Isoquercetin Pretreatment Protects Hippocampal Neurons from Oxygen-Glucose Deprivation

Primary hippocampal neurons were placed in an anaerobic chamber (HERAcell 150 incubator; Thermo Fisher Scientific, Inc.) and the partial oxygen pressure was maintained at <2 mmHg). The medium was replaced with a pre-warmed (37°C) glucose-free balanced salt solution. An anaerobic gas mixture comprising 95% N₂ and 5% CO₂ was bubbled through the solution for 30 min. Cell cultures subjected to OGD were incubated in the solution at 37°C for different time periods to produce oxygen deprivation and then re-oxygenated (returned to the normal aerobic environment). Experimental parameters were assayed 4 h following re-oxygenation. The primary cultures of rat hippocampal neurons were pretreated with 25, 50 and 100 µg/ml isoquercetin for 24 h, followed by exposure to OGD for 4 h, then reperfusion for 24 h. The hippocampal neurons cultured in plain medium served as control. At the end of cell treatments, different tests were carried out as described below. PD98059 (HY-12028; MCE, Monmouth Junction, NJ, USA) was the inhibitor of the MEK.