Selective head cooling and NOX inhibition protect the blood–brain barrier in neonatal epilepsy

Epileptic seizures in the immature brain cause oxidative stress and blood-brain barrier (BBB) disruption, contributing to long-term cerebrovascular and neurodevelopmental deficits. New antioxidant-based strategies are urgently needed to prevent seizure-induced complications. Our previous work identified NADPH oxidase (NOX) as a major enzymatic source of reactive oxygen species (ROS) in the cerebral endothelium following seizures, and demonstrated that selective head cooling reduces brain oxidative stress and prevents cerebrovascular dysfunction in epileptic newborn pigs. Here, we evaluated the effects of selective head cooling and NOX-targeting antioxidants on BBB integrity using in vivo and in vitro models of seizure-induced oxidative stress. Epileptic seizures in newborn pigs were induced by bicuculline under normothermic or selective head cooling conditions. BBB permeability was assessed by Evans Blue extravasation in the cerebral cortex 4–48 h after seizures, and brain-derived circulating endothelial cells (BCECs; CD45 - /CD146 + /GluT1 + ) were quantified as peripheral biomarkers of BBB disruption. Seizures under normothermia caused significant BBB leakage, cerebrovascular apoptosis, and elevated BCECs. Selective head cooling during seizures (reducing brain cortex temperature to ~25°C while maintaining body temperature at ~35°C) markedly attenuated these effects. The NOX1/NOX4 inhibitor setanaxib (5 mg/kg i.p.) and a NOX inhibitor sulforaphane (0.4 mg/kg i.p.) also prevented BBB disruption during normothermia. To examine cellular mechanisms, in vitro BBB models, comprising monolayers of primary neonatal porcine or adult human brain endothelial cells, were exposed to seizure-related excitotoxic and inflammatory pro-oxidants, glutamate and TNF-alpha. The in vitro BBB function was assessed via transendothelial electrical resistance (TEER) and paracellular permeability to 3-kDa dextran–Alexa Fluor 488. Dihydroethidium (DHE) fluorescence and lucigenin-enhanced NADPH-dependent superoxide luminescence were used to measure ROS and NOX activity. Glutamate and TNF-alpha increased endothelial NOX activity, elevated ROS, triggered apoptosis, and caused BBB leakage. Endothelial NOX activity was highly sensitive to the inhibitory effects of setanaxib (5–10 µM), sulforaphane (2–5 µM), and moderate hypothermia (< 30˚C). Both pharmacological NOX inhibitors and moderate hypothermia prevented BBB damage caused by excitotoxicity and inflammation. Collectively, these findings demonstrate that neonatal seizure-induced BBB disruption is mediated primarily through NOX-driven oxidative stress and can be effectively prevented by either NOX inhibition or selective head cooling. These results support the translational potential of combining selective head cooling and targeted antioxidant therapy to protect the developing brain from seizure-induced cerebrovascular injury and improve neurological outcomes in epilepsy. Grant Support: R01NS105655, R01NS134659 (Parfenova) This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.