenized in RIPA buffer (C1053, ApplyGene) supplemented with phosphoprotease inhibitors. After centrifugation, the supernatant was collected for protein quantification via a BCA protein assay (P1511, ApplyGene). The proteins were then separated by SDS‒PAGE (P1200, Solarbio) and transferred onto PVDF membranes (ISEQ00010, Millipore) for immunoblotting. The membranes were incubated with primary antibodies against (1:500, Immunoway, YT5382), caspase-1 (1:500, Immunoway, YT5743), GSDMD (1:1000, Immunoway, YT7991), and β-actin (1:5000, Immunoway, YM3028), followed by visualization via a chemiluminescent substrate (P1010, ApplyGene). The developed films were digitally processed to quantify protein band intensities via ImageJ software.

Statistical analysis

The data are presented as the means ± SDs. For quantitative variables, one-way analysis of variance (ANOVA) was employed, followed by the least significant difference (LSD) method for pairwise comparisons. A random effects model was applied to analyse repeated measurements. In this study, we compared the differences in various indicators between the two heatstroke groups (HS+NS vs. HS+LR) and between the two normal control groups (NC+NS vs. NC+LR). This was done to more clearly delineate the differences in the effects of NS and LR under different pathophysiological conditions. Statistical analyses were performed via Empower software and GraphPad Prism 9.0.0. A P value less than 0.05 was considered to indicate statistical significance.