A physiologically-validated rat model of term birth asphyxia with seizure generation after, not during, brain hypoxia

Abstract

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...

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

“…These seizures occurred repeatedly over a period of 0.5‐8.97 minutes (mean = 3.7 minutes) postasphyxia, after which the animals resumed normal behavior. In agreement with previous findings 16 and as shown in Table 2, the predominant seizure type was generalized convulsive (stage III‐V) seizures, particularly generalized tonic‐clonic (stage V) seizures. The average latency to convulsive stage III‐V seizures was 2.2 minutes (range = 1.0‐3.88 minutes).…”

“…The present study illustrates the antiseizure efficacy of clinically used ASDs in a recently described novel rat model of birth asphyxia, 16 whereas the NKCC1 inhibitor bumetanide is ineffective. To our knowledge, this is the first rodent model of postasphyxic neonatal seizures that allows testing of drugs after asphyxia, because, as in human neonates, seizures occur during establishment of normocapnic conditions (see Introduction).…”

“…The authors concluded that “these data provide preclinical support for clinical trials of bumetanide in human neonates at risk for hypoxic encephalopathy and seizures.” However, in human asphyxiated neonates, seizures occur mainly during the recovery period following perinatal asphyxia, 2,3 not during the hypoxic episode associated with complicated birth. Asphyxia is, by definition, a combination of hypoxia and hypercapnia, and recent work has shown the elevation in brain CO 2 levels has robust brain‐sparing effects, 15 whereas the recovery to normocapnia (and the associated rise in pH) promotes seizures in the model used in the present study 16 …”

“…The onset of epileptiform activity is fast, starting around 2 minutes after the end of asphyxia. Although this is a clear advantage over noninvasive hypoxia‐only models, in which seizures occur during hypoxia, 16 the short latency between asphyxia and onset of seizures in the present model complicates drug testing for pharmacokinetic reasons. This limits the range of ASDs that can be tested for antiseizure efficacy after asphyxia to drugs such as midazolam, which penetrates extremely fast into the brain, 30 whereas drugs such as phenobarbital do not reach sufficiently high levels at their brain targets within the time frame necessary.…”

“…After asphyxia, the average time of onset and duration of seizure activity and the average duration of observation of the rat pups are indicated. For details, see Ala‐Kurikka et al 16 Bum, bumetanide; MDZ, midazolam; PB, phenobarbital…”