Timing of interventions to control neuronal chloride elevation in a model of neonatal seizures after hippocampal injury

Objective: Following hypoxic-ischemic (HI) brain injury, neuronal cytoplasmic chloride concentration ([Cl^-]_i) increases, potentially contributing to depolarizing γ-aminobutyric acid (GABA) responses, onset of seizures, and the failure of antiepileptic drugs that target inhibitory chloride-permeable GABA_A receptors. Post-HI seizures characteristically begin hours after injury, by which time substantial accumulation of [Cl^-]_i may have already occurred. In immature neurons, a major pathway for Cl^- influx is the reversible Na⁺-K⁺-2Cl^- cotransporter NKCC1.

Methods: Spontaneous neuronal network, neuronal [Cl^-]_i, and GABA activity were determined in hippocampal preparations from neonatal Clomeleon and SuperClomeleon/DLX-cre mice to test whether blocking NKCC1 earlier after oxygen-glucose deprivation (OGD) injury would more effectively ameliorate the increase in [Cl^-]_i, ictallike epileptiform discharges (ILDs), and the failure of the GABAergic anticonvulsant phenobarbital.

Results: In vitro, murine intact hippocampi were free of ILDs for 12 h after preparation. Transient OGD resulted in a gradual increase in [Cl^-]_i, depolarizing action of GABA, and facilitation of neuronal network activity. Spontaneous ILDs began 3-5 h after injury. Blocking NKCC1 with 2-10 μmol·L^-1 bumetanide reduced [Cl^-]_i equally well when applied up to 10 h after injury. Whereas phenobarbital or bumetanide applied separately were less effective when applied later after injury, ILDs were successfully suppressed by the combination of phenobarbital and bumetanide regardless of the number of prior ILDs or delay in application.

Significance: The present age-specific group studies demonstrate that after OGD, NKCC1 transport activity significantly contributes to progressive [Cl^-]_i accumulation, depolarizing action of GABA, and delayed onset of ILDs. In this neonatal model of neuronal injury and ILDs, earlier treatment with bumetanide alone more efficiently recovered control baseline [Cl^-]_i and depressed epileptiform discharges. However, there was no time dependency to the anti-ictal efficacy of the combination of phenobarbital and bumetanide. These in vitro results suggest that after perinatal injury, early pre-emptive treatment with phenobarbital plus bumetanide would be as efficacious as late treatment after seizures are manifest.