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

Abstract

Background Mild brain hypothermia (32°C–34°C) after human neonatal asphyxia improves neurodevelopmental outcomes. Astrocytes but not neurons have pyruvate carboxylase (PC) and an acetate uptake transporter. 13C NMR 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 (TCA cycle) entry via pyruvate dehydrogenase (PDH) and PC. Methods Neonatal rat cerebrocortical slices receiving a 13C-acetate/glucose mixture underwent a 45-min asphyxia simulation via oxygen-glucose-deprivation (OGD) 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 OGD start), and delayed hypothermia (32°C for 3.75 h, beginning 15 min after OGD 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 (LASSO). 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 to ratios for the Delayed (1.12 ± 0.12) and Normothermia group (0.94 ± 0.06), implying a higher PC/PDH ratio for glutamine formation. LASSO found the most important metabolites associated with adenosine triphosphate preservation: [3,4-13C]glutamate—produced via PDH entry, [2-13C]taurine--an important osmolyte, 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 OGD, compared with delayed starting or no hypothermia, has higher PC throughput, suggesting that better glial integrity is one important neuroprotection mechanism of earlier hypothermia.

Exploring Perinatal Asphyxia by Metabolomics

“…Almost 10 years later, Liu et al extensively investigated murine models of neonatal asphyxia, focusing on the NMR analysis of brain tissues [26][27][28][29]. In particular, after inducing hypoxia-ischemia in neonatal mice and rats, they tried to identify the metabolomic signatures associated to the brain injury, distinguishing normothermic from mild TH recoveries.…”

“…In particular, after inducing hypoxia-ischemia in neonatal mice and rats, they tried to identify the metabolomic signatures associated to the brain injury, distinguishing normothermic from mild TH recoveries. Most of these experiments were performed ex vivo on neonatal rat cerebrocortical slices [26][27][28]. Asphyxia was simulated via oxygen-glucose-deprivation (OGD) and different thermal treatments were applied for recovery: normothermia (37 • C), hypothermia (32 • C immediately after OGD), and delayed hypothermia (32 • C, 15 min after OGD).…”

Neuroprotective Treatments after Perinatal Hypoxic-Ischemic Brain Injury Evaluated with Magnetic Resonance Spectroscopy

“…For example, in a neonatal-rat brain slice model of oxygen-glucose deprivation, there was an increase of [2-13 C]glutamine in the hypothermia group after the injection of a mixture of [1-13 C]glucose and [1,2-13 C] acetate [36] . [1-13 C]glucose is converted into [2-13 C] glutamine via the enzyme pyruvate carboxylase, which is only present in astrocytes.…”

Metabolomic Profiling in Perinatal Asphyxia: A Promising New Field

“…Metabolomic data thus far is consistent with the perturbation of energy metabolism and mitochondrial injury in hypoxia-ischaemia. In particular both human and animal work have described the alteration of Krebs cycle metabolites [ 23 – 27 , 58 ], amino acids [ 24 , 26 , 31 , 58 , 59 ], and constituents of the cell membrane [ 25 , 35 , 59 ]. These metabolic changes are consistent with the current understanding of injury mechanisms, evidence of strong biological plausibility.…”

“…Several studies have employed animal models of asphyxia/hypoxia to study metabolomic alterations and a number of putative metabolomic fingerprints have been proposed. The accumulation and delayed recovery of Krebs cycle intermediates (e.g., fumarate, succinate, malate, and alpha ketoglutarate) have been described in a range of models [ 24 – 27 ]. These energy metabolites are a product of the shift toward anaerobic conditions, illustrated by the accumulation of lactate [ 22 , 28 , 29 ], and mitochondrial dysfunction [ 30 ] leading to disturbance of the Krebs cycle.…”