A preliminary multimodal forensic assessment of footpad skin in a rat model of fatal heatstroke.

The forensic determination of heatstroke (HS) death remains challenging due to the lack of specific autopsy findings and histopathological changes. The diagnostic biomarkers reported in previous studies have not yet been effectively applied in forensic practice. The skin forms the primary interface between the body and the external environment and is central to heat exchange and thermoregulation. Therefore, the skin may capture both local thermal injury and peripheral responses associated with fatal heatstroke, suggesting its potential value as a biological sample for forensic investigation. Additionally, individual biomarkers are often insufficient to fully reflect the complex processes underlying death, whereas integrating multimodal information may improve diagnostic accuracy. Herein, we conducted a preliminary multimodal analysis to evaluate the forensic potential of skin in a rat model of fatal heatstroke, using functional assessment, HE staining, toluidine blue staining, and immunohistochemical detection of HSP72, Caspase-3, and AQP5. HE staining revealed enlarged secretory lumens and apparent shrinkage of sweat gland secretory cells in HS rats, while toluidine blue staining showed increased dermal mast-cell degranulation. Immunohistochemistry demonstrated increased HSP72, total Caspase-3, and AQP5 immunoreactivity in sweat gland secretory cells, with HSP72 and total Caspase-3 also elevated in the epidermis. PCA suggested clearer group separation when morphological and immunohistochemical variables were considered jointly. Among the individual variables, the mast-cell degranulation ratio showed the best discriminatory performance, with an AUC of 0.9792 (95% CI, 0.9309-1.000). An exploratory random forest classifier integrating seven prespecified variables achieved an internally cross-validated AUC of 1.000 (95% CI, 1.000-1.000) using leave-one-out cross-validation. Overall, this study provides preliminary evidence that skin changes may contribute to forensic assessment of fatal heatstroke.