Abstracts

Rationale: Unregulated neuro-inflammation mediates epileptogenesis. CD40, an immune and inflammatory regulator in systemic disorders, mediates neurite organization during brain development. Previously, our laboratory showed that CD40 deficiency downregulates seizure severity, increases seizure latency, and reduces seizure frequency in an experimental model of acute seizures. Therefore, the goal of this research was to determine the level of CD40 and CD40L expression during development of epilepsy using the pilocarpine induced status epilepticus model. Methods: Status epilepticus (SE) was induced in adult male mice (C57BL/6J, 25 -30 grams, n=17) using Pilocarpine (280 mg/kg, intraperitoneal:ip) an hour after Scopolamine administration (1 mg/kg, ip). Control mice were administered a saline solution (ip) instead of Pilocarpine. Racine stage 3-4 was sustained for ninety minutes and then Midazolam (8 mg/kg, ip) was administered. In addition, in a group of animals, a silicon probe with sixteen microelectrodes (CM16x703) was implanted in the dorsal hippocampus ten days prior to SE induction. Subsequently, simultaneous video and local field potentials (V-LFP) were recorded before, during, and after SE. Spontaneous locomotor activity was analyzed using a video-recoding tracking system; then spontaneous seizures and aberrant behaviors were quantified daily over four weeks. LFP was analyzed for frequency, electrical seizures, and spike units using signal analysis software. Mice were then randomly selected at days 1, 7, 14, and 22 after SE for brain tissue collection as listed;: for each animal, half of the brain was dissected to determine levels of CD40-CD40L, total and phosphorylated p38 as signaling activation of CD40 from hippocampus and cortex using western blotting (WB). While the other half of the brain was processed to conduct cresyl-violet, Fluoro-Jade C staining to determine neuronal damage. Immunohistochemistry (IHC) was performed to determine the localization of CD40, GFAP, and TMEM positive cells, along with analyzing changes in astro- and microgliosis. Data from those procedures was collected and analyzed using a Li-Cor Odyssey CLx for WB and a bright and fluorescence microscope (Olympus BX53) for cell quantification and immunoreactivity analysis using ImageJ. Results: Preliminary results show levels of CD40 and CD40L markedly increase (50% and 40 % respectively) in expression from Day 1 to Day 22 after SE. When compared with control mice, higher levels of CD40 and CD40L expression were noted in the cortex as opposed to hippocampal tissue. This expression was positively correlated with an increase of p38 and phosphorylated-p38, neuronal damage, gliosis, spontaneous seizures, and occurrence of aberrant behavior such as straub tail. Conclusions: These preliminary findings indicate that up-regulation of CD40 and CD40L could mediate epileptogenesis by influencing inflammatory mechanisms that involve and propagate seizure-induced neuronal damage. These observations pave the way to understanding the role of chemokines and cytokines during the development of epilepsy, especially after brain injuries. Funding: Commonwealth Virginia Research Board