Abstracts

Rationale: Neonatal seizures that exhibit refractoriness to 1st line AED’s like Phenobarbital (PB) are a significant clinical issue without clear evidence based management protocols. This study investigated PB efficacy in a post-natal day 7 CD-1 mouse model, with neonatal seizures induced by chemoconvulsant, pentylenetetrazole (PTZ). Bumetanide (BTN) a Na-K-Cl cotransporter 1 (NKCC1) antagonist has been previously proposed as a novel anti-seizure adjunct therapy for refractory neonatal seizures. We therefore also investigated anti-seizure efficacy of BTN as an adjunct follow-on treatment to PB, in the same model. TrkB pathway mediated chloride co-transporter, KCC2, degradation has been shown to underlie emergence of PB refractoriness in a model of ischemia induced refractory neonatal seizures. We investigated expression levels of KCC2 and the TrkB pathway in the new PTZ model of neonatal seizures. Methods: Neonatal seizures in P7 CD-1 pups were induced with PTZ (80 mg/kg; IP) and EEG seizure burdens were quantitated using quantitative video-electroencephalograms (qEEG) over a 3h period following PTZ injection. To test the efficacy of a loading dose of PB (25 mg/kg; IP) followed by BTN (0.1-0.2 mg/kg; IP), as an adjunct therapy, pups in each litter were randomly assigned to four treatment groups: 1.) Control (no drug), 2.) PTZ+saline+saline, 3.) PTZ+PB+saline, 4.) PTZ+PB+BTN. Western blot analysis quantitated expression of KCC2, pKCC2, TrkB, pTrkB, PLCγ, and pPLCγ. Results: A single dose of PTZ in P7 CD-1 pups induced seizure burdens that were typical for chemoconvulsant-induced status epilepticus (mean sz burden = 2152 ± 69 seconds/ hour). Over the 3h period of EEG recording, seizure burdens for each hour remained stable and were not significantly different (1st h v. 2nd h p value = 0.151, 2nd h vs. 3rd h p value = 0.299). PB was an effective anti-seizure agent when given at one hour (P < 0.001). However, BTN given at the end of 2nd hour failed to act as an anti-seizure adjunct to PB. Contradictory to its proposed anti-seizure role for neonatal seizures, BTN aggravated PB-suppressed seizures in the 3rd hour (p < 0.01). These findings were associated with an upregulation of KCC2 with no associated TrkB pathway activation at 24h in the same pups. This was in significant contrast to ischemic seizures at P7 in CD-1 pups where a downregulation of KCC2, activation of the TrkB pathway and emergence of PB-resistance have been reported. Conclusions: Although seizure burdens between the ischemia and PTZ neonatal seizures in CD1 P7 pups were comparable, significant model dependent differences in PB, BTN efficacies were noted with the PTZ seizures. Therefore the mechanism by which you induce seizures may not only dictate the efficacy of GABA agonists like PB following neonatal seizures but also of any novel agent being tested as adjunct therapy. Funding: 1 R01 HD090884 (SDK)