Protective effects of the free radical scavenger edaravone against glutamate neurotoxicity in nearly pure neuronal culture

Edaravone, a free radical scavenger, has shown neuroprotection in both animals and humans. To evaluate the mechanism of this protection, we examined the effect of edaravone on neurons themselves against glutamate neurotoxicity. Neurons were collected from 18-day fetal rat brains and a culture of almost pure neurons was obtained after 14-day culture. The neurons were exposed to 50 mu M glutamate for 10 min, followed by normal culture for 24 h. Edaravone was added to the medium during the glutamate insult (prophylactic effect) or after the insult (treatment effect). First, the cell survival rate was measured by staining with trypan blue. Second, the cells were stained with 6-carboxy-2', 7'-dichlorodihydrofluorescein diacetate, di-(acetoxymethyl ester) (C-DCDHF-DA) and the relative amount of reactive oxygen species (ROS) was measured by flow cytometry. Third, the cells were stained with Hoechst 33342 and propidium iodide and the numbers of apoptotic and necrotic cells were counted. A dose-dependent prophylactic effect was observed and the cell survival rate in 500 mu M edaravone was significantly higher than that without it. However, there was no treatment effect beyond 2 h after the insult. The amount of ROS under 500 mu M edaravone at 4 h after the glutamate insult was significantly lower than the control amount. Necrosis, but not apoptosis, was significantly inhibited by edaravone. Edaravone mainly showed a prophylactic effect on neurons against glutamate neurotoxicity, possibly through the inhibition of necrosis via the suppression of ROS production. However, for a protective effect, a higher, supraclinical concentration was required, compared to the concentrations producing a protective effect in glial and endothelial cells in previous studies.