Phenobarbital and midazolam suppress neonatal seizures in a noninvasive rat model of birth asphyxia, whereas bumetanide is ineffective

Objective: Neonatal seizures are the most frequent type of neurological emergency in newborn infants, often being a consequence of prolonged perinatal asphyxia. Phenobarbital is currently the most widely used antiseizure drug for treatment of neonatal seizures, but fails to stop them in similar to 50% of cases. In a neonatal hypoxia-only model based on 11-day-old (P11) rats, the NKCC1 inhibitor bumetanide was reported to potentiate the antiseizure activity of phenobarbital, whereas it was ineffective in a human trial in neonates. The aim of this study was to evaluate the effect of clinically relevant doses of bumetanide as add-on to phenobarbital on neonatal seizures in a noninvasive model of birth asphyxia in P11 rats, designed for better translation to the human term neonate. Methods: Intermittent asphyxia was induced for 30 minutes by exposing the rat pups to three 7 + 3-minute cycles of 9% and 5% O-2 at constant 20% CO2. Drug treatments were administered intraperitoneally either before or immediately after asphyxia. Results: All untreated rat pups had seizures within 10 minutes after termination of asphyxia. Phenobarbital significantly blocked seizures when applied before asphyxia at 30 mg/kg but not 15 mg/kg. Administration of phenobarbital after asphyxia was ineffective, whereas midazolam (0.3 or 1 mg/kg) exerted significant antiseizure effects when administered before or after asphyxia. In general, focal seizures were more resistant to treatment than generalized convulsive seizures. Bumetanide (0.3 mg/kg) alone or in combination with phenobarbital (15 or 30 mg/kg) exerted no significant effect on seizure occurrence. Significance: The data demonstrate that bumetanide does not increase the efficacy of phenobarbital in a model of birth asphyxia, which is consistent with the negative data of the recent human trial. The translational data obtained with the novel rat model of birth asphyxia indicate that it is a useful tool to evaluate novel treatments for neonatal seizures.Peer reviewe

Deletion of the Na-K-2Cl cotransporter NKCC1 results in a more severe epileptic phenotype in the intrahippocampal kainate mouse model of temporal lobe epilepsy

Increased neuronal expression of the Na-K-2Cl cotransporter NKCC1 has been implicated in the generation of seizures and epilepsy. However, conclusions from studies on the NKCC1-specific inhibitor, bumetanide, are equivocal, which is a consequence of the multiple potential cellular targets and poor brain penetration of this drug. Here, we used Nkcc1 knockout (KO) and wildtype (WT) littermate control mice to study the ictogenic and epileptogenic effects of intrahippocampal injection of kainate. Kainate (0.23 ?g in 50 nl) induced limbic status epilepticus (SE) in both KO and WT mice with similar incidence, latency to SE onset, and SE duration, but the number of intermittent generalized convulsive seizures during SE was significantly higher in Nkcc1 KO mice, indicating increased SE severity. Following SE, spontaneous recurrent seizures (SRS) were recorded by continuous (24/7) video/EEG monitoring at 0-1, 4-5, and 12-13 weeks after kainate, using depth electrodes in the ipsilateral hippocampus. Latency to onset of electrographic SRS and the incidence of electrographic SRS were similar in WT and KO mice. However, the frequency of electrographic seizures was lower whereas the frequency of electroclinical seizures was higher in Nkcc1 KO mice, indicating a facilitated progression from electrographic to electroclinical seizures during chronic epilepsy, and a more severe epileptic phenotype, in the absence of NKCC1. The present findings suggest that NKCC1 is dispensable for the induction, progression and manifestation of epilepsy, and they do not support the widely held notion that inhibition of NKCC1 in the brain is a useful strategy for preventing or modifying epilepsy.Peer reviewe

Long-term outcome in a noninvasive rat model of birth asphyxia with neonatal seizures : Cognitive impairment, anxiety, epilepsy, and structural brain alterations

Objective: Birth asphyxia is a major cause of hypoxic-ischemic encephalopathy (HIE) in neonates and often associated with mortality, neonatal seizures, brain damage, and later life motor, cognitive, and behavioral impairments and epilepsy. Preclinical studies on rodent models are needed to develop more effective therapies for preventing HIE and its consequences. Thus far, the most popular rodent models have used either exposure of intact animals to hypoxia-only, or a combination of hypoxia and carotid occlusion, for the induction of neonatal seizures and adverse outcomes. However, such models lack systemic hypercapnia, which is a fundamental constituent of birth asphyxia with major effects on neuronal excitability. Here, we use a recently developed noninvasive rat model of birth asphyxia with subsequent neonatal seizures to study later life adverse outcome. Methods: Intermittent asphyxia was induced for 30 min by exposing male and female postnatal day 11 rat pups to three 7 + 3-min cycles of 9% and 5% O-2 at constant 20% CO2. All pups exhibited convulsive seizures after asphyxia. A set of behavioral tests were performed systematically over 14 months following asphyxia, that is, a large part of the rat's life span. Video-electroencephalographic (EEG) monitoring was used to determine whether asphyxia led to the development of epilepsy. Finally, structural brain alterations were examined. Results: The animals showed impaired spatial learning and memory and increased anxiety when tested at an age of 3-14 months. Video-EEG at similar to 10 months showed an abundance of spontaneous seizures, which was paralleled by neurodegeneration in the hippocampus and thalamus, and by aberrant mossy fiber sprouting. Significance: The present model of birth asphyxia recapitulates several of the later life consequences associated with human HIE. This model thus allows evaluation of the efficacy of novel therapies designed to prevent HIE and seizures following asphyxia, and of how such therapies might alleviate long-term adverse consequences.Peer reviewe