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

Department of Neurology, Children's Hospital Boston, Boston, Massachusetts, United States of America.
PLoS ONE (Impact Factor: 3.23). 08/2013; 8(3):e57148. DOI: 10.1371/journal.pone.0057148
Source: PubMed


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.

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    • "To assess the possible involvement of NKCC1-dependent, GABAA receptor-mediated depolarization/excitation in the genesis of the sevoflurane PAHBs, we took the approach of examining the effect on the PAHBs of bumetanide administered before sevoflurane anesthesia. While systemic administration of bumetanide has been used in earlier studies to inhibit neuronal Cl− uptake via NKCC1 and to thereby reduce depolarizing actions of GABA (Edwards et al., 2010; Dzhala et al., 2005), it was found more recently that brain tissue levels of bumetanide remain negligible compared to plasma levels (Brandt et al., 2010; Cleary et al., 2013). Considering the dose, route, and timing of administration of bumetanide (5 μmol/kg body weight, i.p.; 5 min before sevoflurane anesthesia), it is possible that the effects of bumetanide on the PAHBs were not mediated by inhibition of NKCC1 in neocortical neurons. "
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    ABSTRACT: Hyperexcitatory behaviors occurring after sevoflurane anesthesia are of serious clinical concern, but the underlying mechanism is unknown. These behaviors may result from the potentiation by sevoflurane of GABAergic depolarization/excitation in neocortical neurons, cells implicated in the genesis of consciousness and arousal. The current study sought to provide evidence for this hypothesis with rats, the neocortical neurons of which are known to respond to GABA with depolarization/excitation at early stages of development (i.e., until the second postnatal week) and with hyperpolarization/inhibition during adulthood. Employing behavioral tests and electrophysiological recordings in neocortical slice preparations, we found: 1) sevoflurane produced post-anesthetic hyperexcitatory behaviors (PAHBs) in postnatal day (P)1-15 rats, whereas it failed to elicit PAHBs in P16 or older rats, 2) GABAergic PSPs were depolarizing/excitatory in the neocortical neurons of P5 and P10 rats whereas mostly hyperpolarizing/inhibitory in the cells of adult rats, 3) at P14-15, <50% of rats had PAHBs and, in general, the cells of the animals with PAHBs exhibited strongly depolarizing GABAergic PSPs whereas those without PAHBs showed hyperpolarizing or weakly depolarizing GABAergic PSPs, 4) bumetanide (inhibitor of the Cl- importer NKCC) treatment at P5 suppressed PAHBs and depolarizing GABAergic responses, and 5) sevoflurane at 1% (i.e., concentration <1 minimum alveolar concentration) potentiated depolarizing GABAergic PSPs in the neurons of P5 and P10 rats and of P14-15 animals with PAHBs, evoking action potentials in ≥50% of these cells. On the basis of these results, we conclude that sevoflurane may produce PAHBs by potentiating GABAergic depolarization/excitation in neocortical neurons.
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    ABSTRACT: The treatment of neonatal seizures has not changed significantly over the last 50 years despite advances in antiepileptic drug (AED) development for older children and adults. Recently new drugs have emerged some of which address age-specific challenges or mechanisms and will be discussed in this review. The loop diuretic bumetanide blocks the neuronal NKCC1 co-transporter and is thought specifically to supress seizures in the immature brain. Levetiracetam has been used in children and infants with good efficacy, an excellent safety profile, and near-ideal pharmacokinetic characteristics. Randomised controlled trials are now underway to test the efficacy of some newer AEDs for neonatal seizures. Topiramate has been shown to have neuroprotective properties in addition to its antiepileptic action and trials in babies with hypoxic-ischaemic encephalopathy are now planned. There is an urgent need to develop age-specific AEDs for preterm and term babies. These drugs must be evaluated with multicentre, collaborative trials using innovative methods and high ethical standards to overcome age-specific challenges with the ultimate aim of improving the outcome for neonates with seizures.
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