ventricle at the speed of 0.2 ml /min. The epileptic rats were further used for experiments. Tissue processing Rats were deeply anesthetized with chloral hydrate (40 mg/kg body weight, i.p.). For Timm’s staining rats were perfused with 100–150 ml sodium sulfide followed by post-fixation in 4% paraformaldehyde (PFA) for 10 h and then brains were transferred into cryo-protection solution (30% sucrose). For Nissl staining, HE staining and immunohistochemistry rats were perfused with phosphate-buffered saline (PBS) followed by post-fixation in 4% PFA for 72 h and brains were embedded with paraffin. Nissl staining The surviving neurons in the hippocampus were visualized by Nissl staining [76], [77]. The mounted sections were rehydrated in distilled water, and submerged in 0.5% cresyl violet solution for 10 min until the desired depth of staining was achieved. GFAP staining After deparaffinization, brain sections were incubated in 2% bovine serum albumin (BSA) in PBS at room temperature for 30 min, and subsequently incubated with mouse anti-GFAP antibody (1:160 diluted in PBS; sigma) at 4uC overnight. After washing in PBS, sections were incubated in peroxidase blocking solution for 10 min at room temperature. Sections were then sequentially incubated with biotin-conjugated anti-mouse immunoglobulin G (1:100 dilution; sigma) for 10 minutes, streptavidin-HRP in PBS for 30 min, and DAB solution for 1- 3 min. Sections were washed in PBS between each step and all these incubation were performed at room temperature. Sections were then counterstained with HE, rinsed in distilled water, dehydrated through 95% ethanol for 2 min, 100% ethanol for 263 min and then cleared in xylene for 265 min for coverslip mounting. Timm’s staining Serial coronal sections (8 mm) were collected with a cryocut (Leica VT 1000S). Every fifth section was collected on slides and dried at room temperature overnight. For the visualization of MFS, sections were developed using the following solution: 120 ml of 50% gum arabic, 10 ml of 47% sodium citrate buffer, 10 ml of 51% citrate buffer, 60 ml of 5.78% hydroquinone and 212.25 mg silver nitrate. The physical development was performed in the dark at room temperature for 120 min. The sections were rinsed in tap water, then dehydrated, cleared, and coverslipped in Entellan (107961.0100; Merck). Evaluation of Timm’s staining was performed according to the method introduced by Shetty and Turner [78]. Both the average width and the density of the MFS in the DSGL were measured in all regions of the DG (The upper and lower dentate blades and the crest). The scores were recorded for the DG in coronal sections from the ipsilateral dorsal hippocam- pus by an observer who was blind to the groups. ELISA ELISA kit (Promega Corp. Madison, WI) was used to deter- mine the protein concentration and the procedure was performed according to the instructions provided by the company. Lysis buffer was added to the tissue sample and the protein was obtained by centrifuging the solution at 14000 rpm for 12 min at 4uC. The expression of IL-1b, TNF-a, and IL-6 at 3 days after SE were measured using equal amounts of lysate from hippocampus tissues. Simvastatin Restrains Exacerbation of Epilepsy Electrode implantation At 4 months after KA-lesion, the rats were again anesthetized with chloral hydrate (40 mg/kg body weight, i.p.) and fixed into stereotaxic apparatus (WPI, Stoelting, USA). A bipolar electrode, consisting of two twisted strands of stainless steel wire, was stereotactically implanted into the right hippocampus using the following coordinates: AP = 25.6 mm, L = 4.5 mm and V = 2.6 mm. A single stainless steel wire was inserted into the left frontal cortex acting as earth electrode. The electrodes were attached to male connector pins, which were inserted into a connector strip. Stainless steel screws were threaded into the right and left parietal cranium to help fix the electrode. The electrode assembly was then fixed to the skull with acrylic resin before the scalp was sutured. EEG examination and analysis Starting at 3 days after electrode implantation, all rats underwent 3 EEG examination sessions per week with 4 h of continuous monitoring per session. Recordings were performed in the afternoon to minimize potential circadian variations, following the habituation of the rats to the test cage. For spike evaluation, the EEG from the hippocampus of rats consisted of waves with a frequency ranging from 0 to 75 Hz. The data were collected to evaluate average spikes at 4 to 6 months post KA-lesion. Image analysis Imaging analysis was performed according to method describe previously [79]. A series of every fifth section of each animal were used for analysis. For neuron counting, in each section, the contour of CA3 pyramidal cell layers, and DH was first delineated with the tracing function of StereoInvestigator(Microbrightfield Inc.,Williston, VT). After this, the optical fractionator component was activated, and the number and location of the counting frames and the counting depth for that section were determined by entering parameters such as the grid size (2006200 mm for CA3 cell layers, and 60660 mm for the DG), the thickness of the guard zone (4 mm), and the optical dissector height (8 mm). A computer- driven motorized stage hen allowed the section to be analyzed at each of the counting frame locations. All nissl-positive cells that were present in the 8 mm section depths were counted in each of the chosen serial sections. The StereoInvestigator program calculated, based on the above-described parameters and cell counts, the total number nissl-positive cells in each of the above- mentioned hippocampal regions. For GFAP counting, similar method was used and the total number of GFAP-positive astrocytes was quantified in DH and CA3 area. Statistical analysis All data were presented as mean6SEM. One-way analysis of variance (ANOVA) with Newman-Keuls multiple comparisons test was employed for comparison. The operations were performed using StatView software (version5.0, SASinstitute Inc., Cary, NC, USA). Statistical significance was set at P,0.05. Acknowledgments We are grateful for Ms. Qi Huang (Harbin Medical University) for her assistance with experiments. Author Contributions Conceived and designed the experiments: ZL CX JS. Performed the experiments: CX JS WQ YS. Analyzed the data: MN XH. Wrote the paper: CX JS. Contributed to writing ‘‘Materials and Methods’’: DL LW. PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e24966