Primary Neuronal Culture and Oxygen-Glucose Deprivation

Primary cultured neurons were prepared from 17- or 18-day-old mouse embryos. The cortex of embryos was dissected in Hank’s buffered saline solution and digested with EDTA-free trypsin at 37°C for 15 min. Neurons were dissociated with DMEM/F12 medium containing 10% (vol/vol) fetal bovine serum (FBS) and plated onto poly(D-lysine)-coated (Sigma, St. Louis, MO, United States) coverslips in a 12-well plate or tissue culture dish. Cells were maintained in a humidified incubator (37°C, 5% CO₂) for 2 h, and the medium was changed to a maintenance medium [Neurobasal containing 2% (vol/vol) B27 and 1% (vol/vol) GlutaMAX]. Half of the medium was replaced with a fresh maintenance medium [Neurobasal containing 2% (vol/vol) B27] every 3 days.

After 14 days in vitro (DIV), primary cultured neurons were challenged with OGD. The medium was first treated with deoxygenated glucose-free Hanks’ balanced salt solution (Invitrogen). The culture plates were then transferred to a hypoxic chamber (37°C, 5% CO₂, and 95% N₂) for 1 h. The medium was subsequently changed to a glucose-containing medium containing 10% (vol/vol) FBS and maintained under normoxic conditions for 24 h (37°C, 5% CO₂).