The mechanism by which MCI-186 (3-methyl-1-phenyl-2-prazolin-5-one) exerts protective effects during cerebral infarction, other than its function as a radical scavenger, has not been fully elucidated. Here, we found that MCI-186 stimulates intracellular survival signaling in vivo and in vitro. In a rat infarction model, the infarct area was significantly smaller and the degree of edema was reduced in MCI-186-treated animals. In the MCI-186-treated rats, the number of single stranded (ss) DNA-positive damaged cells in the peri-infarct area was decreased compared with the control, suggesting that MCI-186 protects cerebral tissues from cell damage. To clarify the mechanisms underlying the effect of MCI-186, we also examined the survival-promoting effect of this agent on cultured cortical neurons. In this in vitro system, MCI-186 blocked serum-free induced neuronal cell death. Interestingly, an increase in the activation of both Akt (a component of the PI3 kinase pathway) and ERK (a component of the MAP kinase pathway) was observed in the cortical cultures after MCI-186 exposure. Furthermore, the MCI-186-dependent survival effect in vitro was blocked by U0126, an MEK (an upstream of ERK) inhibitor, and also by LY294002, a PI3 kinase inhibitor. We also observed similar increases in the activation of Akt and ERK in the in vivo model, further suggesting that the antiapoptotic role of MCI-186 is mediated via the PI3 kinase and MAP kinase signaling pathways. We therefore conclude that, in addition to its role as a free radical scavenger, MCI-186 functions as an antiapoptotic factor by enhancing intracellular survival signaling.

Following endoplasmic reticulum (ER) stress, cerebral infarctions have been reported to involve an apoptotic process, including the activation of the caspase cascade. To confirm whether fragmented caspase-12, which is activated by cleavage and is detectable during ER stress, is also involved in embolic cerebral infarctions in rats, we adopted an autologous blood clot model for the analysis of cerebral infarctions. We performed experiments in rats with brain infarctions, which are closely related to embolic cerebral infarctions. We utilized a homologous blood clot, i.e., natural materials, to form the infarct area. Our findings reveal that caspase-12 is fragmented when infarct areas form in cerebral cortical neurons. Interestingly, we observed that these fragments translocated to the nuclei of not only cerebral cortical neurons but hippocampal neurons. We further found that glucose-regulated protein 78 (GRP78), a marker of ER stress, is up-regulated in both cerebral cortical and hippocampal neurons during cerebral infarction. This result suggests that the fragmentation of caspase-12 and the subsequent nuclear translocation of these fragments are involved in the brain infarction process in rats.

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