tion in neurodegenerative conditions due to its high blood-brain- barrier penetration capacity and cholesterol-lowering effect on neurons [29]. Study from the same group has also suggested that simvastatin is the most effective statin protecting against kainate- induced excitotoxicity and memory impairment [30], which might be related to the role of statins in regulating NMDA receptors [31]. In addition to anti-excitotoxic effect, simvastatin has also shown anti-inflammation effect via reducing expression of interleukin-1 [32–35]. Although studies of the efficacy of statins (atorvastatin and simvastatin) in animal model of TLE have shown an anti-seizure effect, only transient outcome (ranging from 3 days to 2 weeks after SE) of statins treatment were evaluated [30], [35]. The long-term effect of simvastatin treatment on seizure development has not been studied. In this study, we evaluated the long-term effect of a 14-day administration of simvastatin on epileptic rat at 4 to 6 months after KA-induced SE. Results KA-lesion Induced Behavioral Deficits in Rat Model Within 20–30 min after KA injection (i.c.v), all animals developed status epileptic (SE) and convulsive seizures lasted at least for 1–2 h. Seizure behavior was characterized by animal’s rolling toward one side, rotating, and stiffing of limbs and tail. Epileptic rats showed no movement during the interphase of spasms. After a relatively quiescent period (ranging from 10 days to 2 months), 75% rats developed spontaneous seizures that were often evoked by grasping and/or arousing the rats. In the control group, rats with saline injection (i.c.v) did not exhibit any of these abnormal behaviors. Simvastatin Down-Regulated the Expression of IL-1b and TNF-a without Altering Expression of IL-6 in the Injured Hippocampus We first investigated the effect of simvastatin treatment on the expression of cytokines including IL-1b, TNF-a, and IL-6 in the KA-injured hippocampus. Three days after KA-lesion, significant increases in expression of IL-1b (99.34611.10 vs 22.1262.30 pg/ mg at normal level), TNF-a (18.1061.69 vs 4.1260.68 pg/mg), Simvastatin Restrains Exacerbation of Epilepsy and IL-6 (163.11623.45 v.s. 45.1266.93 pg/mg) were observed in hippocampus (Fig. 1). Compared to saline treatment, 3-day administration of simvastatin significantly suppressed KA-induced up-regulation of IL-1b (31.4767.10 pg/mg) and TNF-a (10.136 1.19 pg/mg) in hippocampus (Fig. 1A and B), while it showed no effect on lesion-induced up-regulation of IL-6 (154.13616.97 pg/ mg) (Fig. 1C). GFAP-positive Astrocytes Reduced after Simvastatin Treatment In response to brain injury, astrocytes become reactive and express various inflammatory mediators that play important roles in the secondary injury. KA-lesion induced reactive astrocytes are often identified by increased immunoreactivity of glial fibrillary acidic protein (GFAP). In the normal brain, only a few astrocytes expressed GFAP and GFAP-positive astrocytes showed slim morphology as well as thin and long processes (Fig. 2A1 and A2). Two-week simvastatin treatment attenuated this lesion- induced GFAP immunoreactivity at 4 months in the epileptic rats. GFAP-positive astrocytes also lost fine processes (Fig. 2B1 and B2). In contrast, 4 months after KA-lesion, saline-treated group showed a robust increase in GFAP immunoreactivity and hypertrophic morphology of astrocytes. (Fig. 2C1 and C2). Quantitative data showed that the density of GFAP-positive cells in dentate hilar (DH) area was reduced compared to saline-treated epileptic rats (Fig. 2D). We also evaluated the reactive astrocytosis at 5 and 6 month post KA-lesion. Our data showed that the simvastatin-mediated suppression in reactive astrocytosis lasted until 6 month post-lesion (Fig. 2E). Simvastatin Administration Restrained KA-induced injury in Hippocampal Cytoarchitecture Hippocampal cytoarchitecture was evaluated using Nissl staining in different groups at 4 months after the KA-lesion. In this study, the number of neurons in the CA3 and DH region was counted and compared among each group (Fig. 3). Out results showed that KA-lesion induced dramatic degeneration of neurons in CA3 and DH in ipsilateral hippocampus at 4 months. However, the number of neurons in the simvastatin-treated group was Figure 1. Simvastatin altered the expression level of IL-1b and TNF-a. Bar graphs showed the level of IL-1b (A), TNF-a (B), and IL-6 (C) in the hippocampus 3 days post KA-lesion. KA-injured rats were treated with simvastatin or saline for 3 days. Data were presented as means6standard deviation. *P,0.05 versus control group; #P,0.05 versus the saline group. IL-1b and TNF-a expression was decreased at day 3 after simvastatin treatment compared with that in saline-treated group. However, simvastatin did not suppress the expression of IL-6 compared with the saline-treated group. doi:10.1371/journal.pone.0024966.g001 PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24966