Abstracts

Rationale: Traumatic brain injury is a major cause of acquired epilepsy. Phenytoin prevents acute post-traumatic seizures but not post-traumatic epilepsy. We studied the effects of chronic application of phenytoin on epileptogenesis, seizures, and cell death to elucidate the origins of phenytoin resistance and develop new therapeutic strategies. Methods: We used organotypic hippocampal slice cultures as a model of severe traumatic brain injury. Slices were incubated in medium containing phenytoin, the broad-spectrum glutamate antagonist kynurenic acid, the calcineurin antagonist FK-506, or vehicle for up to six weeks. Electrophysiological activity and cell death were monitored via electrode arrays and confocal imaging of propidium iodide uptake, respectively. Sub-cellular localization of NFATc4 was determined with immunohistochemistry as an assay for calcineurin activation. Results: Continuous electrical recordings revealed progressive development of spontaneous epileptiform discharges, including interictal spikes, ictal-like tonic-clonic seizure activity and electrical status epilepticus over the first 3 weeks of culture. We found that seizures accelerated neuronal death, and that cell death was highly correlated with Ca2 entry and activation of calcineurin. Cell death was dramatically reduced by blockade of glutamatergic transmission with kynurenic acid, by eliminating ictal-like discharges and status epilepticus with phenytoin, and by inhibiting calcineurin with FK-506. Removal of kynurenic acid or phenytoin from incubation medium was followed by a sharp increase in seizure activity. We also found that after four weeks, chronic incubation with phenytoin was no longer effective at controlling seizures or preventing cell death. Conclusions: Our experimental data supports clinical findings indicating that chronic anticonvulsant therapy improves seizure control but does not prevent post-traumatic epileptogenesis, and does not prevent drug-resistant epilepsy. We extend the clinical data by showing that despite a lack of effect on epileptogenesis, seizure control reduces neuronal death. We will use this model to explore post traumatic epileptogenesis, mechanisms of medical intractability, and neurodegeneration in intractable epilepsy.