Male C57/BL6J mice (age 10 weeks) were used in this study. All animals were purchased from SLC Japan Inc. (Shizuoka, Japan). The mice were allowed free access to food and water and were maintained on a 12-h light/dark cycle at room temperature (24 ± 2 °C) with constant humidity (40 ± 15%). All experimental procedures involving animals and clinical data were approved and overseen by the Institutional Animal Care and Use Committee of Showa University (#09022, 02003), which adhered to the ARRIVE guidelines. All methods were performed in accordance with the relevant guidelines and regulations.
Video of wobbling after human heatstroke and head MRI in patients with heatstroke were obtained from the participants. All experimental protocols were approved and overseen by the Clinical Trial Review Board of Showa University (F2019C83), which adhered to the CIOMS Ethical Guidelines for Biomedical Research. Informed consent was obtained from all participants and/or their legal guardians. Research has been performed in accordance with the principles of the Declaration of Helsinki.
Protocol for heatstroke
The high AT and RH, resembling temperate/tropical summers, heatstroke model was used, which was based on our previous study19. A semi-enclosed heatstroke chamber (200 × 340 × 300 mm) made of acrylic was created by vertically stacking the animal cages in a greenhouse-like construction. An ultrasonic humidifier (USB-68, Sanwa, Japan) and a digital thermo-hygrometer (AD-5696; CA&D Company, Japan) were used for the humidification and monitoring of AT, RH, and WBGT. The heatstroke chamber was placed in an incubator (Bio-chamber, BCP-120F; TITEC, Japan) and preheated to the desired experimental temperature for ≥ 3 h. The humidifier was started 3 h before heatstroke to create a hot and humid environment. Meanwhile, the mice were allowed 3 h of water restriction, and the mildly dehydrated mice were placed in the heatstroke chamber and exposed to high AT (41 °C) and RH (> 99.0%) for 60 min. Subsequently, they were returned to the animal cage, where they could access food and water. Mice that were not heat-exposed were used as controls (CTL group).
Behavioral study (rotarod test)
The rotarod test was performed according to a previous report34. A rotarod treadmill (Muromachi Kikai, Japan) consisted of a plastic rod (diameter, 3 cm; length, 10 cm) flanked by four large round plates (diameter, 57 cm). The rod rotates at a constant speed of 4 rpm at the beginning and continuously accelerates to a speed of 40 rpm for 5 min. The time each mouse spent on the rod was measured. Mice were trained with rotarod test once a week for five times before heatstroke. The behavior test in a mouse was performed twice within an interval of 5 min each and was expressed as the average of the trials. The animals were divided into two groups (HS and CTL) according to the scores of the last training (n = 36 in each group). The HS group mice were subjected to heat for 1 h. The CTL group (without HE) mice were prepared as a control. The HS and CTL groups were subjected to behavioral tests at 1, 3, 5, 7, and 9 weeks after heatstroke (Supplementary Fig. S2a).
Under sodium pentobarbital (50 mg/kg, i.p.) anesthesia, the mice (HS, n = 9, per time course in each group) at 1, 3, and 9 weeks after heatstroke were transcardially perfused with 0.9% sodium chloride, followed by 10% neutralized formalin. The brain was removed and divided into two parts along the longitudinal cerebral fissure. Paraffin-embedded specimens of the right hemisphere of the brain were prepared. Subsequently, eight sagittal sections were sliced at a thickness of 5 μm at every 200 μm interval from the cerebral longitudinal fissure for histological examination, as described below. Age-matched CTL group mice were prepared to eliminate the influence of senescence (Supplementary Fig. S2b).
Semi-quantification of cerebellar white matter demyelination
Cerebellar white matter demyelination was examined with the KB method without Nissl staining33,35. Eight sagittal sections of the cerebellar hemispheres were prepared at 200 µm intervals at the thickness 5 µm, stained with KB. These eight sections were tile scanned of the entire cerebellar hemisphere at 400 × magnification in each mouse using 9 animals per time course. The Luxol fast blue intensity, which stains myelin (fast blue) in the white matter, was then semi-quantified to evaluate demyelination. These areas were manually traced with blinded investigators and converted to black and white; black areas were semi-quantified using Scion Image for Windows (Scion Corporation, USA). The pixels of the black myelinated regions were divided by the pixels from the total traced areas and expressed as percentages. Eight serials of sagittal sections were averaged for each animal. These procedures were performed by two investigators (H.Y. and K.Y.) who were blinded to the experimental groups.
Immunochemical staining and counting of Purkinje cells
Another serial series of eight sagittal sections were immunostained with antibodies against calbindin D-28k (calbindin) and used for counting Purkinje cells36. After removing the paraffin, using a series of xylene/alcohol solutions, the sections were incubated in 10 mM sodium citrate buffer (pH = 5.0) for 25 min to retrieve heat-induced antigen and immersed in 0.3% hydrogen peroxide/methanol for 30 min to block the endogenous peroxidase reaction. The sections were then incubated with a mouse Ig blocking reagent (M.O.M.; Vector, USA), followed by 5% goat serum wash to diminish the mouse endogenous immunoglobulin and non-specific reactions. The sections were then incubated overnight with a monoclonal mouse anti-calbindin antibody (1:2000; Swant, Switzerland) and incubated for 90 min with a biotinylated goat anti-mouse IgG the next day (1:200, DAKO, CA, USA). The immunoreactions were visualized by incubating with an avidin–biotin complex solution (Vector, USA) and diaminobenzidine (Sigma, USA). The calbindin-immunopositive Purkinje cells were determined and counted using the CellSens Standard software (Olympus, Japan). The number of Purkinje cells in the molecular layer of the cerebellum was counted twice manually in all lobules of the same section, and the average was calculated. Cell counting was repeated in the eight sagittal sections. This was also performed by an investigator (H.Y. and K.Y.) who was blinded to the experimental groups.
Immunostaining of synaptic markers
Post- and presynaptic markers were co-stained in the cerebellum at 1, 3, and 9 weeks after heatstroke to estimate the synapse around Purkinje cells using four of the nine mice. After heat-induced antigen retrieval and blocking according to the protocol as mentioned above, the sections were incubated with either monoclonal rabbit anti-calbindin antibody (1:100, Cell Signaling, USA) or polyclonal rabbit anti-synaptophysin antibody (1:200, Proteintech, USA) with monoclonal mouse anti-postsynaptic density 95 (PSD95) antibody (1:400, BD, USA) overnight at 4 °C. After washing, the sections were incubated with Alexa 488-conjugated goat anti-rabbit IgG antibody (1:400; Thermo Fisher Scientific, USA) and Alexa 546-conjugated goat anti-mouse IgG antibody (1:400; Invitrogen, USA). Subsequently, cell nuclei were stained with 4,6-diamidine-2-phenylindole dihydrochloride (1:10,000; Roche, Germany) and incubated in 1.0 mM CuSO4 in 50 mM ammonium acetate buffer (pH = 5.0) to diminish autofluorescence37,38. Fluorescence was detected using an Axio Imager optical sectioning microscope with ApoTome II (Carl Zeiss, Germany). For control staining, the same steps were performed except incubation with primary antibodies.
Data are reported as mean ± standard error of the mean. Student’s t test was performed for comparisons between the two groups. A repeated measures analysis of variance and Tukey–Kramer test were performed for multiple comparisons. Statistical significance was set at a P-value of < 0.05.
All experimental procedures involving animals were approved and overseen by the Institutional Animal Care and Use Committee of Showa University, which adhered to the ARRIVE guidelines. All human research protocols were approved and overseen by the Clinical Trial Review Board of Showa University, which adhered to the CIOMS Ethical Guidelines for Biomedical Research. Informed consent was obtained from all participants and/or their legal guardians. The research has been performed in accordance with the Declaration of Helsinki.
Approval for animal experiments
All experimental procedures involving animals and clinical data were approved and overseen by the Institutional Animal Care and Use Committee of Showa University (#09022, 02003), which adhered to the ARRIVE guidelines.
Approval for human experiments
All human research protocols were approved and overseen by the Clinical Trial Review Board of Showa University (F2019C83), which adhered to the CIOMS Ethical Guidelines for Biomedical Research. Informed consent was obtained from all participants and/or their legal guardians. Research has been performed in accordance with the principles of the Declaration of Helsinki.