Hypoxia-Induced Dysfunctions and Injury of Astrocytes in Primary Cell Cultures

The effects of severe hypoxia were studied in a primary culture of astrocytes prepared from newborn rat cerebral cortex. Hypoxia was created by placing cultures in an airtight chamber that was flushed with 95% N₂/5% CO₂ for 15 min before being sealed. The hypoxic environment was maintained constant for up to 24 h. During the first 12 h of hypoxia, astrocytes showed no morphological changes by phase-contrast microscopy. After 18 h of hypoxia, some astrocytes in culture became swollen and started to detach from the culture dish. All cells in the culture were destroyed after 24 h of hypoxia. The lactate dehydrogenase level in the culture medium increased more than tenfold between 12 and 24 h of hypoxia. Glutamate uptake was inhibited 80% by similar hypoxic conditions. The cell volume of astrocytes, as measured by 3-O-methyl-[¹⁴C]-D-glucose uptake, was increased. These observations suggested cell membrane dysfunction. The malondialdehyde level of hypoxic cultures increased twofold after 24 h of hypoxia. Verapamil (0.5 mM), furosemide (1 mM), indomethacin (1 mM), MgCl₂ (10 mM), and mannitol (10 mM) reduced but never completely abolished the release of lactate dehydrogenase from hypoxic astrocytes. These data suggest multifactorial causes for severe injury in hypoxic astrocytes.