Evaluation of autophagy in neonatal rat glial cells in an in vitro model of hypoxic-ischemic injury.

The autophagy process is crucial for cell functioning, yet it is still understudied in glial cells during neurodevelopment. To address this, cultures of the main glial cell types in the central nervous system (CNS), including astrocytes, microglia, oligodendrocyte progenitors, and differentiating oligodendrocytes, were created to examine the impact of an in vitro hypoxia-ischemia (HI) model on autophagy. The HI insult was mimicked by applying temporal oxygen-glucose deprivation (OGD). Since neonatal hypoxic-ischemic insults primarily affect the brain's white matter, the study predominantly focused on oligodendrocytes at different stages of maturation: progenitor cells versus cells that express myelin components (e.g. MBP). The results show that the different glial fractions exhibit varying sensitivity to the applied conditions. Maturing oligodendrocytes were found to be more sensitive to OGD conditions than the progenitor fraction. The OGD procedure was proven to impact the expression of autophagy markers, indicating the activity of this process in response to injury. Western blot analysis of oligodendrocyte progenitor cells (OPCs) showed that the autophagy substrate marker p62 increased after six hours, which may suggest transient inhibition and subsequent activation of autophagy. To verify the involvement of autophagy in the differentiation of neonatal oligodendrocytes, the process was modulated using chloroquine (CQ) treatment. CQ is recognised as an inhibitor of autophagic flux because it disrupts lysosomal acidity and prevents the breakdown of autophagosomes. CQ treatment resulted in the accumulation of autophagosomes. The results suggest that abnormalities in the functioning of glial cells, particularly oligodendrocytes, in response to hypoxic-ischaemic (HI)-like conditions might be associated with altered autophagic flux in response to cellular stress. Transient alterations in autophagy were observed within 24 h of limiting oxygen and glucose supply, and these alterations may contribute to subsequent disorders in oligodendrocyte differentiation. This is recognised as one of the major issues in the pathogenesis of neonatal hypoxia-induced damage. Therefore, modulation of autophagy could be a promising therapeutic approach to prevent these adverse changes.