Stroke induces neuronal cell death through ischemic injury and it is thought that protecting neurons is an important therapeutic strategy for this disease. It has been reported that DNA methylation, which is epigenetic gene expression mechanism, can lead to cell death. Therefore, we investigated about relationships between neuronal cell death and DNA methylation after ischemic injury.
 At first, we performed immunostaining for 5-methylcytosine (5mC), epigenetic marks of DNA methylation, after middle cerebral artery occlusion and reperfusion (MCAO/R). The number of 5mC-positive cells were increased with time in the ischemic core until 1 day after MCAO/R. Furthermore, we determined 5mC-positive cells were colocalized with neuronal marker 1 day after MCAO/R.
Therefore, we focused on the changes in cultured neurons in subsequent experiments. We revealed that the number of 5mC-positive neurons were increased until 1 h after start of N-methyl-D-aspartate (NMDA) treatment and then decreased to the control level. Next, we examined whether DNA methyltransferase (DNMT) inhibitors were able to protect neurons against NMDA treatment. NMDA-treated neurons were protected by different types of DNMT inhibitors, zebularine and RG108 that were accompanied by inhibition of DNA methylation. These results indicated that DNA methylation after ischemic injury may have a role for initiator of neuronal cell death.