Hypoxic/ischaemic cell damage in cultured human NT-2 neurons

Postmitotic neurons were generated from the human NT-2 teratocarcinoma cell line in a novel rapid differentiation procedure. These neurons were used to establish an in vitro assay system that allows the investigation of hypoxic/ischaemic cell damage and the development of neuroprotective strategies. In experiments of simulated ischaemia, the neurons were subjected to anoxia and hypoglycaemia. The viability of NT-2 neuronal cells was significantly reduced by anoxia especially in the presence of glutamate, reflecting the cellular vulnerability to excitotoxic conditions. The addition of the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 reduced glutamate-induced neuronal damage. Calcium imaging showed that NT-2 neurons increased cytosolic calcium levels in response to stimulation with glutamate or NMDA, an effect that was abolished in calcium free medium and at low pH values. The NMDA receptor antagonists MK-801, AP 5 and ketamine reduced the NMDA-induced response, suggesting the presence of functional NMDA receptors in the human neuronal cells. The mitochondrial potential of neurons was estimated using the fluorescent dye rhodamine 123 (R123). The fluorescence imaging experiments indicated an energetic collapse of mitochondrial functions during anoxia, suggesting that the human NT-2 neurons can be used to investigate subcellular processes during the excitotoxic cascade.