Supplementary Material for: Characterisation of an ischaemia-reperfusion model for the formation of a stage II pressure injury in diabetic mice

Introduction: Pressure injuries (PIs) in patients with diabetes mellitus (DM) still impacts patients' health and places a heavy burden on healthcare systems. Stage I and stage II PIs are particularly prevalent among individuals with diabetes. Without timely and appropriate interventions, these injuries can progress to more severe stages, requiring prolonged recovery periods. Thus, the development of preclinical animal models that can mimic stage I or II pressure injuries in diabetic patients is urgently needed to understand the mechanisms of injury formation and healing. Methods: In this study, magnets were used to compress the dorsal sides of mice for 2 hours (h), 4 h, 8 h, or 16 h according to the ischaemia-reperfusion principle, and the changes in compressed skin in diabetic (db/db) and nondiabetic (WT) mice were compared at different ischaemia exposure times and cycle times. Results: After 2 h of ischemia, there was no significant injury in WT and db/db mice. On the third day following 4 h of ischemia, both db/db and WT mice exhibited characteristics resembling human stage II pressure injuries, with damage primarily confined to the epidermis and upper dermis. Ischemia durations of 8 and 16 h resulted in more severe full-thickness skin defects, including exposed subcutaneous adipose tissue and inward contraction of wound margins. After ischaemia (I) for 4 h and reperfusion (R) for 24 h, the morphology of fibroblasts in the compressed skin area of db/db mice changed, and the expression of TGF-β1 decreased significantly compared with those in WT mice. On day 5, epidermal–dermal separation and pronounced infiltration of inflammatory cells were evident in both groups. On day 10, db/db mice exhibited delayed wound closure, as well as impaired regeneration of the panniculus carnosus and dermis, with significantly decreased mRNA levels of VEGF and HSP90. Conclusion: Ischaemia lasting 4 h is the appropriate duration for generating stage II pressure injuries in diabetic mice, which may be applicable to generate a reproducible model of stage II pressure injury caused by ischaemia-reperfusion injury. This model offers a valuable experimental tool for in-depth investigation of the pathogenesis of diabetic pressure injuries and for the development of novel therapeutic strategies.