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As a potent free radical scavenger and antioxidant, melatonin protects brain tissue against ischemia–reperfusion injury, partly via suppression of ischemia-induced production of nitric oxide, when given before ischemia–reperfusion or within 2 hr of onset of ischemia. In this study, we examined the neuroprotective effect of melatonin in an in vitro model of ischemia. Primary cultured astrocytes were subjected to 4 or 8 hr of oxygen–glucose deprivation (OGD), and cultured SHSY5Y human neuronal cells were exposed to 1 hr of OGD. Melatonin was added to the medium at the commencement of OGD to achieve different final concentrations, and cell death was quantified using the measurement of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) at 24 hr after reversion of OGD. Treatment with melatonin did not affect the astrocytic cell death following 4 or 8 hr of OGD. The relative MTT values of the neuronal cells were (as mean ± S.E.M.) 59.1 ± 2.4% in the vehicle-treated OGD group and 80.1 ± 2.7%, 82.5 ± 2.9%, 74.1 ± 2.3%, 64.2 ± 2.3%, 62.7 ± 2.8%, and 61.0 ± 3.9% in the OGD groups treated with melatonin at 10−3, 10−4, 10−5, 10−6, 10−7, and 10−8 M, respectively. Reduction in cell death was significant following treatment with melatonin at 10−3, 10−4, or 10−5 M. Reverse transcription-polymerase chain reaction showed that human mt1 and MT2 membrane receptors were not expressed in the cultured neuronal cells. Our results show that melatonin co-treatment protects cultured neuronal cells but not astrocytes against OGD-induced cell death in a dose-dependent manner and that the neuroprotection is independent of its known membrane receptors.