Aim: 

Nervous system cells are highly dependent on an adequate tissue oxygenation and are very susceptible to hypoxia which causes mitochondrial dysfunctions involved in apoptosis and necrosis. The aim of this study was to evaluate the effect of hypoxia on the physiological behaviour of differentiated NT2N cells.

Methods: 

NT2 cells were differentiated in NT2N cells with 10 micromolar retinoic acid in DMEM for 30 days. After differentiation cells appeared and have a physiological behaviour like neonatal neurons. Cells were distributed in 96 well plates (6x10⁴cells/ml) and placed in a humidified incubator at 37 °C in hypoxia (2% O₂: 5% CO₂: 93% N₂) for 12 and 24 h. Controls were incubated in normoxia. After hypoxia cells were reoxiyenatet for 24 h. At the end of hypoxia and reoxygenation period we evaluated nitric oxide (NO) production, intracellular Ca²⁺ concentrations [Ca²⁺]_i, the mitochondrial membrane potential, the proliferation and the MAP kinases (ERK1 and ERK2).

Results: 

After 12 and 24 h hypoxia, all cells were viable, NO and [Ca²⁺]_i significantly increased, the mitochondrial membrane was slightly depolarized and, after the reoxigenation period, the values returned similar to controls. Finally phosphorylated ERK1/2 proteins remained fairly constant after hypoxia but, after the following reoxygenation period, decreased.

Conclusion: 

Our results indicate that NT2N, having a physiological behaviour of neonatal neurons, are susceptible to mild hypoxia. Moreover it is well known that reperfusion after brief periods of global cerebral ischemia is accompanied by hyperoxidation phenomena and we observed a decrease of phosphorylated ERK 1/2 proteins after reoxygenation.