Perfusion system for studying dynamic metabolomics in rat brain slices exposed to oxygen-glucose deprivation using proton and phosphorus nuclear magnetic resonance.

The aim of the current study was to establish a controlled and reproducible model to study metabolic changes during oxygen-glucose deprivation (OGD) in rat brain using a nuclear magnetic resonance (NMR)-compatible perfusion system. Rat brains were cut into 400-μm thick slices and perfused with artificial cerebrospinal fluid (aCSF) in a 10-mm NMR tube inside a 600-MHz NMR spectrometer. Four experimental conditions were tested: (1) continuous perfusion with aCSF with glucose and normoxia, and (2) 30-, (3) 60-, or (4) 120-min periods of OGD followed by reperfusion of aCSF containing glucose and normoxia. The energetic state of perfused brain slices was measured using phosphorus ( 31 P) NMR and metabolite changes were measured using proton ( 1 H) NMR. aCSF samples were collected every 30 min and analyzed using 1 H NMR. The sample temperature was maintained at 36.7 ± 0.1°C and was checked periodically throughout the experiments. Brain slice histology was compared before and after OGD in the perfusion system using hematoxylin-eosin-saffron staining. NMR data clearly distinguished three severity groups (mild, moderate, and severe) after 30, 60, and 120 min of OGD, respectively, compared with the control group. 31 P NMR spectra obtained from controls showed that phosphocreatine levels were stable for 5 h inside the perfusion system. Control 1 H NMR spectra showed that lactate, N-acetylaspartic acid, glutamate, γ-aminobutyric acid, and creatine metabolite levels were stable over time, with lactate levels having a tendency to gradually increase due to the recirculation of the aCSF in the perfusion system. A controlled and reproducible perfusion system was established to study the energetic and metabolic changes in rat brain slices during and after OGD of varying severity.