Icing treatment is commonly accepted as the gold standard for the first aid of sports injuries. However, we have previously demonstrated that icing treatment immediately following skeletal muscle injury impairs muscle regeneration; for example, it promotes the development of fibrosis during the regenerative process. Although it has been suggested that the icing treatment delays both the timing of appearance and disappearance of transforming growth factor β1, a cytokine known to stimulate collagen production, during the course of muscle regeneration, the genes that may initiate the promotion of fibrosis in response to icing, as well as the mechanisms and regulatory changes underlying their expression, remain unclear. Therefore, the aim of the present study was to investigate the impact of icing treatment on regenerative responses at early phase of regeneration after skeletal muscle injury. Male Wistar rats, aged 10 weeks, were used for this study. In order to induce muscle injury, bupivacaine was injected into the plantaris muscle bilaterally. Half of the rats received icing treatment (applied ice packs to the injured area) for 20 min immediately following the injury. Six hours after the bupivacaine injection, plantaris muscles were dissected, and then western blot, RNA-sequencing (RNA-seq), and gene ontology (GO) analyses were performed. Six hours after the plantaris muscle injury, the plantaris-to-body weight ratio tended to increase similarly in both the icing and non-icing groups. This response paralleled the early elevation in myeloperoxidase protein expression, a neutrophil marker, suggesting equivalent early neutrophil infiltration between the groups. In contrast, cytoskeletal (Desmin), mitochondrial (Pyruvate dehydrogenase E1 component subunit alpha: PDH), and cytosolic (Myoglobin) protein expressions were decreased regardless of icing treatment. However, the icing treatment partially attenuated the injury-related reductions in PDH and Myoglobin. RNA-seq analysis identified 195 upregulated and 46 downregulated genes in response to the icing treatment. GO enrichment analysis indicated that the upregulated genes were associated with fibrosis-related biological processes, including collagen fibril organization and extracellular matrix organization. In contrast, no GO biological process terms were significantly enriched among the downregulated genes. These results suggest that icing treatment delays the degradation process and disrupts the early regulation of fibrosis-related genes after plantaris muscle injury, which may contribute to impaired muscle regeneration at later stages. This research was supported by JSPS KAKENHI (TS: 23K10584), Kanazawa University “HOZUMINE” project for promotion of research (TS), and Yamaha Motor Foundation for Sports (TS). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Shibaguchi et al. (Fri,) studied this question.