Prolonged Neonatal Seizures Exacerbate Hypoxic-Ischemic Brain Damage: Correlation with Cerebral Energy Metabolism and Excitatory Amino Acid Release

BACKGROUND: Perinatal hypoxia-ischemia (HI) is the most common precipitant of seizures in the first 24-48 h of a newborn's life. In a previous study, our laboratory developed a model of prolonged, continuous electrographic seizures in 10-day-old rat pups using kainic acid (KA) as a proconvulsant. Groups of animals included those receiving only KA, or HI for 15 or 30 min, followed by KA infusion. Our results showed that prolonged electrographic seizures following 30 min of HI resulted in a marked exacerbation of brain damage. We have undertaken studies to determine alterations in hippocampal high-energy phosphate reserves and the extracellular release of hippocampal amino acids in an attempt to ascertain the underlying mechanisms responsible for the damage promoted by the combination of HI and KA seizures. METHODS: All studies were performed on 10-day-old rats. Five groups were identified: (1) group I--KA alone, (2) group II--15 min of HI plus KA, (3) group III--15 min of HI alone, (4) group IV--30 min of HI plus KA, and (5) group VI--30 min of HI alone. HI was induced by right common carotid artery ligation and exposure to 8% oxygen/balance nitrogen. Glycolytic intermediates and high-energy phosphates were measured. Prior to treatment, at the end of HI (both 15 and 30 min), prior to KA injection, and at 1 (onset of seizures), 3, 5 (end of seizures), 7, 24 and 48 h, blood samples were taken for glucose, lactate and beta-hydroxybutyrate. At the same time points, animals were sacrificed by decapitation and brains were rapidly frozen for subsequent dissection of the hippocampus and measurement of glucose, lactate, beta-hydroxybutyrate, adenosine triphosphate (ATP) and phosphocreatine (PCr). In separate groups of rats as defined above, microdialysis probes (CMA) were stereotactically implanted into the CA2-3 region of the ipsilateral hippocampus for measurement of extracellular amino acid release. Dialysate was collected prior to any treatment, at the end of HI (15 and 30 min), prior to KA injection, and at 1 (onset of seizures), 3, 5 (end of seizures), 7 and 9 h. Determination of glutamate, serine, glutamine, glycine, taurine, alanine, and GABA was accomplished using high-performance liquid chromatography with EC detection. RESULTS: Blood and hippocampal glucose concentrations in all groups receiving KA were significantly lower than control during seizures (p < 0.05). beta-Hydroxybutyrate values displayed the inverse, in that values were significantly higher (p < 0.01) in all KA groups compared with pretreatment controls during seizure activity. Values returned to control by 2 h following the cessation of seizures. Lactate concentrations in brain and blood mimicked those of beta-hydroxybutyrate. ATP values declined to 0.36 mmol/l in both the 15 and 30 min hypoxia groups compared with 1.85 mmol/l for controls (p < 0.01). During seizures, ATP and PCr values declined significantly below their homologous controls. Following seizures, ATP values only for those animals receiving KA plus HI for 30 min remained below their homologous controls for at least 24 h. Determination of amino acid release revealed elevations of glutamate, glycine, taurine, alanine and GABA above pretreatment control during HI, with a return to normal prior to KA injections. During seizures and for the 4 h of recovery monitored, only glutamate in the combined HI and KA group rose significantly above both the 15 min of HI plus KA and the KA alone group (p < 0.05). CONCLUSION: Under circumstances in which there is a protracted depletion of high-energy phosphate reserves, as occurs with a combination of HI- and KA-induced seizures, excess amounts of glutamate become toxic to the brain. The latter may account for the exacerbation of damage to the newborn hippocampus, and serve as a target for future therapeutic intervention.

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