Exploring different mechanisms of reactive oxygen species formation in hypoxic conditions at the hippocampal CA3 area.

Abstract

Hypoxia can lead to severe consequences for brain function, particularly in regions with high metabolic demands such as the hippocampus. Excessive production of reactive oxygen species (ROS) during hypoxia can initiate a cascade of oxidative stress, evoking cellular damage and neuronal dysfunction. Most of the studies characterizing the formation of ROS are performed in the context of ischemia induced by oxygen-glucose deprivation, thus, the role of hypoxia in less severe conditions requires further clarification. The aim of this work was to identify the major mechanisms of ROS generation and assess flavoprotein autofluorescence changes. For ROS detection, the slices were incubated with the indicator H₂DCFDA, while intrinsic FAD-linked autofluorescence was recorded from indicator free slices. All signals were measured under hypoxia, at the hippocampal mossy fiber synapses of CA3 area, which were chemically stimulated using 20 mM KCl. The results suggest that ROS is formed in the mitochondria, during moderate hypoxia. The blockage of mitochondrial complexes I, III and IV with rotenone, myxothiazol and sodium azide, respectively, and of the mitochondrial calcium uniporter with Ru265, led to the abolishment of ROS changes and to an increase of FAD-linked autofluorescence (with the exception of the complexes III and IV). The blockage of the enzyme oxidases NADPH and xanthine oxidase also impaired ROS formation and rose FAD-linked autofluorescence. Thus, the blockage of any of the steps of the process of ROS formation, namely the activation of critical MRC complexes, calcium entry into the mitochondria, or enzyme oxidases activity, ceases the production of ROS.