Abstracts

Rationale: Neonatal seizures after hypoxia-ischemia (HI) are common in newborns and associated with significant mortality, chronic epilepsy, and other 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 system to track neuronal survival and seizures after oxygen-glucose deprivation (OGD). We used sustained emission of transgenically-expressed fluorescent proteins as a robust biomarker of neuronal viability (Arrasate et al. Nature 2004, PMID: 15483602) and genetically encoded calcium indicators to follow seizure activity in real-time for 2-5 weeks following OGD.

Methods: Organotypic hippocampal slices prepared from neonatal mice expressing a neuronal red fluorescent protein and green calcium indicator (GCaMP6s) underwent 20 min of OGD. Chronic high resolution two-photon 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). All analyses were performed in ImageJ.

Results:
1) After OGD, calcium imaging demonstrated recurrent seizures (duration 23±1 sec (mean±SE))(Figure 1).
2) Neurons dying after OGD could be separated into two cohorts based on time course: immediate (< 24 hours) and later death (1 – 14 days).
3) OGD induced an excess of acute and chronic neuronal death compared to controls (Figure 2).
4) Neurons that died late after OGD participated in seizure activity and did not demonstrate caspase activation acutely post-injury.
5) Neurons that died late after OGD had similar ictal calcium activity compared to neurons that survived.

Conclusions: This is the first study to longitudinally measure neuronal death and seizures in real-time after perinatal acute injury. We found that neurons died with time courses consistent with both early necrotic and later apoptotic deaths. Neurons that eventually underwent apoptosis participated in seizure activity after OGD, and their participation was largely indistinguishable from the participation of neurons that survived. We will next perform in vivo studies with suppression of seizure activity in a subpopulation of neurons to test whether neuronal survival after injury improves. In the future, these novel methods will permit rapid, quantitative measurements with cellular resolution of the neuroprotective utility versus neurotoxicity of new treatment interventions that can be applied to a wide array of epilepsy and brain injury models.

Funding: Please list any funding that was received in support of this abstract.: NIH/NINDS funding for M.A.M (5R25NS065743, 1K08NS121599-01).