Fig 4. Mmp-9 upregulation during epilepsy development is strictly dependent on epileptogenesis-evoked demethylation of its gene promoter.

To block epilepsy development, we used dizocilpine (the NMDA receptor antagonist displaying anticonvulsant activity). 0.1 mg/kg of dizocilpine or saline was intraperitoneally injected to rats 30 min before each PTZ dose administration (30 mg/kg). (A) Dizocilpine treatment effectively suppresses the development of PTZ-evoked epilepsy in rats. Animals were observed up to 2 h after each PTZ injection and seizures were scored according to a modified Racine’s scale. Values are means ± SEM (*, p<0.05; n = 11). (B) PTZ kindling-evoked upregulation of the Mmp-9 mRNA expression is fully inhibited by dizocilpine administration in the rat hippocampus. Dizocilpine administration suppresses the PTZ kindling–evoked augmentation in the hippocampal Mmp-9 mRNA expression, whereas repeated PTZ treatment without dizocilpine injections leads to significant upregulation of Mmp-9 mRNA level. For RT-qPCR analysis equal amounts of RNA isolated from naive (control), PTZ-treated (saline + PTZ), and dizocilpine-treated (dizocilpine + PTZ) rat hippocampi were used. Data is presented as fold change in mRNA expression. Values are means ± SEM (*, p<0.05; ***, p<0.001; n = 8). (C) Dizocilpine treatment completely inhibits the PTZ kindling-dependent Mmp-9 proximal promoter demethylation in the rat hippocampus. Mmp-9 proximal promoter methylation level was evaluated using qPCR in DNA samples obtained by immunoprecipitation of methylated DNA from naive (control), PTZ-treated (saline+PTZ), and dizocilpine-treated (dizocilpine+PTZ) rat hippocampi. Data is presented as a percent of input. Values are means ± SEM (*, p<0.05; n = 5).