Cell culture, oxygen glucose deprivation/reoxygenation (OGD/R) injury and cell viability determination

The experiment was performed according to the previously described method with a few modifications (Huang et al., 2015). The primary cultures of cortical neurons were harvested from E15-18 embryos of pregnant Spraguee-Dawley rats. Then neurons were digested in 1000 mL cysteine (0.25%), stopped by adding 200 mL fetal bovine serum (FBS) and gently resuspended in MEM medium containing 10% (v/v) FBS, and dissociated properly. After filtration, the cortical tissues were centrifuged at 1000 rpm for 5 min, and then resuspended in MEM + 10% (v/v) FBS. After the supernatant was discarded, cortical neuron cells were plated at 1 × 10⁶ g/ml onto 24 or 6 well culture plates pre-coated with cell adhesion solution (0.5%; Applygen Technologies Inc, Beijing, China). The culture plates were stored in a 5% CO2 humidified incubator for 6 h at 37°C (Thermo, Waltham, MA, USA). Then, the culture media was replaced by Neurobasal Media supplemented with 2% B27 (v/v). Neurons were routinely cultured and maintained for 7–8 days for using in the following experiments.

The OGD/R model was carried out to mimic I/R injury in vivo according to previously established method (Fan et al., 2014) with a few modifications. After 7–8 days in culture, the cortical neurons were refreshed with glucose-free DMEM medium and placed in an anaerobic chamber fully flushed with 80% N₂ and 20% CO₂ (pH 6.8) to mimic the acidic environment of ischemic brain in vivo at 37°C for 2 h. Subsequent reoxygenation was carried out by exposing neurons to normal medium and further incubating for 24 h. After OGD/R induction, cortical neurons were treated with or without AR (3, 10, and 30 μM) in the presence or absence of PD98095 (20 μM, a specific ERK1/2 inhibitor) and/or GF109203X (5 μM, a specific PKC inhibitor) for indicated time in the following experiments. Cell viability was evaluated by MTT assay as described previously (Fan et al., 2014) at 8 h after incubation.