Icing following skeletal muscle injury alters fibro/adipogenic progenitor responses and results in excessive collagen deposition in male rats

Icing is a common acute intervention following skeletal muscle injuries in sports medicine; however, previous studies in rodent models indicated that icing impairs muscle regeneration and promotes collagen deposition in regenerating muscles. As fibrosis decreases muscle function and increases the risk of reinjury, it poses a significant challenge for athletes. To address the fibrosis-related impairment of muscle function, we examined the mechanisms underlying icing-induced fibrosis. Specifically, we focused on the response of fibro/adipogenic progenitors (FAPs), key players in muscle fibrosis, during muscle regeneration. Using a crush-injury model in male rats, we investigated the effects of icing on the FAP response and fibrosis-related outcomes. Icing inhibited the early-phase increase in FAPs within injured muscles. Moreover, icing upregulated the expression of transforming growth factor-β1 (TGF-β1), which promotes the differentiation of FAPs into myofibroblasts, particularly 4 and 5 days postinjury, when FAP accumulation was abundant. Consequently, an increased number of α-smooth muscle actin (α-SMA)-positive myofibroblasts and an expanded collagen fiber area were observed during the later stages of icing-treated muscle regeneration, indicating excessive muscle fibrosis. Taken together, our results suggest that icing after muscle injury may promote fibrosis by altering FAP behavior and its surrounding environment, which warrants careful reevaluation of its clinical effectiveness.<b>NEW & NOTEWORTHY</b> Previous studies have examined the effects of icing on macrophages and muscle stem cells during muscle regeneration. Here, we identified FAPs as another cell population affected by icing and involved in excessive fibrosis. Icing delayed FAP proliferation and increased their number when TGF-β1 was markedly elevated, resulting in excessive collagen deposition. These results reveal how icing contributes to muscle fibrosis by affecting FAP behavior and highlight the importance of FAP dynamics in muscle injury management.