Cell culture and establishment of the oxygen-glucose deprivation/reperfusion (OGD/R) model

PC12 neurons (rat adrenal pheochromocytoma cells) were obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. The cells were cultured in DMEM supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and maintained at 37˚C in a humidified atmosphere with 5% CO₂. The proliferation of the cells was observed regularly, and cells were passaged when required. Cells in the logarithmic phase were collected for use in subsequent experiments.

Treatment of PC12 cells with OGD/R was performed to simulate cerebral IRI in vitro. The detailed treatment protocol is described in a previously published study (16). Briefly, PC12 cells were cultured in glucose-free DMEM without FBS at 37˚C in an incubator containing 95% N₂ and 5% CO₂ for 3 h. Subsequently, the medium was replaced with glucose-containing DMEM supplemented with 10% FBS and reoxygenated for 24 h under normoxic conditions. Following successful establishment of the cell model, the cells were used for subsequent experiments.