C NMR Metabolomic Evaluation of Immediate and Delayed Mild Hypothermia in Cerebrocortical Slices after Oxygen–Glucose Deprivation

Background: Mild brain hypothermia (32°–34°C) after human neonatal asphyxia improves neurodevelopmental outcomes. Astrocytes but not neurons have pyruvate carboxylase and an acetate uptake transporter. 13 C nuclear magnetic resonance spectroscopy of rodent brain extracts after administering [1- 13 C]glucose and [1,2- 13 C]acetate can distinguish metabolic differences between glia and neurons, and tricarboxylic acid cycle entry via pyruvate dehydrogenase and pyruvate carboxylase. Methods: Neonatal rat cerebrocortical slices receiving a 13 C-acetate/glucose mixture underwent a 45-min asphyxia simulation via oxygen–glucose-deprivation followed by 6 h of recovery. Protocols in three groups of N = 3 experiments were identical except for temperature management. The three temperature groups were: normothermia (37°C), hypothermia (32°C for 3.75 h beginning at oxygen–-glu-cose deprivation start), and delayed hypothermia (32°C for 3.75 h, beginning 15 min after oxygen–glucose deprivation start). Multivariate analysis of nuclear magnetic resonance metabolite quantifications included principal component analyses and the L1-penalized regularized regression algorithm known as the least absolute shrinkage and selection operator . Results: The most significant metabolite difference ( P < 0.0056) was [2- 13 C]glutamine’s higher final/control ratio for the hypothermia group (1.75 ± 0.12) compared with ratios for the delayed (1.12 ± 0.12) and normothermia group (0.94 ± 0.06), implying a higher pyruvate carboxyl-ase/pyruvate dehydrogenase ratio for glutamine formation.