Abstracts

Rationale: The incidence of seizure is highest in the neonate, and hypoxia is the most common cause of neonatal seizures. Neonatal seizures associated with hypoxia increase the risk of developing epilepsy, but the mechanisms by which such epileptogenesis occurs are not well understood. Therefore, there are no therapeutic strategies to prevent epilepsy following neonatal seizure-inducing hypoxia. Recent studies indicate that neuroinflammatory processes may play a critical role in epileptogenesis consequent to experimental seizures through alterations of neuroanatomical circuits and possibly direct modulation of synaptic transmission. Additionally, neuroinflammatory responses may be developmentally regulated and differ substantially between the neonatal and adult brains. Methods: To examine the role of neuroinflammation in neonatal hypoxic seizures (HS) we first aimed to characterize the cytokine response in the hippocampus of rats after HS. In P10 Long-Evans rat pups, HS were induced by exposure to 5-7% oxygen for 14-16min. Control littermate animals were handled identically but exposed to room air for the same period. Animals were sacrificed at 2, 6, 24, and 72hrs or 7 days after hypoxia treatment, the brains removed, and the hippocampi rapidly dissected out and frozen in isopentane on dry ice for later homogenization. Hippocampi were homogenized, centrifuged, and the supernatant collected for multi-plexing immunoassays using Milliplex™ map cytokine panels and a BioRad Bioplex-200 system. This allowed us simultaneously to measure concentrations of the cytokines IL-1β, IL-2, IL-9, IL-10, and TNFα. Results: There were no significant changes at 2 and 6hrs after HS. At 24hrs after HS we observed significantly increased expression of the pro-inflammatory cytokine IL-1β, and modulatory cytokines IL-2 and IL-9, whereas TNFα and IL-10 were unchanged compared to controls. At 72hrs after HS there were significant increases in all cytokines measured. Interestingly, only IL-1β remained significantly increased at seven days after HS, thus persisting days after the apparent cessation of spontaneous seizures. Preliminary GFAP immunohistochemistry experiments at 72 hours after HS also indicated marked astrocytic hypertrophy characteristic of neuroinflammation in hippocampal area CA1 with an accompanying constriction of the pyramidal cell layer. Conclusions: These results show that there is a dynamic neuroinflammatory response in the hippocampus after neonatal HS. This is evident by alterations in the pattern and amount of cytokines present at specific times accompanied by changes in structural anatomy and astrocytic morphology. This study provides a basis to examine further the specific roles of each of these cytokines in neuroanatomical and functional perturbations to hippocampal development and whether these may offer accessible therapeutic targets to prevent epileptogenesis after neonatal HS.