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Post\u2010HI seizures characteristically begin hours after injury, by which time substantial accumulation of [Cl<jats:sup>\u2212<\/jats:sup>]<jats:sub>i<\/jats:sub> may have already occurred. In immature neurons, a major pathway for Cl<jats:sup>\u2212<\/jats:sup> influx is the reversible Na<jats:sup>+<\/jats:sup>\u2010K<jats:sup>+<\/jats:sup>\u20102Cl<jats:sup>\u2212<\/jats:sup> cotransporter NKCC1.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Spontaneous neuronal network, neuronal [Cl<jats:sup>\u2212<\/jats:sup>]<jats:sub>i<\/jats:sub>, and GABA activity were determined in hippocampal preparations from neonatal Clomeleon and SuperClomeleon\/DLX\u2010cre mice to test whether blocking NKCC1 earlier after oxygen\u2013glucose deprivation (OGD) injury would more effectively ameliorate the increase in [Cl<jats:sup>\u2212<\/jats:sup>]<jats:sub>i<\/jats:sub>, ictallike epileptiform discharges (ILDs), and the failure of the GABAergic anticonvulsant phenobarbital.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>In vitro, murine intact hippocampi were free of ILDs for 12\u2009h after preparation. Transient OGD resulted in a gradual increase in [Cl<jats:sup>\u2212<\/jats:sup>]<jats:sub>i<\/jats:sub>, depolarizing action of GABA, and facilitation of neuronal network activity. Spontaneous ILDs began 3\u20135\u2009h after injury. Blocking NKCC1 with 2\u201310\u2009\u03bcmol\u00b7L<jats:sup>\u22121<\/jats:sup> bumetanide reduced [Cl<jats:sup>\u2212<\/jats:sup>]<jats:sub>i<\/jats:sub> equally well when applied up to 10\u2009h 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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Significance<\/jats:title><jats:p>The present age\u2010specific group studies demonstrate that after OGD, NKCC1 transport activity significantly contributes to progressive [Cl<jats:sup>\u2212<\/jats:sup>]<jats:sub>i<\/jats:sub> 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<jats:sup>\u2212<\/jats:sup>]<jats:sub>i<\/jats:sub> and depressed epileptiform discharges. However, there was no time dependency to the anti\u2010ictal efficacy of the combination of phenobarbital and bumetanide. These in\u00a0vitro results suggest that after perinatal injury, early pre\u2010emptive treatment with phenobarbital plus bumetanide would be as efficacious as late treatment after seizures are manifest.<\/jats:p><\/jats:sec>","DOI":"10.1111\/epi.18108","type":"journal-article","created":{"date-parts":[[2024,8,30]],"date-time":"2024-08-30T12:59:51Z","timestamp":1725022791000},"page":"3391-3405","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Timing of interventions to control neuronal chloride elevation in a model of neonatal seizures after hippocampal injury"],"prefix":"10.1111","volume":"65","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-2875-4352","authenticated-orcid":false,"given":"Volodymyr I.","family":"Dzhala","sequence":"first","affiliation":[{"name":"Department of Neurology, Massachusetts General Hospital Harvard Medical School  Boston Massachusetts 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