Effect of mild hypothermia and barbiturate for cerebral ischemia-Evaluation by using primary neuronal culture

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It has been reported that mild hypothermia and anesthetics such as barbiturates are neuroprotecitve. However, the mechanism of this protective effect has not been well clarified. We evaluated the effect of hypothermia and pentobarbital on glutamate neurotoxicity in rat cortical neurons.Rat embryonic cortical neurons were obtained from timed pregnant rats at 18 days gestation. They were maintained in serum-free medium (Neurobasal medium supplemented with B-27 and N-2) for 6 days. The nearly pure neuronal population was obtained by this culture system. After exposure to 50 μM glutamate for 30 min, hypothermia or pentobarbital was induced. After 24 hours, neurotoxicity was evaluated by a leakage assay of lactic dehydogenase. Neuroprotective effect was seen in hypothermia (33 and 30 ℃), but not in clinical doses of pentobarbital. However, induction of hypothermia only during exposure to glutamate did not show any protective effect. Second, production of ROS (measured by flow cytometry with a ROS-specific fluorogen, C-DCDHF-DA) and caspase-3 activation (measured by DEVD-p-nitroaniline cleavage assays) was not inhibited by hypothermia after exposure of glutamate.Our data indicated that hypothermia is neuroprotective against exposure to glutamate by inhibiting any cascade after the exposure, but not by inhibiting direct effect of glutamate during exposure. However, we did not make clear what cascade was inhibited by hypothermia in this study.