Changes in reactive oxygen species and autofluorescence under hypoxia at the hippocampal CA3 area: Role of calcium and zinc influxes

Reactive oxygen species (ROS) have an important role in cellular biology, being involved, in a way that depends on their levels, in cell signaling processes or in oxidative stress, probably associated with neurodegenerative and other diseases. Most of the studies about ROS formation were performed in ischemic conditions, and thus, there is limited knowledge about ROS formation in less severe hypoxic conditions. This study investigates neuronal ROS generation and autofluorescence changes in hypoxic conditions, focusing on the involvement of calcium and zinc. Using hippocampal slices from Wistar rats, ROS production was monitored by the permeant fluorescent indicator H₂DCFDA under different oxygenation levels. Moderate hypoxia (40% O₂) led to a small ROS increase, while severe hypoxia (0% O₂) showed a more pronounced rise. KCl-induced depolarization significantly enhanced ROS formation, particularly under severe hypoxia. Inhibition of NMDA receptors reduced ROS generation without affecting autofluorescence, while chelation of zinc ions decreased ROS production and increased flavin adenine dinucleotide (FAD) autofluorescence. These findings suggest that, in hypoxic conditions, ROS formation is mediated by calcium entry through NMDA receptors and also by zinc influxes. Thus, these ions play a crucial role in oxidative stress, which may be related with neurodegenerative diseases associated with ROS dysregulation.