13C NMR metabolomic evaluation of immediate and delayed mild hypothermia in cerebrocortical slices after oxygen-glucose deprivation.

Background: Mild brain hypothermia (32 degrees-34 degrees C) after human neonatal asphyxia improves neurodevelopmental outcomes. Astrocytes but not neurons have pyruvate carboxylase and an acetate uptake transporter. C-13 nuclear magnetic resonance spectroscopy of rodent brain extracts after administering [1-C-13]glucose and [1,2-C-13]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 C-13-acetate/glucose mixture underwent a 45-min asphyxia simulation via oxygen-glucose-deprivation followed by 6h of recovery. Protocols in three groups of N = 3 experiments were identical except for temperature management. The three temperature groups were: normothermia (37 degrees C), hypothermia (32 degrees C for 3.75h beginning at oxygen--glucose deprivation start), and delayed hypothermia (32 degrees C for 3.75h, beginning 15min 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-C-13]glutamine's higher final/control ratio for the hypothermia group (1.750.12) 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-C-13]glutamateproduced via pyruvate dehydrogenase entry, [2-C-13]taurinean 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.