Abstracts

Rationale: Current antiepileptic drugs suppress symptomatic seizures, but lack a preventive effect on epileptogenesis. Understanding mechanisms of epileptogenesis is essential to develop novel drugs that could prevent or modify the disease. Emerging data from human and rodent studies have shown that acute and/or chronic activation of cytokine-related pathways such as IL-1β, TNFα, IL-6 and HMGB1 contribute to seizure mechanisms and may play a role in the epileptogenic process evolving from an inciting event. In this study, we aimed to evaluate the role of innate inflammation in an in vitro model of post-traumatic epileptogenesis. We investigated cytokine release and development of epileptiform activity to understand the impact of modulating inflammatory pathways on the epileptogenic process.Methods: Hippocampi were dissected out from the brain of 6-9 day-old Sprague-Dawley rats and cut into slices. Organotypic hippocampal slice cultures (OHSCs) were prepared on multi-electrode arrays (MEAs) and cultured for 7-21 days. Released cytokines (IL-1β, IL-6 and TNFα) and lactate dehydrogenase (LDH) were measured from culture supernatants. Field potential recordings, performed for 1 hour at different days in vitro (DIVs), allowed the development of epileptiform activities throughout the hippocampal areas to be analyzed. Immunohistochemical staining was performed in OHSCs at 14 DIV to examine glial cell activation and IL-1β expression.Results: Field potential recordings from OHSCs (n=69) on the MEA electrodes revealed that epileptiform activity spontaneously develops in this slice preparation recorded over 40 minutes. The epileptiform activity was composed of ictal-like and interictal-like discharges that were well-synchronized in DG, CA3 and CA1 hippocampal sub-regions. The ictal-like activity was already observed at 3 DIV and developed progressively with the age of the culture. OHSCs at 3 DIV displayed 1.8±0.2 ictal events that lasted for 1.7±0.2 min (n=13), whereas cultures at 21 DIV displayed 18.3±2.0 ictal events that lasted for 31.7±2.4 min (n=9). Cytokine levels peaked within first 3 days concomitantly with cell damage, as reflected by increased LDH. After 3 DIV, the cytokine levels declined progressively until another increase was observed after 14 DIV. We also found activation of MAC-1 positive microglia and GFAP-positive astrocytes as well as expression of IL-1β in all hippocampal sub-regions at 14 DIV.Conclusions: These results indicate that epileptic activity rapidly and progressively develops in the OHSCs model. Pro-inflammatory cytokines and LDH levels were significantly increased by 3 DIV. Additional experiments are being performed to investigate whether modulating selected inflammatory pathways affects the development of epileptiform activity as well as on the cytokine release in this model. UCB Pharma-sponsored