Abstracts

Rationale: Acquired epilepsy (AE) is a common disorder that develops after a neurological insult such as status epilepticus (SE). AE develops in three stages: neuronal injury, epileptogenesis or latency, and chronic epileptic phase. Understanding the alterations that occur during the generation of AE can lead to the development of new therapeutic targets to prevent long-term sequelae. We have demonstrated one such pathophysiologic change in the rat pilocarpine model of SE-induced AE. During SE, intracellular Ca2+ ([Ca2+]i) in hippocampal neurons rises 8-fold when compared to control neurons. A Ca2+ plateau is generated wherein [Ca2+]i remains elevated for several days following SE and leads to changes in gene expression and plasticity leading to the development of epilepsy. While [Ca2+]i decreases slightly during the chronic phase, animals that later display epilepsy exhibit elevated [Ca2+]i for at least a year post-injury unlike control animals or those animals that fail to develop epilepsy. The purpose of this study was to examine whether the Ca2+ plateau observed in vivo also occurred in vitro and if so, to screen Ca2+-lowering agents for the ability to restore baseline Ca2+ when administered post-injury. Methods: Cultured hippocampal neurons were treated with a low-Mg2+ solution for 3 h during which time electrophysiologic activity (in vitro SE) consistent with clinical SE was observed. Following the return of the cells to Mg2+ containing media, spontaneous, recurrent epileptiform discharges (SREDs) were exhibited for the life of the neurons in culture. Ca2+ imaging was performed using the fluorescent indicator Fura-2AM. Results: During in vitro SE, cultured hippocampal neurons showed significantly elevated [Ca2+]i. Following the cessation of in vitro SE, the neurons exhibited SREDs and demonstrated a Ca2+ plateau consistent with that observed in vivo. [Ca2+]i remained significantly elevated for up to 6 h post-SE and then decreased gradually over time, but still remained significantly elevated in comparison to controls for the life of the cultures. After 6 h, [Ca2+]i stabilized, but at a concentration still greater than that recorded from control neurons. The use of the NMDA antagonist MK-801 (10uM) during SE did not reduce the electrographic severity of SE but prevented the Ca2+ plateau and the development of SREDs. We were able to lower [Ca2+]i to baseline levels by treating the cultures post-SE with inhibitors of intracellular Ca2+ release, including dantrolene (50uM) and levetiracetam (100uM). Conclusions: In the in vitro model of SE-induced AE, [Ca2+]i followed a similar trend to that observed in the in vivo pilocarpine model. Blocking NMDA receptors prior to SE prevented the generation of the Ca2+ plateau, whereas, inhibitors of intracellular Ca2+ release were able to block the plateau when administered post-SE. The ability of the in vitro model to mimic the Ca2+ trends recorded in vivo makes this model useful in the screening of pharmacologic agents to inhibit the Ca2+ plateau after injury, and in doing so prevent epileptogenesis.