Melatonin Acts in Synergy with Hypothermia to Reduce Oxygen-Glucose Deprivation-Induced Cell Death in Rat Hippocampus Organotypic Slice Cultures

Background: Hypoxic-ischemic encephalopathy is a major cause of neonatal morbidity. Therapeutic hypothermia, while beneficial, still leaves many treated infants with lifelong disabilities. Thus, adjunctive therapies, such as melatonin, are needed to provide additional neuroprotection. Objectives: The aim of this study was to determine a range of melatonin concentrations that could result in neuroprotective synergy with hypothermia. Methods: Hypoxia-ischemia was simulated by transient oxygen-glucose deprivation (OGD) in organotypic hippocampal slice cultures derived from neonatal rats. Cell damage was quantified by propidium iodide (PI) labeling. Results: Melatonin reduced OGD-induced cell death in a concentration-dependent manner (1-100 mu M) with an EC50 of about 25 mu M. Hypothermia attenuated cell death in a time-dependent manner, with a nearly full protection upon 24-h exposure (78%) and partial protection (40%) upon 6-h exposure. When submaximal effective concentrations of melatonin (25 or 50 mu M, resulting in 54 and 64% protection) were combined with 6 h of hypothermia, nearly full protection (73 and 78%, respectively; p < 0.05 and p < 0.01) was observed. Conclusion: Melatonin acts in synergy with hypothermia in attenuating OGD-induced damage in organotypic hippocampal cultures. This reductionist approach allows the determination of a range of concentrations of melatonin capable of enhancing hypothermic neuroprotection. This information, coupled with pharmacokinetic data, will help to define the therapeutic dosage of melatonin in vivo and, ultimately, in patients.