NMR studies on energy metabolism of immobilized primary neurons and astrocytes during hypoxia, ischemia and hypoglycemia

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Abstract
Changes in high‐energy phosphate metabolites (ATP and phosphocreatine) were monitored, in real time, by 31P‐nuclear magnetic resonance in primary cell cultures of neurons and astrocytes during periods of hypoxia, ischemia and hypoglycemia, and also during the recovery periods following the re‐establishment of standard conditions. Cells were immobilized in basement membrane gel threads and perfused with oxygen‐depleted medium (oxygen concentration below 30 µM), to create hypoxic conditions, or with aerobic medium (oxygen concentration approximately 460 µM) containing different concentrations of glucose (hypoglycemia). Ischemic conditions were imposed by stopping perfusion for different periods of time (15 min to 2 h). The experimental set‐up enabled the acquisition of 31P‐spectra with high signal‐to‐noise ratio within 10–20 min for both cell types. The effect of hypoxia on glucose metabolism was assessed by 13C‐NMR using [1‐13C]glucose as substrate. The levels of ATP and PCr in astrocytes were unaffected during hypoxia (up to 2 h), but decreased notably under ischemia. In neurons, hypoxic periods caused a sharp drop of the ATP and PCr levels, and considerable damage to the capacity of neurons to replenish the ATP and PCr pools upon returning to normoxic conditions. However, neurons were remarkably less sensitive to ischemic conditions, the ATP and PCr pools being restored quickly, even after 2 h under challenging conditions. The data show that neurons were more resistant to ischemia than astrocytes, and suggest that the capacity to sustain the pools of ATP and PCr was part of the neuronal protective strategy. Copyright © 2000 John Wiley & Sons, Ltd. Abbreviations used: BME Eagle's basal medium BMG basement membrane gel DMEM Dulbecco's modified Eagle's medium FBS foetal bovine serum GFAP glial fibrillary acidic protein HRP horseradish peroxidase Pi inorganic phosphate PBS phosphate‐buffered saline PCr phosphocreatine PME phosphomonoesters.