Depolarization and hypoxia-induced cell damage in serum-free cultures of the rat cortex, and related extracellular glutamate changes

Abstract

Experiments were carried out to clarify the influence of K⁺-induced chronic membrane depolarization on cytotoxicity and changes in extracellular glutamate, as induced by hypoxia in serum-free cortical cultures. Excitotoxic cell death was examined by measuring lactic dehydrogenase (LDH) activity released into the culture medium. In cultures grown in the presence of 25 mM K⁺, morphological injury occured during a 4 h exposure to hypoxia, together with a substantial efflux of LDH. In hypoxic cultures, extracellular glutamate concentrations were elevated and these responses were absent in cultures grown in physiological medium (K⁺=5.4 mM), even with 16 h of hypoxia. In cultures at 25 mM K⁺, the cytotoxicity induced by hypoxia was attenuated by NMDA receptor antagonists, in a concentration dependent manner. We also examined the effects of excitatory amino acids, agonists of the main glutamate receptor classes (glutamate, NMDA, kainate, and AMPA). In both 5.4 mM and 25 mM K⁺ cultures, a dose dependent release of LDH was induced by a long exposure to glutamate receptor agonists, although the release of LDH in the 5.4 mM K⁺ was less than that in the 25 mM K⁺ cultures. Despite of the expression of the glutamate receptor in the 5.4 mM K⁺ cultures, hypoxic neuronal damage did not occur. These results suggest that when cultures grown in a chronically depolarizing environment are exposed to hypoxia, they are damaged by an excitotoxic mechanism in which the main cause seems to be the glutamate released into the medium at high extracellular levels.