Abstracts

To study the time course and nature of biochemical mechanisms accompanying neuronal death in hippocampal subregions following status epileticus (SE) in an animal model.
Self-sustaining limbic SE was induced in freely moving rats by perforant path stimulation as previously described. The SE was terminated at 5 hours with diazepam and the rats allowed to recover. Samples from sham (S: electrode implantation, no stimulation) and Status (SE) rats were analysed blindly in parallel. Rats were sacrificed at 4 (n=5S+5SE), 16 (n=10S+8SE) and 44 (n=11S+8SE) hours after SE. The hippocampi were removed and microdissected into 3 regions corresponding to dentate gyrus, including hilar cells(D+H), CA3 and CA1. Regions from both hippocampi were pooled for homogenization and stored at -80[deg]C for analysis within 4 days. Reduced glutathione (GSH, nmols/mg protein) was measured using HPLC, and activities of aconitase, citrate synthase (CS), [alpha]-ketoglutarate dehydrogenase ([alpha]KD)(nmols/min/mg protein), and mitochondrial respiratory chain (MRC) complexes (Cx)I-IV (expressed as CS ratios) assayed using standard spectrophotometric techniques. Statistical analysis was performed using multifactorial ANOVA and non-parametric tests. Both total hippocampal and individual regional comparisons were made.
Previous studies (Cock, 2002) confirmed that neither the anaesthestic for electrode implantation nor the diazepam used to terminate seizures had any effects on the biochemical parameters studied. At all time points after SE there were significant decreases in GSH in SE hippocampi (pooled regions) compared to Sham: 4h: S 18.0[plusmn]0.75 (SEM) vs SE 15.5[plusmn]0.5, p=0.037; 16h S 13.5[plusmn]0.42 vs SE 11.1[plusmn]0.23, p[lt]0.001; 44h S 15.9[plusmn]0.71 vs 13.3[plusmn]0.71, p=0.017. There was no significant decrease in aconitase activity until 44h: S 34.7[plusmn]0.86 vs 29.5[plusmn]1/09, p=0.001. Other activities were unremarkable. At 4h subregional analysis revealed significant decreases in GSH in D+H (S 19.6[plusmn]1.34 vs SE 16.7[plusmn]0.38, p=0.013) and in CA3 (S 19.12[plusmn]1.02 vs SE 16.41[plusmn]0.48, p=0.018); CxI in CA1 (S 0.15[plusmn]0.01 vs SE 0.10[plusmn]0.02, p=0.009) in [alpha]KDH in DG/H (S 17.2[plusmn]0.82 vs SE 13.0[plusmn]1.72, p=0.032). At 16 hours CxI activity remained significantly reduced in CA1, and also now in CA3 (data not shown). GSH was further reduced, and significant in all subregions, most marked in D+H (S 14.0[plusmn]0.63 vs SE 10.6[plusmn]0.38, p[lt]0.001). At 44 hours aconitase was reduced in all subregions (data not shown), and glutathione was reduced in CA3 and D+H but not CA1.
Previous studies have confirmed ongoing neuronal death up to 44 hours after SE. The results presented are consistent with free radical (FR) damage, known to specifically affect both GSH and CxI activity, and the time course suggests ongoing FR production after SE has terminated. The additional involvement of aconitase and [alpha]KD at some timepoints may suggest peroxynitrite in particular. Aggressive antioxidant therapy in the hours following SE may reduce neuronal damage.
[Supported by: Wellcome Trust Advanced Fellowship to Dr H R Cock]