Abstracts

Rationale: C57BL6/J (B6) mouse genetic background is widely used in transgenic mice production. B6 mice are resistant to kainate (KA) induced toxicity and moreover there are reports of inconsistent and inadequate seizure response (inter-animal variability) for the same (single) dose of KA and high mortality rate. Therefore, it has been perceived B6 as inappropriate model for status epilepticus (SE) and epileptogenesis. Our new data from repeated low dose (RLD) of KA, instead of a single high dose (SHD), provide evidence to overcome B6 disadvantages as a model of epileptogenesis. Methods: 45 B6 male mice were used in this study. Five mice each were used for RLD (5mg/kg, i.p; at 30min intervals) and SHD (20mg/kg, single i.p injection) of KA for each timepoint (1wk, 2wk and 4wk) and another five mice (each for RLD and SHD) that were implanted with DSI-transmitters for continuous video-EEG recording for the duration of the experiment. Five remaining mice served as na ve control. All mice were anesthetized with pentobarbitone (100mg/kg) and perfuse-fixed in sodium sulfide + paraformaldehyde solution and processed for immunohistochemistry and Timm staining. The temporal change of spike rate was displayed graphically and Student s t-tests were performed, with a Bonferroni correction for multiple samples, to compare mean spike rates between SHD and RLD groups. Results: RLD reduced inter-animal variation, increased the duration of convulsive motor seizure activity and a significant reduction in the mortality rate was achieved. Telemetric EEG analysis of SE and up to 7days showed consistently higher spike frequency (p=0.01; n=5) and the number of EEG seizure-like episodes lasting for >5sec increased during the first few weeks after SE in RLD group. The pattern of astrogliosis and microgliosis and their distribution in different regions of the hippocampus and the dentate gyrus varied between RLD and SHD group during early and late phase of epileptogenesis. Reactive astrocytes and microglia dominated amongst the dispersed CA3 pyramidal cells and the hilus of the dentate gyrus, and NeuN immunoreactivity demonstrated marginally decreased number of hilar cells in RLD group at first week of post-SE (n=5, p= 0.032). However, the number of ramified microglia and astrocytes were increased in the hilus, perhaps as a compensatory mechanism, suggesting their supportive role for degenerating hilar neurons. Interestingly, the neuroblast marker, doublecortin cells significantly increased and they migrated to the hilus and the dentate granule cell layer in large numbers in RLD group (n=5, p < 0.01). The intense dark brown Timm staining seen at 7d post-SE in the supragranular layer of the dentate gyrus in RLD group was decreased at week 2 and reappeared at 4 weeks post-SE. Such changes were not seen in SHD group or na ve control.Conclusions: The results presented here from integrated behavioral, electrographic, histological and immunohistochemical analyses confirm that RLD method of KA in B6 mice produces a reliable and better mouse model of SE and epileptogenesis, and also significantly reduces the mortality rate.