Ischemia–Reperfusion Injury in Free Flaps: Molecular Mechanisms and Protective Effects of Remote Ischemic Preconditioning

Ischemia-reperfusion injury remains a major challenge in free flap surgery, contributing to oxidative stress, inflammation, and cell death that impair tissue viability and outcomes. Remote ischemic preconditioning (RIPC) has emerged as a potential protective strategy by modulating cellular stress responses, but its molecular mechanisms in free flaps remain incompletely understood. We prospectively enrolled 36 female patients undergoing autologous breast reconstruction with mainly deep inferior epigastric perforator (DIEP) free flaps, randomised into three groups: No RIPC, Early RIPC (24 h preconditioning), and Late RIPC (1 h preconditioning). Tissue samples were collected pre-ischemia and post-reperfusion for immunohistochemical and multiplex protein analyses. RIPC did not reduce oxidative stress markers, as 4-hydroxynonenal (4-HNE) levels were comparable across groups, while 3-nitrotyrosine levels paradoxically increased after RIPC. Early RIPC selectively modulated cell death pathways, with decreased expression of mitochondrial apoptotic marker caspase 9 and reduced necroptotic activation of mixed lineage kinase domain-like protein (MLKL) after reperfusion. Caspase 8 showed a transient modulation, suggesting effects on apoptosis-necroptosis crosstalk. Cyclophilin A levels were elevated after reperfusion in RIPC groups, indicating an adaptive stress response. These findings suggest that early RIPC exerts selective protection by modulating apoptosis and necroptosis, rather than broadly reducing oxidative stress. RIPC may represent a targeted strategy to improve free flap survival in reconstructive surgery.