Efficacy of HET0016 for improving delayed mild hypothermia-induced protection of rat neurons subjected to oxygen-glucose deprivation and restoration in vitro

Objective To evaluate the efficacy of HET0016 for improving the protection of rat neurons subjected to oxygen-glucose deprivation and restoration (OGD/R) by delayed mild hypothermia. Methods The hippocampal neurons were isolated from healthy neonatal Sprague-Dawley rats and then cultured.The neurons were seeded in 6-well plates at a density of (2-3)×105 cells/ml, and randomly divided into 4 groups (n=12 each) using a random number table: control group (group C), group OGD, OGD/R-mild hypothermia group (group OGD/R-MH), and OGD/R-mild hypothermia-HET0016 group (group OGD/R-MH-HET). In group C, the neurons were cultured in the normal culture medium.In group OGD, the neurons were subjected to OGD for 2 h. In group OGD/R-MH, the neurons were subjected to OGD for 2 h followed by restoration of oxygen-glucose for 4 h, and then mild hypothermia was applied for 24 h. In group OGD/R-MH-HET, HET0016 with the final concentration of 1 μmol/L was added immediately after OGD (before restoration of oxygen-glucose), and the procedures else were same as those in group OGD/R-MH.The neurons were then continuously cultured for 24 h. After 24 h of culture, the viability of neurons was measured using methyl thiazolyl tetrazolium assay, and the survival rate was measured using PI/FDA double staining. Results Compared with group C, the viability of neurons was significantly weakened, and the survival rate was significantly decreased in group OGD (P<0.05). Compared with group OGD, the viability of neurons was significantly enhanced, and the survival rate was significantly increased in OGD/R-MH and OGD/R-MH-HET groups, especially in group OGD/R-MH-HET (P<0.05). Conclusion HET0016 improves delayed mild hypothermia-induced protection of isolated rat neurons subjected to OGD/R to some extent. Key words: Hypothermia, induced; Hypoxia-ischemia, brain; Infant, newborn; Neurons; HET0016