13C NMR Metabolomic Evaluation of Immediate and Delayed Mild Hypothermia in Cerebrocortical Slices after Oxygen–Glucose Deprivation

Abstract

Abstract 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. 13C nuclear magnetic resonance spectroscopy of rodent brain extracts after administering [1-13C]glucose and [1,2-13C]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 13C-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–-glucose 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 [2-13C]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 carboxylase/pyruvate dehydrogenase ratio for glutamine formation. Least Absolute Shrinkage and Selection Operator found the most important metabolites associated with adenosine triphosphate preservation: [3,4-13C]glutamate—produced via pyruvate dehydrogenase entry, [2-13C]taurine—an important osmolyte and antioxidant, and phosphocreatine. Final principal component analyses scores plots suggested separate cluster formation for the hypothermia group, but with insufficient data for statistical significance. Conclusions: Starting mild hypothermia simultaneously with oxygen–glucose deprivation, compared with delayed starting or no hypothermia, has higher pyruvate carboxylase throughput, suggesting that better glial integrity is one important neuroprotection mechanism of earlier hypothermia.