All animal experiments were followed a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the Gyeongsang National University. Male Sprague-Dawley rats (210-230 g, n=48) were purchased from Samtako Co. (Animal Breeding Center, Osan, Korea). Animals were maintained on 12 h light/dark cycle with free access to food and water. Animals were randomly divided into following three groups: sham-operated group, vehicle-treated group, and EGb 761-treated group (n=16 per group). Animals were intraperitoneally administrated EGb 761 (100 mg/kg, Yuyu, Seoul, Korea) in EGb 761-treated group and vehicle in the vehicle-treated group at 1 h before the onset of MCAO [9].

Focal cerebral ischemia was induced by the method of MCAO [14]. Before MCAO operation, animals were anesthetized with sodium pentobarbital (30 mg/kg). a midline incision was made and The right common carotid, external carotid artery and internal carotid arteries were exposed. The external carotid artery was tied and cut. A 4/0 monofilament nylon with its tip rounded by heating over a flame was advanced from the external carotid artery into lumen of the internal carotid artery, thereby occluding blood flow through the middle cerebral artery. At 24 h after the onset of permanent occlusion, brain tissues were removed.

Brains were cut into coronal slices of 2 mm in thickness. These slices were stained for 20 min in a 2% triphenyltetrazolium chloride (TTC; Sigma, St. Louis, MO, USA) and fixed in 10% formalin. The stained slices were photographed by a Nikon CoolPIX990 digital camera (Nikon, Tokyo, Japan) and measured for the ischemic lesion by Image-ProPlus 4.0 software (Media Cybernetics, Silver Spring, MD, USA). The ischemic lesion percentage of each slice was calculated by the ratio of the infarction area to the whole slice area.

The right cerebral cortices were dissolved in lysis buffer (8 M urea, 4% CHAPS, ampholytes, and 40 mM Tris-HCl). After centrifuge at 16,000 g for 20 min at 4℃, the total protein concentration was determined using the Bradford method (Bio-Rad, Hercules, CA, USA) according to the manufacturer's protocol. Briefly, prior to isoelectric focusing (IEF), the immobilized pH gradients (IPG, pH 4-7, pH 6-9, 17 cm, Bio-Rad) gel strips were re-hydrated in buffer (8 M urea, 2% CHAPS, 20 mM DTT, 0.5% IPG buffer, bromophenol blue) for 13 h. The protein samples (100 µg) were loaded on IPG strips. IEF was followed a multi-step protocol: 250 V (15 min), 10000 V (3 h), and then 10000 V to 50000 V using Protean IEF Cell (Bio-Rad). After equilibration of the isoelectric focusing strips, IPG strips were applied onto gradient gels (7.5~17.5%) and electrophoresed at 10 mA for 10 h using Protein-II XI electrophoresis equipment (Bio-Rad) at 10℃. The gels were fixed in a solution (12% acetic acid, 50% methanol) for 2 h, washed twice with 50% ethanol for 20 min, and then sensitized in 0.2% sodium thiosulfate for 1 min. The gels were washed with deionized water, incubated in a silver solution (0.2% silver nitrate, 0.75 mL/L formaldehyde) for 20 min. The gel was developed in a solution (0.2% sodium carbonate, 0.5 mL/L formaldehyde) and the reaction was stopped by adding 1% acetic acid. The silver stained gels were scanned by Agfar ARCUS 1200™ (Agfar-Gevaert, Mortsel, Belgium) and analyzed by a standard protocol for PDQuest software (Bio-Rad). The analyzed data were expressed as mean±S.E.M. Relative intensity of spots in each group was analyzed and compared by Student's t-test. Alteration of protein expression was identified in vehiclel- and EGb 761-treated animals. The gel pieces containing the desired protein spots were manually cut out from the gels and processed for MALDI-TOF.ed and distained. Detained and dehydrated gel pieces were incubated with trypsin-containing digestion buffer for 24 h at 37℃. The peptides were extracted and analyzed in a Voyager-DE™ STR biospectrometry workstation (Applied Biosystem, Forster city, CA, USA) for MALDI-TOF mass spectrometric analysis. the acquired peaks were identified using search programs MS-Fit and ProFound. SWISS-PROT and NCBI were used as the protein sequence databases.

RNA was extracted using Trizol reagent according to the manufacturer's instruction (Invitrogen, Carlsbad, CA, USA). Total RNA (1 µg) was reverse transcribed using superscript III first-strand system for RT-PCR (Invitrogen) based on the manufacturer's protocol. The primers sequences of parvalbumin are 5'-AAGAGTGCGGATGATGTGAAG-3' (forward primer) and 5'-AGCCATCAGCGTCTTTGTTT-3' (reverse primer). The primers sequences of actin are 5'-GGGTCAGAAGGACTCCTACG-3' (forward primer) and 5'-GGTCTCAAACATGATCTGGG-3' (reverse primer). The PCR reaction was carried out as followed: 5 min at 94℃, 30 sec at 94℃, 30 sec at 54℃, 1 min at 72℃ and 10 min at 72℃. The samples were amplified 30 cycles. PCR product was run in a 1% agarose gel and visualized under UV light.

Western blot analysis were performed as previously described [10]. The proteins of right cerebral cortex were extracted by homogenization in buffer [1% Triton X-100, 1 mM EDTA in 1×PBS (pH 7.4)] containing 200 µM phenylmethylsulfonyl fluoride and 10 µM leupeptin. The lysates were centrifuged at 12,000 rpm for 20 min at 4℃. The supernatants were collected and the protein concentration of each lysate was determined using the bicinchoninic acid (BCA) kit (Pierce, Rockford, IL, USA). Total protein (30 µg) was run on to 10% SDS-polyacrylamide gel and subsequently transferred to a poly-vinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and membranes were blocked with 5% skim milk in 0.1% Tween-20 (TBST). After washing membranes with TBST, membranes were incubated with the following antibodies: a rabbit polyclonal anti-rat parvalbumin IgG (diluted 1:1000, Cell Signaling Technology, Beverly, MA, USA) and a mouse monoclonal anti-rat actin IgG (diluted 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membrane was incubated with secondary antibody (1:5000, Pierce, Rockford, IL, USA) and the ECL Western blot analysis system (Amersham Pharmacia Biotech, Piscataway, NJ, USA) was used for detection according to the manufacturer's protocol.

Brains were fixed in 4% paraformaldehyde in 0.1M phosphate-buffered saline (PBS, pH 7.4) and embedded in paraffin. Three sections were selected in each group for the immunohistochemical staining. Sections were blocked with 1% normal goat serum in PBS for 1 h and incubated with a rabbit polyclonal anti-rat parvalbumin IgG (diluted 1:200, Cell Signaling Technology, Beverly, MA, USA) at 4℃ for 15 h. After washing with PBS, sections were incubated with biotin-conjugated goat anti-rabbit IgG (1:200 in PBS) for 1 h, followed by incubation with an avidin-biotin-peroxidase complex for 1 h from a Vector ABC Elite kit (Vector Laboratories Inc., Burlingame, CA, USA), incubated with DAB solution with 0.03% hydrogen peroxidase. Sections were counterstained with hematoxylin, dehydrated in graded alcohol. For the positive cell counting, three fields for cortical region were selected from right cerebral cortex. The total cell number and parvalbumin-positive cell number were obtained in each field. The index of positive cells was described as the percentage of the number of parvalbumin-positive cells to the total number of cells in each field.

All data are expressed as mean±S.E.M. The intensity analysis of Western blot analysis was carried out using SigmaGel 1.0 (Jandel Scientific, San Rafael, CA, USA) and SigmaPlot 4.0 (SPSS Inc., Point Richmond, CA, USA). The results in each group were compared by one-way analysis of variance (ANOVA) followed by Student's t-test. The difference for comparison was considered significant at ^*P<0.05.