Mild hypothermia fails to protect infant macaques from brain injury caused by prolonged exposure to Antiseizure drugs.

Barbiturates and benzodiazepines are GABAA-receptor agonists and potent antiseizure medications. We reported that exposure of neonatal macaques to combination of phenobarbital and midazolam (Pb/M) for 24 h, at clinically relevant doses and plasma levels, causes widespread apoptosis affecting neurons and oligodendrocytes. Notably, the extent of injury was markedly more severe compared to shorter (8 h) exposure to these drugs. We also reported that, in the infant macaque, mild hypothermia ameliorates the apoptosis response to the anesthetic sevoflurane. These findings prompted us explore whether mild hypothermia might protect infant nonhuman primates from neuro- and gliotoxicity of Pb/M. Since human infants with seizures may receive combinations of benzodiazepines and barbiturates for days, we opted for 24 h treatment with Pb/M. Neonatal rhesus monkeys received phenobarbital intravenously, followed by midazolam infusion over 24 h under normothermia (T > 36.5 °C-37.5 °C; n = 4) or mild hypothermia (T = 35 °C-36.5 °C; n = 5). Medication doses and blood levels measured were comparable to those in human infants. Animals were euthanized at 36 h and brains examined immunohistochemically and stereologically. Treatment was well tolerated. Extensive degeneration of neurons and oligodendrocytes was seen at 36 h in both groups within neocortex, basal ganglia, hippocampus and brainstem. Mild hypothermia over 36 h (maintained until terminal perfusion) conferred no protection against the neurotoxic and gliotoxic effects of Pb/M. This is in marked contrast to our previous findings that mild hypothermia is protective in the context of a 5 h-long exposure to sevoflurane in infant macaques. These findings demonstrate that brain injury caused by prolonged exposure to Pb/M in the neonatal primate cannot be ameliorated by mild hypothermia.

Brain pathology caused in the neonatal macaque by short and prolonged exposures to anticonvulsant drugs.

Barbiturates and benzodiazepines are potent GABAA receptor agonists and strong anticonvulsants. In the developing brain they can cause neuronal and oligodendroglia apoptosis, impair synaptogenesis, inhibit neurogenesis and trigger long-term neurocognitive sequelae. In humans, the vulnerable period is projected to extend from the third trimester of pregnancy to the third year of life. Infants with seizures and epilepsies may receive barbiturates, benzodiazepines and their combinations for days, months or years. How exposure duration affects neuropathological sequelae is unknown. Here we investigated toxicity of phenobarbital/midazolam (Pb/M) combination in the developing nonhuman primate brain. Neonatal rhesus monkeys received phenobarbital intravenously, followed by infusion of midazolam over 5 (n = 4) or 24 h (n = 4). Animals were euthanized at 8 or 36 h and brains examined immunohistochemically and stereologically. Treatment was well tolerated, physiological parameters remained at optimal levels. Compared to naïve controls, Pb/M exposed brains displayed widespread apoptosis affecting neurons and oligodendrocytes. Pattern and severity of cell death differed depending on treatment-duration, with more extensive neurodegeneration following longer exposure. At 36 h, areas of the brain not affected at 8 h displayed neuronal apoptosis, while oligodendroglia death was most prominent at 8 h. A notable feature at 36 h was degeneration of neuronal tracts and trans-neuronal death of neurons, presumably following their disconnection from degenerated presynaptic partners. These findings demonstrate that brain toxicity of Pb/M in the neonatal primate brain becomes more severe with longer exposures and expands trans-synaptically. Impact of these sequelae on neurocognitive outcomes and the brain connectome will need to be explored.