Abstracts

Rationale: Hypoxic-ischemic encephalopathy (HIE) is one of the most common causes of seizures in full-term infants and is frequently associated with epilepsy and cognitive deficits in later life. Studies in both human neonates and animal models suggest that seizures themselves may independently contribute to brain injury and poor neurological outcome. First-line drugs, such as phenobarbital and diazepam, which augment GABA mediated inhibition, are less efficacious in treating neonatal seizures and may also be associated with side effects such as cognitive deficits. Potassium channels play an important role in controlling excitability in the developing brain due to underdeveloped GABAergic inhibition. Our earlier study demonstrated that flupirtine, a potassium channel opener, was more efficacious than diazepam or phenobarbital for the treatment of chemoconvulsant-induced neonatal seizures in rats. Further, flupirtine is neuroprotective in various degenerative disease models and because of its neuroprotective properties it is currently in clinical trials for the treatment of multiple sclerosis. In the current study we examined the efficacy of flupirtine to inhibit neonatal seizures and provide neuroprotection in an animal model of neonatal HIE. Methods: To induce hypoxia-ischemia (HI), the right carotid artery of 7-day-old rats was ligated under isoflurane anesthesia. Two hours after carotid ligation, pups were exposed to hypoxia (8% oxygen) for two hours. Pups were treated with 50mg/kg flupirtine or vehicle either 15 minutes before carotid ligation or 30 minutes after carotid ligation. Treated rats were scored for behavioral seizure intensity during hypoxia. Rat pups were sacrificed 24 hours after HI induction and brain sections were stained with fluoro-jade B or anti active caspase-3 antibody. Fluoro-jade B selectively stains degenerating neurons and caspase-3 is considered as one of the most specific markers of apoptosis. Results: None of the rat pups treated with 50mg/kg flupirtine 15 minutes prior to carotid ligation (n=4) or 30 minutes after carotid ligation (n=3) developed behavioral seizures during hypoxia exposure, whereas all rat pups treated with vehicle (n=9) developed seizures upon exposure to hypoxia (*P<0.005, two-sided Fisher exact test). Robust cell death was found in multiple brain areas such as CA1, CA3 and dentate gyrus region of the hippocampus 24 hours after HI in vehicle treated rats (n=2). Flupirtine treatment given 15 minutes prior to HI induction (n=3) reduced HI-induced acute total cell death and apoptotic cell death in CA1 and dentate gyrus regions of the hippocampus.Conclusions: These studies suggest that treatment with flupirtine before HI induction effectively blocks neonatal HI induced behavioral seizures and reduces HI-induced cell death. Further studies are required to determine the therapeutic time window in which flupirtine is effective for treatment of HI induced seizures and brain injury, and the effects of flupirtine treatment on long-term neurological outcome.