Administration of simvastatin after kainic acid-induced status epilepticus restrains chronic temporal lobe epilepsy

Abstract

In this study, we examined the effect of chronic administration of simvastatin immediately after status epilepticus (SE) on rat brain with temporal lobe epilepsy (TLE). First, we evaluated cytokines expression at 3 days post KA-lesion in hippocampus and found that simvastatin-treatment suppressed lesion-induced expression of interleukin (IL)-1β and tumor necrosis factor-α (TNF-α). Further, we quantified reactive astrocytosis using glial fibrillary acidic protein (GFAP) staining and neuron loss using Nissl staining in hippocampus at 4-6 months after KA-lesion. We found that simvastatin suppressed reactive astrocytosis demonstrated by a significant decrease in GFAP-positive cells, and attenuated loss of pyramidal neurons in CA3 and interneurons in dentate hilar (DH). We next assessed aberrant mossy fiber sprouting (MFS) that is known to contribute to recurrence of spontaneous seizure in epileptic brain. In contrast to the robust MFS observed in saline-treated animals, the extent of MFS was restrained by simvastatin in epileptic rats. Attenuated MFS was related to decreased neuronal loss in CA3 and DH, which is possibly a mechanism underlying decreased hippocampal susceptibility in animal treated with simvastatin. Electronic encephalography (EEG) was recorded during 4 to 6 months after KA-lesion. The frequency of abnormal spikes in rats with simvastatin-treatment decreased significantly compared to the saline group. In summary, simvastatin treatment suppressed cytokines expression and reactive astrocytosis and decreased the frequency of discharges of epileptic brain, which might be due to the inhibition of MFS in DH. Our study suggests that simvastatin administration might be a possible intervention and promising strategy for preventing SE exacerbating to chronic epilepsy.

Figure 1. Simvastatin altered the expression level of IL-1β and TNF-α.

Bar graphs showed the level of IL-1β (A), TNF-α (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 means±standard deviation. *P<0.05 versus control group; #P<0.05 versus the saline group. IL-1β and TNF-α 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.

Figure 2. Immunostaining of GFAP for reactive astrocytes in hippocampus at 4, 5 and 6 months post-lesion.

(A1–A2) In the normal brain, GFAP staining (brown) showed slim astrocyte morphology and HE staining showed normal neuron (blue) number. Higher magnification of insert was presented in A2. (B1–B2) Simvastatin-treated group exhibited astrocytic hypertrophy and moderate reduction in neurons. (C1–C2) Animals with KA-lesion followed by saline administration showed pronounced up-regulation of GFAP expression. GFAP-positive astrocytes exhibited hypertrophy and atrophied processes. The number of neuron was also severely reduced. (D–E) Quantification of GFAP-positive cells in DH area of hippocampus. Scale bar = 400 µm in C1 (applies to A1, B1, C1); scale bar = 100 µm in C2 (applies to A2, B2, C2). (* P<0.05, vs. the control group; # P<0.05, vs. the saline-treated group).

Figure 3. Hippocampal cytoarchitecture visualized with Nissl staining.

A1, A2, A3 and A4 showed hippocampal regions from rats with intracerebroventricular saline injection. B1, B2, B3 and B4 showed regions of hippocampus from KA-injured rat followed by simvastatin treatment. C1, C2, C3 and C4 showed hippocampal regions from KA-injured rat followed by saline-treatment. D showed quantitative data of the number of neurons in DH and CA3. scale bar = 400 µm in A1 (applies to B1, C1, A3, B3, C3); scale bar = 100 µm in A2 (applies to B2, C2, A4, B4, C4 ). (* P<0.05, vs. control group, # P<0.05, vs simvastatin-treated group).

Figure 4. Comparison of KA-lesion induced MFS using Timm's staining in DH.

The extent of aberrant MFS in saline-treated group with severe hippocampal injury (A1and A2), simvastatin-treated group with moderate hippocampal injury (B1 and B2) and in comparison with rats with intracerebroventricular saline injection (C1 and C2), visualized by Timm's histochemical staining. Quantitative data for width and density of sprouting into DSGL were presented in (D and E). GCL, granule cell layer, UB, upper blade; LB, lower blade. Scale bars = 500 µm in A1 and 200 µm in A2. (*P<0.05, vs. the simvastatin-treated group).

Figure 5. Simvastatin-treatment decreased the frequency of abnormal spikes of epileptic brain.

Representative EEG recordings from saline-treated group and simvastatin-treated group at 4 months (A), 5 months (B) and 6 months (C). Quantitative data was presented in (D). *P<0.05, vs. the saline-treated group.

Figure 6. Simvastatin attenuated KA-induced seizure behavior in rats.

The rats were orally treated with simvastatin (1 mg/kg) for 0.5 h after KA injection (1.0 µl of KA. i.c.v). The seizure behavior test (seizure score) was performed during 4–6 months after KA injection (1.0 µl of KA. i.c.v). Simvastatin attenuated the seizure score. Data were presented as means±standard deviation. *P<0.05 versus the saline group.