Bumetanide Enhances Phenobarbital Efficacy in a Rat Model of Hypoxic Neonatal Seizures

Abstract

Neonatal seizures can be refractory to conventional anticonvulsants, and this may in part be due to a developmental increase in expression of the neuronal Na+-K+-2 Cl− cotransporter, NKCC1, and consequent paradoxical excitatory actions of GABAA receptors in the perinatal period. The most common cause of neonatal seizures is hypoxic encephalopathy, and here we show in an established model of neonatal hypoxia-induced seizures that the NKCC1 inhibitor, bumetanide, in combination with phenobarbital is significantly more effective than phenobarbital alone. A sensitive mass spectrometry assay revealed that bumetanide concentrations in serum and brain were dose-dependent, and the expression of NKCC1 protein transiently increased in cortex and hippocampus after hypoxic seizures. Importantly, the low doses of phenobarbital and bumetanide used in the study did not increase constitutive apoptosis, alone or in combination. Perforated patch clamp recordings from ex vivo hippocampal slices removed following seizures revealed that phenobarbital and bumetanide largely reversed seizure-induced changes in EGABA. Taken together, these data provide preclinical support for clinical trials of bumetanide in human neonates at risk for hypoxic encephalopathy and seizures.

Chloride cotransporter NKCC1 inhibitor bumetanide protects against white matter injury in a rodent model of periventricular leukomalacia

“…Developing neocortical neurons are potentially vulnerable to the direct and indirect effects of depolarizing GABA receptors due to developmental regulation of NKCC1 as well as glutamate receptors, as previously discussed. In termequivalent rats, bumetanide has been shown to augment the neuroprotective efficacy of phenobarbital plus hypothermia following HI-and hypoxia-induced seizures without causing apoptosis (12,19). Taken together with our findings showing protection of white and gray matter following ischemic injury in preterm equivalent rats, these data indicate that NKCC1 is a modifiable molecular target in the developing brain.…”

“…While NKCC2 expression is limited to the kidney, NKCC1 is expressed more widely throughout the brain. Bumetanide effectively crosses the blood-brain barrier (12). Previously, we have shown that NKCC1 facilitates epileptiform discharges in the neonatal rodent brain, and that bumetanide attenuates epileptiform activity and enhances phenobarbital efficacy in a rat model of neonatal hypoxic seizures (10,12).…”

“…Bumetanide effectively crosses the blood-brain barrier (12). Previously, we have shown that NKCC1 facilitates epileptiform discharges in the neonatal rodent brain, and that bumetanide attenuates epileptiform activity and enhances phenobarbital efficacy in a rat model of neonatal hypoxic seizures (10,12). Currently, bumetanide is under study in a phase I/II clinical trial as a single add-on therapy to existing phenobarbital treatment for neonatal seizures in infants 33-44 wks of age at dosages of 0.1-0.3 mg/kg (clinicaltrials.gov, NCT00830531).…”

“…Bumetanide reduces neuronal and astrocytic injury in adult rodent models of ischemia, with protective mechanisms related to maintenance of intracellular ion gradients, reduced edema, and preservation of Ca 2+ homeostasis. Additionally, bumetanide attenuates neonatal seizures in a term-equivalent rodent model of hypoxia, and can augment the neuroprotective effects of phenobarbital and hypothermia in a rat model of term hypoxic-ischemic encephalopathy (10,12,19). Here, we investigated the relationship between NKCC1 expression and OL and neuronal HI injury in our preterm rodent model of PVL.…”

“…NKCC1 expression is developmentally regulated in the human and rodent brain, peaking on neurons and astrocytes in early perinatal development before declining in adulthood (10,11). The developmental regulation of NKCC1 on neurons is thought to contribute in part to the increased excitability in the developing brain and to the pathophysiology of neonatal seizures (10,12). Further, in adult animals, NKCC1 is upregulated on neurons and astrocytes following focal ischemia (13,14), with NKCC1 knockout mice exhibiting significantly reduced gray and white matter injury (15), suggesting…”