Abstracts

RATIONALE: Stroke is a major cause of acquired epilepsy in neurons that survive the initial injury. Despite the importance of understanding the relationship between stroke and epilepsy, little is known concerning the pathophysiology of stroke induced epileptogenesis. Strokes have been shown to cause excessive increases in extracellular glutamate, resulting in neuronal cell death. The purpose of this study was to develop an in vitro model of glutamate injury induced epileptogenesis to model the clinical setting of stroke induced epilepsy. We hypothesized that hippocampal neurons surviving a glutamate injury analogous to a stroke would develop spontaneous, recurrent epileptiform discharges in culture.
METHODS: Hippocampal neurons prepared from 2 day postnatal rats were plated on confluent glial beds at a density of 10⁵ cells/ml. At 12 days in vitro, cultures were exposed to 5 [mu]M glutamate for 30 minutes. Whole-cell current clamp recordings were made during glutamate exposure, as well, as 1-8 days after glutamate exposure to monitor the acute and chronic effects of glutamate injury on neuronal physiology. Fura-2 calcium imaging was used to monitor changes in free intracellular calcium during the glutamate injury. In addition, fluorescein diacetate-propidium iodide microfluorometry was utilized to assess neuronal swelling acutely and neuronal death 24 hours after glutamate injury.
RESULTS: Neuronal injury caused by prolonged glutamate exposure was characterized by a reversible neuronal membrane depolarization associated with decreased membrane input resistance, loss of synaptic potentials, neuronal swelling and significant increases in free intracellular calcium. This type of neuronal injury is similar to the reversible injury observed in the penumbra of a stroke. 24 hours after glutamate injury, neurons either underwent delayed neuronal death (29%) or survived and exhibited synchronized, spontaneous, recurrent epileptiform discharges (SREDs) characterized by paroxysmal depolarizing shifts with high frequency spike firing for the life of the culture (8 days post exposure). SREDs were defined as spontaneous epileptiform discharges lasting longer than 20 sec and having a spike frequency greater than 3 Hz. The surviving neurons exhibited synchronized SREDs that were terminated in a reversible manner by treatment with phenobarbital.
CONCLUSIONS: Neurons surviving glutamate injury characterized by reversible neuronal swelling, membrane depolarization, and increased free intracellular calcium similar to neuronal injury produced by ischemia or stroke, expressed spontaneous epileptiform discharges not seen in sham controls. This study demonstrates a novel in vitro model of glutamate injury induced epileptogenesis that may help elucidate the mechanisms that underlie the clinical setting of stroke induced epilepsy.
Support: RO1-NS23350, P50-NS25630 to R.J. DeLorenzo and NSO7288, 2000 Glenn/AFAR to D.A. Sun