Feasibility of Deuterium Metabolic Magnetic Resonance Spectroscopy for the Investigation of Ischemia and Reperfusion in Rat Brain Slices Perfused Ex Vivo

Abstract

Investigating glucose metabolism in the brain using [6,6-²H₂]glucose (²H₂-Glc) and deuterium-based NMR spectroscopy has shown promise for noninvasive monitoring of the fate of this labeled compound. This approach has already been applied in vivo in small animals and human subjects. A model of perfused rat brain slices recently showed promise for the investigation of the metabolic consequences of acute ischemic stroke, which is a significant cause of death and morbidity worldwide. The current study aimed to implement the deuterium-based glucose metabolism monitoring approach to study the metabolic consequences of ischemia and reperfusion in the rat brain ex vivo. In agreement with previous studies, we found that deuterated lactate (²H₂-Lac) was immediately formed in the brain upon administration of ²H₂-Glc to the perfusion medium. This metabolite remained the predominant metabolic fate observed in the ²H-NMR spectra. Upon perfusion arrest, ²H₂-Lac quickly built up to the same amount of ²H₂-Glc eliminated from the medium engulfing the slices, reaching fivefold to sixfold its baseline level (n = 6, three animals, and two ischemic conditions in each). Upon reperfusion, ²H₂-Lac decreased to its level before the ischemic condition, and ²H₂-Glc returned to its baseline. ²H₂-Lac washout to the medium amounted to 2.2% of the ²H₂-Lac signal associated with the slices after about 5 h of perfusion with ²H₂-Glc, suggesting that the ²H₂-Lac signal observed during the experiments was predominantly intracellular. These results demonstrate the utility of ²H₂-Glc and ²H-NMR in monitoring the consequences of ischemia and reperfusion in the perfused rat brain slices model.

FIGURE 1

The glycolysis pathway, followed by the lactate dehydrogenase reaction, shows the fate of the two‐deuteron label of ²H₂‐Glc. This scheme provides the basis for quantifying the deuterated‐lactate signal relative to the signals of the natural abundance of deuterium in water and the deuterated glucose in the ²H NMR spectra. D (red) in the compound's name marks the label with two deuterons (²H₂, in a single molecule, not to be confused with dextrorotatory). This scheme illustrates that each ²H₂‐Glc (D‐Glc) molecule yields one ²H₂‐Lac (D‐Lac) molecule and one unlabeled lactate molecule. The red arrow on ²H₂‐fructose‐6‐phosphate (D‐fructose‐6‐phosphate) marks the cleavage sites of phosphofructokinase. Encircled “P” marks a PO₃ moiety. Abbreviations: Glc, glucose; Lac, lactate; D‐Glc, [6,6‐²H₂]D‐glucose; D‐Glc‐6‐phosphate, [6,6‐²H₂]D‐glucose‐6‐phosphate; D‐fructose‐6‐phosphate, [6,6‐²H₂]fructose‐6‐phosphate; D‐fructose‐1,6‐bisphosphate, [6,6‐²H₂]fructose‐1,6‐bisphosphate; D‐glyceraldehyde‐3‐phosphate, [3,3‐²H₂]glyceraldehyde‐3‐phosphate, the numbers on the left mark the original carbon position in D‐Glc, which are also marked on D‐fructose‐1,6‐bisphosphate; D‐1,3‐biphosphoglycerate, [3,3‐²H₂]1,3‐biphosphoglycerate; D‐3‐phosphoglycerate, [3,3‐²H₂]3‐phosphoglycerate; D‐2‐phosphoglycerate, [3,3‐²H₂]2‐phosphoglycerate; D‐phosphoenol pyruvate, [3,3‐²H₂]phosphoenol pyruvate; D‐pyruvate, [3,3‐²H₂]pyruvate; D‐Lac, [3,3‐²H₂]lactate.

FIGURE 2

Stacked deuterium NMR spectra of a typical experiment with two ischemic periods. The H²HO, ²H₂‐Glc, and ²H₂‐Lac signals are consistently observed and allow the calculation of the concentration changes during the ischemic conditions. The time scale is in hh:mm (h, hour; m, min). The addition of ²H₂‐Glc to the external perfusion medium was taken as the starting time (00:00). The durations of continuous perfusion and flow arrest are marked as Perfusion and Ischemia, respectively. H²HO, the natural abundance of deuterium in water; ²H₂‐Glc, [6,6‐²H₂]D‐glucose; ²H₂‐Lac, [3,3‐²H₂]lactate.

FIGURE 3

Individual time courses of ²H₂‐Glc and ²H₂‐Lac in brain slices on three experimental days (A, B, and C). The results show ²H₂HO (HDO), ²H_2‐Glc, and ²H₂‐Lac concentrations in mM. The Y‐axis is the concentration in the NMR tube in mM for the three species. Breaks in ²H‐NMR acquisitions were taken to record ³¹P spectra.

FIGURE 4

Stacked deuterium NMR spectra of an experiment with three ischemic conditions. For clarity, only the third ischemic duration is shown. In addition to the H²HO, ²H₂‐Glc, and ²H₂‐Lac signals, which are consistently observed, the signal of ²H_x‐Glx can also be observed. The data are shown at 2‐min intervals starting at the beginning of the third ischemic condition and before reperfusion. The top spectrum, marked Spectrum 21, was acquired at the end of the third ischemic condition, 22 min after the spectrum marked 20, and after 62 min of ischemia. H²HO, the natural abundance of deuterium in water; ²H₂‐Glc, [6,6‐²H₂]D‐glucose; ²H₂‐Lac, [3,3‐²H₂]lactate; ²H_x‐Glx, deuterium‐labeled glutamine and glutamate.