*2.6. Activation VEGF*/*VEGFR2 Regulates the MEK*/*ERK Signaling Pathway in the Hippocampal Neurons Following Experimental Status Epilepticus*

VEGF/VEGFR2 activates the MEK/ERK signaling pathway as evidenced by an increase in ERK and MEK phosphorylation levels [36,37]. To determine the regulatory role of VEGE/VEGFR2 in the MEK/ERK pathway, we examined the protein levels of phosphorylated ERK (p-ERK) and MEK (p-MEK), and total ERK and MEK. Rats received pre-treatment with resveratrol or siRNA against *pgc-1*α and these proteins were analyzed by western blotting 6 h after KA-induced experimental status epilepticus. The ratio of p-ERK/total ERK and p-MEK/total MEK were increased 6h after microinjection of DMSO and KA into the left hippocampal CA3 region compared with control animals (Figure 7a,b). Additionally, the ratios of p-ERK/total ERK and p-MEK/total MEK were significantly increased 6 h after microinjection of resveratrol (100 μmol) followed by KA administration into the left hippocampal CA3 (Figure 7a,b). Otherwise, the ratio of p-PI3K/total PI3K and p-AKT/total AKT were increased 6 h after administration of control siRNA with KA into the left hippocampal CA3 region compared with control rats (Figure 7c,d). However, the ratio of p-PI3K/total PI3K and p-AKT/total AKT were reduced

in the hippocampal CA3 area 24 h before pre-treated microinjection of siRNA against *pgc-1*α (2 μg) followed by KA microinjection into the hippocampus (Figure 7c,d).

**Figure 7.** (**a**) Changes of phosphorylated mitogen activated protein kinase (MEK) (p-MEK), total MEK, phosphorylated MEK (p-ERK), and total ERK expression, and (**b**) ratio of phosphorylation level for MEK protein levels and total MEK and phosphorylation of ERK in the CA3 tissue of the hippocampus 6 h after application of kainic acid (KA; 0.5 nmol) with pre-treated microinjection of 3% DMSO or resveratrol (Res; 100 μmol) into the hippocampal CA3 subfield. (**c**) Changes of p-MEK, total MEK, p-ERK, and total ERK expression in the hippocampus CA3 region 6 h after the microinjection of KA (0.5 nmol) into the left CA3 region with pre-treatment of application into the bilateral CA3 subfield with control siRNA or siRNA for *pgc-1*α (2 μg) 24 h in advance. (**d**) Ratio of phosphorylation level for MEK protein levels and total MEK and phosphorylation of ERK in the CA3 region of the hippocampus 6 h after the same experiments. Values are mean ± SEM of quadruplicate analyses from six animals per experimental group. \* *p* < 0.05 versus control group, + *p* < 0.05 versus DMSO+KA group, and # *p* < 0.05 versus control siRNA+KA group in the Scheffé multiple-range test.

*2.7. E*ff*ect of VEGF*/*VEGFR2 on Apoptosis and Neuronal Survival in the Hippocampal CA3 Subfield Following Experimental Status Epilepticus*

To further confirm the role of PGC-1α/VEGF/VEGFR2 signaling in neuroprotection from hippocampal damage due to prolonged seizure, we investigated the regulatory role of resveratrol on KA-induced hippocampal neuronal cell death. Significantly, pretreatment with resveratrol (100 μmol) attenuated the extent of cleaved caspases-3 expression in the hippocampal CA3 subfield 7 days after KA-induced status epilepticus (Figure 8a). Otherwise, the level of cleaved caspases-3 was significantly increased in the hippocampal CA3 area 24 h before pre-treated microinjection of siRNA against *pgc-1*α (2 μg) followed by KA microinjection into the hippocampus (Figure 8b). Mitigation of neuronal damage by resveratrol (100 μmol) treatment was demonstrated in the hippocampus in both DNA fragmentation qualitative (Figure 9a) and quantitative (Figure 9b) analyses, indexes for cell death, after inducing status epilepticus for 7 days. On the other hand, the level of qualitative (Figure 9c) and quantitative (Figure 9d) analysis of DNA fragmentation were aggravated in the hippocampal CA3 area 24 h before pre-treated microinjection of siRNA against *pgc-1*α (2 μg), followed by KA microinjection into the hippocampus.

**Figure 8.** (**a**) Changes of cleaved caspase-3 levels 7 days after microinjection of 0.5 nmol of kainic acid (KA) with 24-h pretreatment with resveratrol (Res; 100 μmol) or 3% DMSO into the hippocampal CA3 subfield. (**b**) Increase of cleaved caspase-3 (19 kDa) 7 days after rats were microinjected in the bilateral CA3 subfields with control siRNA or siRNA for *pgc-1*α (2 μg) 24 h before the microinjection of KA into the left hippocampal CA3. Values are mean ± SEM of quadruplicate analyses from four animals per experimental group. \* *p* < 0.05 versus control group, + *p* < 0.05 versus DMSO+KA group, and # *p* < 0.05 versus control siRNA+KA group in the Scheffé multiple-range test.

**Figure 9.** Qualitative (**a**),(**c**) or quantitative (**b**),(**d**) analysis of DNA fragmentation detected in samples harvested from the hippocampus 7 days after status epilepticus with or without Res and *pgc-1*α siRNA. Values are mean ± SEM from six animals per experimental group. (**a**),(**b**) Seven days after microinjection of 0.5 nmol kainic acid (KA) with 24-h pretreatment of microinjection of resveratrol (Res; 100 μmol) into the hippocampal CA3 subfield decreased fragmentation of DNA. Changes of qualitative (**c**) or quantitative (**d**) DNA fragmentation 7 days after microinjected control siRNA or siRNA for *pgc-1*α (2 μg) into bilateral CA3 subfields with 24 h before the microinjection of KA into the left hippocampal CA3. Values are mean ± SEM of quadruplicate analyses from four animals per experimental group. \* *p* < 0.05 versus control group, + *p* < 0.05 versus DMSO+KA group, and # *p* < 0.05 versus control siRNA+KA group in the Scheffé multiple-range test.
