Abstracts

Rationale: Hypoxic encephalopathy is the most common cause of neonatal seizures. We have established a rodent model that mimics the acute seizures and chronic pro-epileptogenic effects of neonatal hypoxia. We have previously shown enhanced LTP and kindling in hippocampal slices following hypoxic seizures, as well as enhanced suscpetibilty to later life “second-hit” seizures. The long term effects of hypoxia-induced early life seizures can be attenuated by early post-insult treatment(1-48h) with the AMPA receptor (AMPAR) antagonists NBQX and topiramate. We recently showed that there is a significant increase in the phosphorylation of AMPA receptors GluR1-S831, GluR1-S845 and GluR2-S880 and a concomitant increase in PKA, PKC and CamK II activity 12-48 hours following a hypoxic seizure at P10 (Rakhade et al, Epilepsia, Oct 2006, 47 (s4), 27.) We hypothesize that AMPAR antagonists may interrupt the acute and sub-acute effects of hypoxia-induced seizures kinase activity as well as AMPAR phosphorylation, thereby mediating the long-term protective effects observed in the rodent model of early-life seizures.Methods: Rat pups were subjected to hypoxia (15 minutes at 4 7% O2) on postnatal day (P) 10. NBQX (20 mg/kg) and TPM (30 mg/kg) were dissolved in PBS and administered intraperitoneally (i.p.) to the pups, within 30 minutes of global hypoxia-induced seizures, vehicle-treated pups (0.1ml PBS) served as controls. Protein extracts were prepared from hippocampal tissue obtained at 1, 6, 12, 24 and 48 hours post hypoxia. PKA, PKC and CamK II kinase activity was assayed using ELISA assays. Immunoblot analysis was performed using phospho-specific antibodies against serine 831 and 845 epitope of AMPAR subunit GluR1. Results: We observe treatment with the AMPAR antagonists NBQX and topiramate significantly decreased the activity of CamK II (128% and 167% n=4, p<0.05 compared to 331% in vehicle-treated subjects), PKA (178% and 199%,n=4, p<0.05; compared to 317% in vehicle-treated subjects) and PKC (155% and 181% , n=4, p<0.05; compared to 191% in vehicle-treated subjects). Furthermore, we also observe a reversal in the increased phosphorylation of GluR1-S831 (133% and 87%, n=5, p<0.05) and GluR1-S845 (110% and 113 %, n=5, p<0.05) following post-hypoxia administration of AMPAR antagonists NBQX and topiramate respectively.Conclusions: : Here we show that early post-hypoxia treatment with the AMPAR antagonists NBQX and topiramate appear to reverse the rapid post-translational modification of the GluR1 AMPAR subunits. Importantly, we have previously shown that identical post-treatment paradigm protect against the long term increases in seizure susceptibility after hypoxia-induced early life seizures (Koh et al Epilepsia, 2004; 45(6):569-75.) and neuronal injury can be attenuated by early post-insult treatment with the AMPAR antagonists NBQX and topiramate. Taken together, these data imply that the early post-translational changes are dependent upon activation of AMPARs, and may contribute to long term epileptogenesis in this model.