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 ~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% O2 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.

Objective Birth asphyxia (BA) is often associated with seizures that may exacerbate the ensuing hypoxic–ischemic encephalopathy. In rodent models of BA, exposure to hypoxia is used to evoke seizures, that commence already during the insult. This is in stark contrast to clinical BA, in which seizures are typically seen upon recovery. Here, we introduce a term‐equivalent rat model of BA, in which seizures are triggered after exposure to asphyxia. Methods Postnatal day 11–12 male rat pups were exposed to steady asphyxia (15 min; air containing 5% O2 + 20% CO2) or to intermittent asphyxia (30 min; three 5 + 5‐min cycles of 9% and 5% O2 at 20% CO2). Cortical activity and electrographic seizures were recorded in freely behaving animals. Simultaneous electrode measurements of intracortical pH, Po2, and local field potentials (LFPs) were made under urethane anesthesia. Results Both protocols decreased blood pH to <7.0 and brain pH from 7.3 to 6.7 and led to a fall in base excess by 20 mmol·L–1. Electrographic seizures with convulsions spanning the entire Racine scale were triggered after intermittent but not steady asphyxia. In the presence of 20% CO2, brain Po2 was only transiently affected by 9% ambient O2 but fell below detection level during the steps to 5% O2, and LFP activity was nearly abolished. Post‐asphyxia seizures were strongly suppressed when brain pH recovery was slowed down by 5% CO2. Significance The rate of brain pH recovery has a strong influence on post‐asphyxia seizure propensity. The recurring hypoxic episodes during intermittent asphyxia promote neuronal excitability, which leads to seizures only after the suppressing effect of the hypercapnic acidosis is relieved. The present rodent model of BA is to our best knowledge the first one in which, consistent with clinical BA, behavioral and electrographic seizures are triggered after and not during the BA‐mimicking insult.

Birth asphyxia (BA) is a pathologic condition that arises from severe perinatal hypoxia and hypercapnia.Recovery following BA is often associated with seizures which may exacerbate the ensuing hypoxicischemic encephalopathy (HIE). Drugs used to treat post-BA seizures are often ineffective and there are concerns over their safety. Therefore, novel seizure-suppressing therapies are urgently needed. Most rodent models of BA-induced seizures are based on exposing neonatal rats or mice to hypoxia (or hypoxiaischemia), and overlook the fact that the hypercapnic acidosis linked to asphyxia has brain-sparing effects by suppressing neuronal excitability and enhancing cerebral blood flow. Thus, the aim of the present study was to investigate the dependence of asphyxia-induced seizures on brain pH and oxygen (Po 2 ) levels in a rodent model of term BA based on postnatal day 11-12 rat pups. Cortical activity and electrographic seizures were recorded in freely-behaving animals using epidural electrodes. Simultaneous measurements of cortical local field potentials as well as intracortical pH and Po 2 were made using microelectrodes and microsensors in urethane-anesthesized animals. The pups were exposed either to steady asphyxia (duration 15 min; with ambient air containing 5 % O 2 plus 20 % CO 2 ) or to intermittent asphyxia (30 min; with repetitive 5 min steps from 9 % to 5 % O 2 at constant 20 % CO 2 ). Both protocols led to acidemia (blood pH <7.0) coupled to a fall in base excess by 20 mmol/l, and to a large increase in plasma copeptin (from 0.2 nM to about 5 nM), a biomarker of BA. Brain pH decreased from 7.3 to 6.7 by the end of intermittent asphyxia.Brain Po 2 was only transiently affected by 9% ambient O 2 , but it fell below the level of detection with steps to 5 % O 2 , during which neuronal activity was near-abolished. The Po 2 steps to 9% were associated with a moderate increase in pH (0.12 units) and a slight recovery (~10 % of baseline) in ongoing neuronal activity.Behavioral seizures spanning the entire Racine scale were triggered after intermittent but not steady asphyxia, and they were tightly associated with neocortical electrographic seizures. The seizures were strongly suppressed when the post-asphyxia brain pH recovery was slowed down by a low level (5 %) of ambient CO 2 . The post-asphyxia overshoot in brain Po 2 (from 30 to 85 mmHg) had no discernible effect on neuronal activity. Our data suggest that the recurring hypoxic episodes during intermittent asphyxia promote neuronal excitability, which becomes established as hyperexcitability and seizures once the suppressing effect of the hypercapnic acidosis is relieved. The present rodent model of BA is to our best knowledge the first one where, consistent with the clinical picture of BA, robust behavioral and electrographic seizures are triggered after and not during the asphyxic insult. HIGHLIGHTS Experimental asphyxia induced severe acidemia and abolished most cortical activity. Cortical activity during asphyxia was closely linked with changes in brai...

Objective Seizures are common in neonates recovering from birth asphyxia but there is general consensus that current pharmacotherapy is suboptimal and that novel antiseizure drugs are needed. We recently showed in a rat model of birth asphyxia that seizures are triggered by the post‐asphyxia recovery of brain pH. Here our aim was to investigate whether carbonic anhydrase inhibitors (CAIs), which induce systemic acidosis, block the post‐asphyxia seizures. Methods The CAIs acetazolamide (AZA), benzolamide (BZA), and ethoxzolamide (EZA) were administered intraperitoneally or intravenously to 11‐day‐old rats exposed to intermittent asphyxia (30 min; three 7+3 min cycles of 9% and 5% O2 at 20% CO2). Electrode measurements of intracortical pH, Po2, and local field potentials (LFPs) were made under urethane anesthesia. Convulsive seizures and blood acid‐base parameters were examined in freely behaving animals. Results The three CAIs decreased brain pH by 0.14–0.17 pH units and suppressed electrographic post‐asphyxia seizures. AZA, BZA, and EZA differ greatly in their lipid solubility (EZA > AZA > BZA) and pharmacokinetics. However, there were only minor differences in the delay (range 0.8–3.7 min) from intraperitoneal application to their action on brain pH. The CAIs induced a modest post‐asphyxia elevation of brain Po2 that had no effect on LFP activity. AZA was tested in freely behaving rats, in which it induced a respiratory acidosis and decreased the incidence of convulsive seizures from 9 of 20 to 2 of 17 animals. Significance AZA, BZA, and EZA effectively block post‐asphyxia seizures. Despite the differences in their pharmacokinetics, they had similar effects on brain pH, which indicates that their antiseizure mode of action was based on respiratory (hypercapnic) acidosis resulting from inhibition of blood‐borne and extracellular vascular carbonic anhydrases. AZA has been used for several indications in neonates, suggesting that it can be safely repurposed for the treatment of neonatal seizures as an add‐on to the current treatment regimen.

Systemic inflammation triggers protective as well as pro-inflammatory responses in the brain based on neuronal and/or cytokine signaling, and it associates with acutely and protractedly disrupted cognition. However, the multiple mechanisms underlying the peripheral–central inflammatory signaling are still not fully characterized. We used intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) in freely moving mice with chronically implanted electrodes for recording of local field potentials (LFP) and electrocorticography (ECoG) in the hippocampus and neocortex, respectively. We show here that a sudden switch in the mode of network activity occurred in both areas starting at 10–15 min after the LPS injection, simultaneously with a robust change from exploration to sickness behavior. This switch in cortical mode commenced before any elevations in pro-inflammatory cytokines IL-1β, TNFα, CCL2 or IL-6 were detected in brain tissue. Thereafter, this mode dominated cortical activity for the recording period of 3 h, except for a partial and transient recovery around 40 min post-LPS. These effects were closely paralleled by changes in ECoG spectral entropy. Continuous recordings for up to 72 h showed a protracted attenuation in hippocampal activity, while neocortical activity recovered after 48 h. The acute sickness behavior recovered by 72 h post-LPS. Notably, urethane (1.3 mg/kg) administered prior to LPS blocked the early effect of LPS on cortical activity. However, experiments under urethane anesthesia which were started 24 h post-LPS (with neuroinflammation fully developed before application of urethane) showed that both theta–supratheta and fast gamma CA1 activity were reduced, DG delta activity was increased, and sharp-wave ripples were abolished. Finally, we observed that experimental compensation of inflammation-induced hypothermia 24–48 h post-LPS promoted seizures and status epilepticus; and that LPS decreased the threshold of kainate-provoked seizures beyond the duration of acute sickness behavior indicating post-acute inflammatory hyperexcitability. Taken together, the strikingly fast development and initial independence of brain cytokines of the LPS-induced cortical mode, its spectral characteristics and simultaneity in hippocampus and neocortex, as well as inhibition by pre-applied urethane, strongly suggest that the underlying mechanisms are based on activation of the afferent vagus nerve and its mainly cholinergic ascending projections to higher brain areas.