Immature oligodendroglia of the developing brain are highly susceptible to hyperoxia. As the in-vivo O2 saturation of the brain parenchyma is about 3–7%, we hypothesised that the 21% O2 commonly sustained in cell culture poses a hyperoxic challenge to immature oligodendroglia affecting their development.
We cultured primary rat oligodendroglial precursor cells (OPC) for 24 h, 48 h and 96 h at 5% and 21% and assessed their developmental progress through analysis of cell numbers of different oligodendroglial stages. For immunocytochemistry, A2B5+, O4+, and O1+ markers were used to label precursor, immature and mature stages respectively, combined with Ki67 for proliferation or TUNEL for apoptosis. qPCR was used to determine gene expression of factors important for development (Olig1, Olig2, Sox9, Sox10) and maturation (MBP, CNP). NRF2 and SOD2 expression was measured to quantify responses to oxidative stress.
Morphology of OLC varied greatly between 21% and 5% at all time points. At 48 h, O4 positive immature oligodendroglia frequently had multiple processes with typical shape at 5% but rarely at 21% oxygen. Gene expression of MBP, CNP, Olig1, and Olig2 was significantly reduced and antioxidant genes NRF2 and SOD2 were significantly up-regulated after 48 h at 21% immunohistochemistry (each p < 0.05). However, there was no difference in cell death as analysed by NG2+TUNEL+ OPCs.
Altogether, these findings indicate that 21% O2 in vitro has negative effects on oligodendroglial development. It has to be debated whether 5% O2 resemble physiological oxygen conditions more appropriately than current standard protocols with 21% for oligodendroglial cultures.