Abstracts

Rationale: Neonatal seizures, with an incidence of 3.5 per 1000 newborns, are one of the common clinical presentations dealt with in the neonatal intensive care unit. Hypoxic-ischemic encephalopathy (HIE) accounts for 50-60% of the reported cases for neonatal seizures, and results in adverse neurodevelopmental morbidity and lethality depending on the severity of the brain injury. HIE-associated neonatal seizures are difficult to treat because of their well-known resistance to 1st-line anti-seizure drugs such as phenobarbital (PB) and benzodiazepines. In our recent study (Kang et al., 2015a), ANA12 (2.5 mg/kg, IP), a small molecule selective TrkB receptor antagonist, significantly rescued PB-resistant seizures at postnatal day 7 (P7) in a CD1 mouse model of neonatal ischemic seizures. ANA12 also rescued the post-ischemic KCC2 downregulation characterized in this model. To investigate the dose-dependent anti-seizure efficacy of ANA12 for ischemic neonatal seizures we used three ANA12+PB treatment groups with graded dosing.Methods: The neonatal ischemic seizure model in CD1 mice develops acute PB-resistant seizures at P7 by unilateral carotid ligation(Kang et al., 2015b). To address the question of whether the rescue of PB-resistance by ANA12 is dose dependent, this study evaluated three increasing doses of ANA-12 (single IP injection doses of 0.5 mg/kg, 2.5 mg/kg, and 5 mg/kg in age-matched littermates for both sexes). The PB dose administered remained the same in all three treatment groups and was 25mg/kg (IP). The main evaluation criteria for the therapeutic benefit of the graded doses of ANA-12 included; 1) quantitative electroencephalographic (EEG) seizure burden and 2) KCC2 protein expression levels 24h after carotid ligation.Results: Increasing doses of the ANA12 at P7 significantly and consistently improved PB-efficacy for PB-resistant neonatal seizures in a dose dependent manner. ANA12+PB reduced seizure burden by 21.8±6.5% at 0.5 mg/kg, 43.5±5.7% at 2.5 mg/kg, and 64.5±5.3% at 5mg/kg dose when given as a single IP injection following onset of ischemia. The seizure burden with the 5mg/kg dose was significantly lower than at 0.5 mg/kg dose (p<0.0001). This reduction in seizure burden was achieved by a significant reduction in the occurrence of ictal events at 5mg/kg dose compared to 0.5 mg/kg dose (p<0.004). Male and female seizure burdens and anti-seizure responses were similar in each dose category. Concomitant western blot data evaluating the levels of KCC2 expression at 24h after ligation showed that the previously documented KCC2 degradation associated with the ischemic insult was rescued at all three doses tested at 24h.Conclusions: Conclusion: A single dose of ANA12 administered after the onset of ischemia was efficacious at rescuing PB-resistant neonatal ischemic seizures in a dose-dependent manner.