Neuronal cultures and oxygen and glucose deprivation (OGD) induced neuronal cell injury

According to a previously described method, cultured neurons were obtained from the cerebral cortices of 1-day-old Sprague-Dawley rats (36,37). Experiments were performed on cultured neurons between 7 and 10 days in vitro. OGD was achieved by inducing hypoxia and aglycemia according to the method (38,39). The OGD medium consisting of Hank's Balanced Salt Solution (HBSS) lacking glucose. The HBSS solution was bubbled with N₂ for 30 min to deplete glucose and oxygen from intracellular stores and extracellular space. Cultured neurons were pretreated with melatonin (10–500 µM) or control (0.1% DMSO) for 30 min, and then were subjected to OGD. After the deprivation period, cultured neurons were incubated in the culture medium under normal conditions (a humidified incubator with 5% CO₂ at 37°C). Each experiment consisted of three samples. Four independent instances were carried out for each experiment.