*2.3. E*ff*ect of Resveratrol and Gene Knock-Down by Small Interfering RNA (siRNA) Against pgc-1*α *on VEGF Expression in the Hippocampus following Experimental Status Epilepticus*

In a previous report [26], we demonstrated that resveratrol is a PGC-1α activator, and it can activate PGC-1α and the following signaling pathways and then promote mitochondrial biogenesis. To determine the causality of PGC-1α in modulation of VEGF on this experimental paradigm, we tested the effects of resveratrol, as a positive control, and siRNA against *pgc-1*α*,* as a negative control, on VEGF expression in the hippocampus following status epilepticus. Microinjection of resveratrol (100 μmol) into the hippocampal CA3 region significantly increased the expression of *vegf* mRNA (Figure 3a) and the level of VEGF protein (Figure 3b) in the CA3 region 6 h after the elicitation of sustained hippocampal seizure activities. On the other hand, the specificity of the gene knock-down strategy by siRNA against *pgc-1*α was tested for VEGF expression in the hippocampus. For confirming the specific effect of *pgc-1*α siRNA on VEGF expression, after a pre-treated microinjection with siRNA against *pgc-1*α (2 μg) into bilateral hippocampal CA3 region, significant decrease of the *vegf* mRNA level was shown in our real-time PCR data (Figure 3c), and western blot analysis also confirmed a drastically decline in the VEGF protein level in the hippocampal CA3 area 6 h after KA-induced status epilepticus (Figure 3d) compared with the pre-treatment of the control siRNA group.

**Figure 3.** (**a**) Changes of *vegf* mRNA expression and (**b**) VEGF protein levels relative to β-actin from the CA3 of the hippocampus 6 h after microinjection of 0.5 nmol kainic acid (KA) or pretreatment with

microinjection of resveratrol (Res; 100 μmol) or 3% dimethyl sulfoxide (DMSO) into the hippocampal CA3 subfield. (**c**) Changes of *vegf* mRNA expression in the CA3 of the hippocampus 6 h after the microinjection of KA (0.5 nmol) into the left hippocampal CA3 subfield, and 24 h before pre-treatment with application into the bilateral CA3 subfield of control siRNA or siRNA for *pgc-1*α (2 μg). (**d**) Changes in VEGF relative to β-actin from the CA3 subfield of the hippocampus 6 h after the same experiments. Values are mean ± SEM of quadruplicate analyses from 6–8 animals per experimental group. \* *p* < 0.05 versus the control group, + *p* < 0.05 versus DMSO+KA group, and # *p* < 0.05 versus control siRNA+KA group in the Scheffé multiple-range test.

To confirm that the changes in expression of VEGF by resveratrol treatment and *pgc-1*α gene knock-down observed in our above biochemical analyses, we applied double immunofluorescence staining to detect the intracellular expression of VEGF in hippocampal CA3 neurons (Figure 4). Within the unified fields of a laser scanning confocal microscope, signals for VEGF were weak in the neurons, which were strongly immunoreactive to the neuron-specific nuclear protein (NeuN) neuronal marker, of hippocampal CA3 in the control animals (Figure 4a–c). In contrast to the control, there was an increase in VEGF immunoreactivity in the neurons from the same field, the CA3 area, 6 h after KA-induced status epilepticus (Figure 4d–f). Pretreatment of resveratrol (100 μmol) augmented the PGC-1α immunoreactivity in the hippocampal CA3 neurons (Figure 4j–l).

**Figure 4.** Confocal microscopic images of the right CA3b subregion of the hippocampus showing cells that were immunoreactive to neuron-specific nuclear protein (NeuN) (red fluorescence) or additionally

stained for VEGF (green fluorescence) in control rats (Figure 4a–c), or 6 h after administration of kainic acid (KA; 0.5 nmol) (Figure 4d–f) or pre-treatment with resveratrol (Res; 100 μmol) 30 min before KA-induced status epilepticus (Figure 4g–i). Induction of status epilepticus by KA in animals that received pre-treatment with application into the bilateral CA3 subfield 24 h after animals received control siRNA (Figure 4j–l) or siRNA against *pgc-1*α (2 μg) (Figure 4m–o). Scale bar, 10 μm.

In addition, in the parallel experiments, many VEGF-positive neurons were detected in the hippocampal CA3 area 6 h after experimental status epilepticus in animals pre-treated with pgc-1α control siRNA. However, reduced VEGF-positive cells were observed in the hippocampal CA3 subfield 6 h after status epilepticus in rats with pre-treated microinjection of control siRNA (Figure 4j–l) or siRNA against pgc-1α (2 μg) (Figure 4m–o) into the bilateral hippocampal CA3 field.
