Acute and Chronic Efficacy of Bumetanide in an in vitro Model of Posttraumatic Epileptogenesis

Abstract

Background Seizures triggered by acute injuries to the developing brain respond poorly to first-line medications that target the inhibitory chloride-permeable GABAA-receptor. Neuronal injury is associated with profound increases in cytoplasmic chloride ([Cl−]i) resulting in depolarizing GABA signaling, higher seizure propensity and limited efficacy of GABAergic anticonvulsants. The Na+-K+-2Cl− (NKCC1) co-transporter blocker bumetanide reduces [Cl−]i and causes more negative GABA equilibrium potential in injured neurons. We therefore tested both the acute and chronic efficacy of bumetanide on early post-traumatic ictal-like epileptiform discharges and epileptogenesis. Methods Acute hippocampal slices were used as a model of severe traumatic brain injury and post-traumatic epileptogenesis. Hippocampal slices were then incubated for three weeks. After a one week latent period slice cultures developed chronic spontaneous ictal-like discharges. The anticonvulsant and antiepileptogenic efficacy of bumetanide, phenobarbital and the combination of these drugs was studied. Results Bumetanide reduced the frequency and power of early post-traumatic ictal-like discharges in vitro and enhanced the anticonvulsant efficacy of phenobarbital. Continuous two-three week administration of bumetanide as well as phenobarbital in combination with bumetanide failed to prevent post-traumatic ictal-like discharges and epileptogenesis. Conclusions Our data demonstrate a persistent contribution of NKCC1 co-transport in post-traumatic ictal-like activity, presumably as a consequence of chronic alterations in neuronal chloride homeostasis and GABA-mediated inhibition. New strategies for more effective reduction in post-traumatic and seizure-induced [Cl−]i accumulation could provide the basis for effective treatments for post-traumatic epileptogenesis and the resultant seizures.

Transient ischemia facilitates neuronal chloride accumulation and severity of seizures

“…D). These effects reveal a significant anticonvulsant effect of phenobarbital in chronic ictal‐like discharges …”

Chloride Dysregulation, Seizures, and Cerebral Edema: A Relationship with Therapeutic Potential

“…This increase in [Cl − ] i should be reduced by CCC inhibitors and there is evidence supporting this idea. For example, the addition of bumetanide (an NKCC1 blocker and FDA-approved diuretic) to phenobarbital reduces seizure activity in the hippocampus and the neocortex in vivo and in vitro [184,44,185,186]. Importantly, inhibiting the NKCC1 transporter decreases the neuron’s [Cl − ] i if they have a high initial [Cl − ] i [25], thus making GABA A Rs activation more inhibitory in this group of neurons.…”

Diazepam effect during early neonatal development correlates with neuronal Cl−

“…Hippocampal organotypic slices develop spontaneous seizures after a short silent period . They can be used to study anticonvulsive medications without applying convulsant conditions such as altered Mg 2+ 41,42 and K + concentration, blocking K + channels with 4‐aminopyridine, or activating kainate receptors .…”