The role of glia in neuronal recovery following anoxia: In vitro evidence of neuronal adaptation

We investigated the effects of 3 h of anoxia on metabolism of neurons and astrocytes, using a robust cell-based model system that mimics closely the living tissue milieu, i.e., in 3D neural aggregates cultured in bioreactors. Cells were incubated simultaneously with [1-13C]glucose and [1,2-13C]acetate; and, the gliotoxin fluorocitrate (FC) was used for glial tricarboxylic acid (TCA) cycle inhibition to assess the role of astrocytes for neuronal metabolism after oxygen deprivation. Results show that culture viability was not compromised by exposure to anoxia with and without FC. Interaction between astrocytes and glutamatergic neurons was altered due to anoxia: labeling in glutamine from [1-13C]glucose was decreased, whereas that in glutamate from [1,2-13C]acetate was increased. In contrast, GABA labeling was not affected by anoxia. It was shown that anoxia did not affect astrocytic capacity to synthesize glutamine in the reoxygenation period. The selective action of FC on astrocytes was confirmed. However, the presence of small amounts of glutamate and GABA labeled from acetate indicated residual activity of the glial TCA cycle. Although major metabolic changes were found due to FC-treatment, the intracellular pool of GABA was kept unchanged. Overall, our data clearly confirm that the glutamate–glutamine cycle depends on astrocytic TCA cycle activity and that mitochondrial impairment of astrocytes will ultimately stop metabolic trafficking between astrocytes and glutamatergic neurons. Additionally, our data suggest a metabolic independence of GABAergic neurons from astrocytes even after situations of complete oxygen depletion.

Research highlights
► First report on the effects of anoxia plus glial TCA inhibition in 3D cultures.
► Our approach provides a robust cell-based model mimicking closely the living milieu.
► Results clearly confirm that Glu–Gln cycle depends on astrocytic TCA cycle activity.
► Shows a metabolic independence of GABAergic neurons and astrocytes even after anoxia.
► Useful to better understand the complexity of metabolic trafficking after anoxia.