Protective effect of mild hypothermia and barbiturate for cerebral ischemia-Evaluation by using neuronal cells-

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It has been reported that mild hypothermia and barbiturate are neuroprotecitve. However, the mechanism of this protective effect has not been well clarified. One possible mechanism is the inhibition of neuronal apoptosis. We evaluated the effect of hypothermia (29-35℃) and pentobarbital on apoptosis in neuronal cells using PC 12 cells, which is derived from rat pheochromocytoma. We further evaluated the mechanism of protective effect from the viewpoints of production of reactive oxygen species (ROS) and caspase activationApoptosis was induced by depriving serum from the medium, which is one of the most representative methods to induce apoptosis in PC 12 cells. First, cytotoxicity (evaluated by a leakage assay of lactic dehydogenase) and the percentage of apoptotic cells (calculated by flow cytometry with propidium iodide) were evaluated 4 days after induction of apoptosis. Second, production of ROS was measured by flow cytometry with a ROS-specific fluorogen, C-DCDHF-DA 3 or 6 hours after induction. Third, caspase-2 and -3 activation was measured by VDVAD p-nitroaniline and DEVD-p-nitroaniline cleavage assays 15 hours after induction.Hypothermia (29 - 35℃) and clinical dose of pentobarbital significantly decreased cytotoxicity and the percentage of apoptotic cells. At 37℃ the production of ROS has already increased almost twice compared to control 3 hours after induction of apoptosis. Clinical dose of pentobarbital significantly decreased ROS production whereas hypothermia did not inhibit it. Caspase-2 and -3 activity was not inhibited by hypothermia and pentobarbital, although the activity increased by the induction of apoptosis.Our data indicated that hypothermia (29 - 35℃) and clinical dose of pentobarbital inhibited induced apoptosis in neuronal cells. Inhibition of ROS production may be partly attributable to this protection by barbiturate. However, production of ROS and caspase activation may not be related to the mechanism of protective effect of hypothermia.