Abstracts

Rationale: It is well established that hypoxic ischemic injury induces cytotoxic edema as neurons swell due to the accumulation of water and sodium and potassium chloride salts.  In vitro data indicate that the chloride accumulation induced by hypoxia-ischemia is progressive in both mature and developing neurons.  This progressive chloride accumulation erodes inhibition by changing the direction of the GABAergic membrane currents that sub-serve synaptic inhibition. Consistent with these processes, seizures become gradually more prevalent over the first 24 hours after human neonatal injury and resistant to GABAergic anticonvulsant such as phenobarbital.  Cation-chloride cotransporters (CCCs) expressed at the blood-brain-barrier and the neuronal membrane facilitate neuronal chloride accumulation after injury. Combining these key results, we hypothesize that early limitation of neuronal chloride uptake preserves GABAergic inhibition and improves the anticonvulsive action of phenobarbital in the neonatal brain.  _x000D_
Methods: High-resolution two-photon fluorescence imaging and non-invasive extracellular field potential recordings were performed in the CA3 and CA1 hippocampal areas in the intact preparation in vitro from neonatal mice expressing the Cl^- sensitive fluorescent protein Clomeleon. Spontaneous neuronal network activity, cell type-specific Cl^- accumulation and GABA action were compared under conditions in which NKCC1 inhibitor bumetanide and GABAergic anticonvulsant phenobarbital were applied immediately after oxygen-glucose deprivation (OGD) vs after seizure-like activity was manifested. 

Results::As previously demonstrated (AES-2021), after a transient period of OGD all intact hippocampi developed spontaneous periodic interictal- and ictal-like epileptiform discharges (PEDs). We report now that: (1) early and sustained bumetanide exposure after OGD significantly delayed the onset and reduced the frequency and power of PEDs; (2) early and sustained application of phenobarbital failed to prevent or abolish OGD-induced recurrent seizures; (3) early and sustained application of bumetanide in combination with phenobarbital more efficiently reduced the rate of neuronal chloride accumulation, preserved the net inhibitory action of GABA and prevented PEDs.    

Conclusions: Our results demonstrate that early block of ongoing neuronal chloride uptake is superior to late block of chloride uptake as measured by control of epileptiform discharges during recovery from OGD.  This has direct implications for the timing of clinical interventions such as bumetanide for neonatal seizures.  Early inhibition of NKCC1 transport by bumetanide enables the anticonvulsive action of phenobarbital in immature brain exposed to OGD.  Our new data validate that early inhibition of neuronal chloride uptake is a uniquely effective anticonvulsant strategy for control of neonatal seizures.

Funding: Research reported in this abstract was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under Award Number R01NS120973.