Bumetanide enhances phenobarbital efficacy in a rat model of hypoxic neonatal seizures.

Ryan T Cleary,Hongyu Sun,Kristopher T Kahle,Frances E Jensen,Delia M Talos,Simon M Manning,Michele Jackson,Alexander Rotenberg,Sanjay N Rakhade,Gerard Berry,Yijun Li,Thanhthao Huynh,Massimo Avoli

doi:10.1371/journal.pone.0057148

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.

Highlights

Neonatal seizures occur most commonly in the setting of perinatal asphyxia and hypoxic-ischemic encephalopathy (HIE), and can be resistant to conventional antiepileptic therapies
Neuronal intracellular Cl2 concentrations are higher than in the adult due to a high Na+-K+-2 Cl2 cotransporter isoform 1 (NKCC1) expression coincident with a low K+-Cl2 cotransporter isoform 2 (KCC2) expression, relative to normal adult expression patterns. [5,17] The expression of NKCC1 mRNA is increased in human forebrain neurons during the perinatal period, relative to later life. [17,18] In humans, this switch is thought to occur in utero after NKCC1 peaks between 31–41 postconceptional weeks, whereas in rats this switch occurs near the end of the second postnatal week, with NKCC1 expression decreasing after postnatal day (P)14
[23] We examined whether seizures acutely altered NKCC1 and KCC2 protein expression in cortex and hippocampus, or GABAA receptor function in ex vivo hippocampal slices following hypoxia-induced seizures (HS) in immature rats

Summary

Introduction

Neonatal seizures occur most commonly in the setting of perinatal asphyxia and hypoxic-ischemic encephalopathy (HIE), and can be resistant to conventional antiepileptic therapies. [5,7,8] In normal adult brain, activation of GABAA receptors results in membrane hyperpolarization due to Cl2 influx through its ion channel, and are inhibitory. [10,11] This switch is thought to be in part due to developmental changes in the expression of two proteins involved in the maintenance of intracellular Cl2 homeostasis in neurons: the Na+-K+-2 Cl2 cotransporter isoform 1 (NKCC1) that transports Cl2 into the cell, and the K+-Cl2 cotransporter isoform 2 (KCC2) that moves Cl2 out of the cell. [5,17] The expression of NKCC1 mRNA is increased in human forebrain neurons during the perinatal period, relative to later life. Neuronal intracellular Cl2 concentrations are higher than in the adult due to a high NKCC1 expression coincident with a low KCC2 expression, relative to normal adult expression patterns. [5,17] The expression of NKCC1 mRNA is increased in human forebrain neurons during the perinatal period, relative to later life. [17,18] In humans, this switch is thought to occur in utero after NKCC1 peaks between 31–41 postconceptional weeks, whereas in rats this switch occurs near the end of the second postnatal week, with NKCC1 expression decreasing after postnatal day (P)14. [17] Other studies have confirmed that the functional correlate of this switch, the appearance of hyperpolarizing GABAA receptors, occurs around P14. [13,14] the caudal to rostral maturation of these transporters [4,17] is thought to contribute to the electroclinical dissociation seen in neonates after treatment with phenobarbital

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Conclusion