Long-term region-specific mitochondrial respiration impairment after perinatal asphyxia is prevented by the NAD⁺ donor nicotinamide riboside: A real-time organotypic metabolic profiling approach

Perinatal asphyxia (PA) is a primary cause of neonatal morbidity, with persistent neurological sequelae, associated with mitochondrial dysfunction and redox imbalance. The lack of experimental approaches for region-specific evaluation of mitochondrial bioenergetics in structurally preserved neural tissues has limited the identification of selective vulnerabilities and redox-based therapeutic targets. Here, we introduce a real-time metabolic profiling strategy that integrates Seahorse XFe96 respirometry with Cytation 5 image-based quality control to quantify mitochondrial respiration and glycolytic flux in organotypic cultures derived from distinct brain regions of neonatal rats exposed to global PA. Using this strategy, we demonstrate that PA induces a profound and region-selective impairment of mitochondrial function, characterized by reduced basal, ATP-linked, maximal, and spare respiratory capacity in the substantia nigra and neostriatum, while neocortical bioenergetics remain largely preserved. These alterations are accompanied by a shift toward glycolysis, reflected by increased extracellular acidification rates and reduced OCR/ECAR ratios. Notably, in vivo administration of the NAD⁺ precursor nicotinamide riboside (0.8 mmol·kg⁻¹, i.p., 1 h after birth) effectively prevented PA-induced mitochondrial respiration deficits, attenuated oxidative stress-induced cell death, and preserved regional neurochemical integrity, particularly within the nigrostriatal neurocircuitry. Together, these findings reveal a heterogeneous mitochondrial redox and bioenergetic vulnerability of the developing brain to perinatal hypoxia. This integrated organotypic-metabolic approach may offer novel opportunities to dissect out region-specific mitochondrial dysfunction and to explore NAD⁺-based therapeutic strategies targeting neonatal brain injury.