*4.4. Gene Knockdown by Microinjection of Small Interfering RNA (siRNA) Against pgc-1*α *into the Hippocampus*

Gene knockdown was conducted with small interfering RNA (siRNA) against the *pgc-1*α gene. Multiple siRNA sequences were synthesized form by GE Healthcare (GE Healthcare, Chicago, IL, USA). All siRNAs were dissolved in an isotonic RNAi buffer (100 mM potassium acetate; 30 mM Hepes-KOH; 2 mM magnesium acetate; 26 mM NaCl, pH 7.4, at 37 ◦C). The siRNA against *pgc-1*α as following blow:


Pooled siRNAs were mixed with Lipofectin reagent (1:1, 18292-011, Invitrogen, Carlsbad, CA, US) and microinjected bilaterally into the CA3 region of the hippocampus 24 h before the microinjected administration of KA. The pooled non-targeting siRNAs containing four scramble sequences were used as control siRNA.

#### *4.5. Collection of Tissue Samples from the Hippocampus*

At time-intervals (1, 3, 6, or 24; or 7 days) after microinjection of KA or PBS into the hippocampus, animals were anesthetized with 3% isoflurane and were perfused intracardially [83,85]. The brain was immediately removed and placed on gauze moistened with 0.9% ice-cold saline. To avoid the confounding effect of KA toxicity, we routinely collected samples from the CA3 region of the right hippocampus (this side is the hEEG recording side, with no KA injection). Our method permitted us to verify that our results from the analyzed samples were directly from sustained seizures (status

epilepticus) and not indirectly due to KA toxicity [5,26,44]. Hippocampal samples were stored at −80 ◦C until use in biochemical analyses.

### *4.6. RNA Isolation and Reverse Transcription Real-Time Polymerase Chain Reaction*

To quantitatively analyze the expression of *pgc-1*α, *vegf*, and *vegfr2 (Flk-1)* mRNA in the tissue of the hippocampal CA3, the brain was rapidly removed after PBS perfusion and total RNA from the hippocampus was extracted with a RNeasy Mini Kit (Qiagen, Duesseldorf, Germany) according to the manufacturer's protocol as in our previous reports [5,26,44]. Reverse transcriptase (RT) reaction was applied with the ImProm IITM Reverse Transcription System (Promega, Madison, WI, USA) to acquire first-strand cDNA synthesis. Real-time polymerase chain reaction (PCR) for amplification of cDNA was performed using a LightCycler® 480 SYBR Green I Master (Roche Diagnostics, Mannheim, Germany). The qPCR of each sample was carried out in duplicate for all cDNA and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the housekeeping gene used as a control [5,26,44]. The primer pairs for amplification of *pgc-1*α*, vegf*, and *vegfr2 (Flk-1)*, and *Gapdh* cDNA used in this study were as follows:


The products of PCR were subsequently subjected to agarose gel electrophoresis for further confirmation of amplification specificity [5,26,44]. Fluorescence signals from the amplified products were quantitatively assessed using the LightCycler software program (version 3.5). The second derivative maximum mode was chosen with baseline adjustment set in the arithmetic mode. The relative change in target mRNA expression was determined by the fold-change analysis [5,26,44], in which fold change = 2−[ΔΔC*t*], where ΔΔC*t* = (C*t*,target mRNA − C*t*,GAPDH).

#### *4.7. Western Blotting Analysis*

Western blot analysis was carried out on proteins extracted from total lysate or cytosolic fractions from hippocampal samples. The primary antiserum used included rabbit monoclonal or polyclonal antibodies against PGC-1α(sc-13067, Santa Cruz Biotechnology, Heidelberg, Germany), VEGF (5365-100, BioVision Huissen, Netherlands), VEGFR2 (9698S, Cell signaling, Danvers, MA, USA), PI3 Kinase (4257S, Cell signaling), Phospho-PI3 Kinase (4228S, Cell signaling), AKT (9272, Cell signaling), Phospho-AKT (4060S, Cell signaling), ERK1/2 (9102S, Cell signaling), Phospho-ERK (4370S, Cell signaling), Cleaved caspase-3 (9664, Cell signaling), and β-actin (ab8227, abcam, Cambridge, UK). The primary antibodies were diluted in 5% skimmed milk in TBST. The conditions for the membrane wash, detection of immunoreactive signals, and quantification of signal intensities on the blots were performed as previously described [5,26,44,46]. This was followed by incubation with horseradish peroxidase-conjugated secondary goat anti-rabbit IgG (111-035-045, Jackson ImmunoResearch, West Grove, PA, USA) to detect the first antibodies for PGC-1α, VEGF, VEGFR2, PI3 Kinase, Phospho-PI3 Kinase, AKT, Phospho-Akt, ERK1/2, Phospho-ERK Cleaved caspase-3, and β-actin. Specific antibody–antigen complexes were detected by an enhanced chemiluminescence western HRP substrate (Merck Millipore, Billerica, MA, USA). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was applied to quantify the amount of proteins and expressed as the ratio relative to β-actin protein.

#### *4.8. Double Immunofluorescence Staining and Laser Confocal Microscopy*

Double immunofluorescence staining [5,26,44,46,84] was carried out using a goat polyclonal antiserum against VEGF (Santa Cruz Biotechnology) and a mouse monoclonal antiserum against a specific marker for neurons, neuron-specific nuclear protein (NeuN) (Chemicon, Temecula, CA, USA). A goat anti-mouse IgG conjugated with Alexa Fluor 568 (Molecular Probes, Eugene, OR, USA) and a goat anti-rabbit IgG conjugated with AlexaFluor 488 (Molecular Probes) served as the secondary antisera. The sections of right hippocampal CA3 tissue were inspected under an epifluorescence microscope (Olympus AX-51; Olympus, Kyoto, Japan). Under the epifluorescence microscope, the immunoreactivity for VEGF exhibited green fluorescence, and NeuN manifested red fluorescence.
