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. Modulating NKCC1 function in immature brains undergoing excitotoxic injury has become a major area of investigation for treating neonatal seizures. However, KCC2 modulation as an alternative target for the development of novel therapeutics for HIE-associated neonatal seizures remains unexplored. In this study, using a previously established ischemic mouse model, we investigated a novel therapeutic strategy of enhancing KCC2 to rescue the occurrence of PB-resistant neonatal seizures. Methods: The ischemic seizure model in CD1 mice develops acute PB-resistant seizures at postnatal day 7 (P7) that are PB-responsive at P10. Using this model, this study addressed the question of whether the PB-resistance can be reversed by the presence of a KCC2 modulator, ANA-12. The therapeutic potential of an acute dosing with ANA-12 (IP; 2.5mg/kg), a small molecule selective TrkB receptor antagonist was investigated at P7 and was compared to P10. The main evaluation criteria for ANA-12's therapeutic benefit included: 1) quantitative electroencephalographic (EEG) seizure burden and 2) KCC2 and NKCC1 protein expression levels with and without its treatment after an acute ischemic-insult. Results: ANA-12 treatment significantly rescued PB-resistant seizures at P7. Additionally, ANA-12 significantly enhanced the PB efficacy at P10. Western blot results showed that ANA-12 + PB treatment significantly prevented the acute post-ischemic KCC2 downregulation documented in the model. NKCC1 expression was not affected either by ischemia or ANA-12 treatment. At both ages investigated, bumetanide not only failed to act as an efficacious adjunct therapy, but resulted in a significant aggravation of the effectively subdued post-PB seizures. Conclusions: This study shows that KCC2 modulation helps reverse the occurrence of PB-resistant seizures recorded in P7 mice that may prove to be a powerful alternative and unexplored strategy in treating this fragile patient population.