Evidence for a nadph oxidase dependent mechanism of superoxide generation by brain microglial cells

The central nervous system contains a network of specialized phagocytes known as microglia which orchestrate the local immune response. They generate oxygen free radicals in response to specific signals and are recently implicated in neuronal injury in a variety of neurodegenerative disorders. The type of oxygen radicals and the mechanisms that trigger their production are unknown. In this study, electron paramagnetic resonance measurements with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) demonstrate that cultured sheep brain microglia when stimulated by phorbol-12-myristale-13-acetale or opsonified zymosan, generate superoxide radicals giving rise to DMPO-OH signal. The EPR signal was quantified by computer simulation of the spectra and comparison with a standard nitroxide radical spectrum. Cu-Zn superoxide dismutase (SOD) abolished > 80% of the signal, while deferoxamine only reduced the observed signal by 10-20%. The NADPH oxidase inhibitor, diphenylene iodonium, blocked the radical production completely suggesting a NADPH oxidase dependent pathway similar to that observed in matched experiments with sheep neutrophils. Western blotting performed with cellular extracts from neutrophils and microglia further confirmed the expression of the NADPH oxidase. These experiments provide evidence that activated microglial cells generate superoxide via a NADPH oxidase dependent pathway.