Abstracts

Rationale: Neonatal seizures after hypoxia-ischemia (HI) are common and associated with poor outcomes. However, definitive data indicating whether they are independently harmful or not after HI is a critical gap in the field. Our central hypothesis is that neonatal seizures independently worsen brain injury after HI. To address this question, we developed a novel system to track neuronal survival and seizures after HI in vitro and in vivo using sustained emission of transgenically-expressed fluorescent proteins as a robust biomarker of neuronal viability and genetically encoded calcium indicators to follow seizure activity in real-time for weeks following injury.

Methods: Organotypic hippocampal slices prepared from neonatal mice expressing a neuronal red fluorescent protein and green calcium indicator (GCaMP6s) underwent 20 min of oxygen-glucose deprivation (OGD). Chronic high resolution two-photon (2P) imaging of the pyramidal cell layer was performed. Neuronal death was quantified using the fluorophore quenching assay (Balena et al. in prep). To assess for timing of caspase activation, slices were incubated with Biotracker NucView 405 Blue Caspase-3 Dye (Sigma-Aldrich). For in vivo experiments, post-natal day 1 (P1) wildtype mouse pups underwent AAV intracerebroventricular injection with AAV1 Syn1-mRuby2-GCaMP6s (Addgene). A cranial window was placed on P8. 2P imaging was completed daily from P8 through P14. All analyses were performed in ImageJ, MatLab, and Excel.

Results:_x000D_ 1) OGD causes recurrent acute seizures, induces an excess of acute and delayed neuronal death compared to controls (n=5 pups per group, p < 0.001), and accelerates apoptosis for weeks after injury (Figure 1)._x000D_ _x000D_ 2) Neurons that die late after OGD participate in acute seizure activity and exhibit robust fluorescence emission up to the time of death. _x000D_ _x000D_ 3) Tracking cortical neurons (Figure 2) and measuring neuronal calcium activity over 7 days in vivo in mouse pups is feasible._x000D_