Measurement and characterization of superoxide generation in microglial cells: evidence for an NADPH oxidase-dependent pathway.

While oxygen free radicals are important mediators of brain injury, questions remain regarding which cell types and enzyme pathways trigger this radical generation. Microglial cells have been hypothesized to be an important source of radical generation; however, the magnitude, kinetics, and mechanism of this process are unknown. Oxygen radical generation by stimulated primary microglia was directly measured and characterized by electron paramagnetic resonance spin trapping. Microglia, when stimulated by phorbol ester or opsonified zymosan, gave rise to EPR spectra characteristic of superoxide. Experiments performed in the presence of superoxide dismutase, catalase, deferoxamine, and dimethyl sulfoxide excluded generation of hydroxyl radicals in significant amounts. Microglial superoxide generation was blocked by the NADPH oxidase inhibitor diphenylene iodonium in a manner similar to that seen in neutrophils, suggesting that a neutrophil like NADPH oxidase was the source of superoxide production. However, microglia produced 20 to 40 times less superoxide compared to a similar number of neutrophils during the first 30 min following stimulation, indicating a marked difference in the regulation of NADPH oxidase activation. Western blots of microglia lysates demonstrated that both large (gp91-phox) and small (p22-phox) NADPH oxidase subunits are expressed in both unstimulated and stimulated microglia. Indirect immunofluorescence demonstrated localization at the membrane surfaces of activated cells. Thus, microglial cells generate superoxide via a neutrophil-like NADPH oxidase but exhibit distinctly different time course and magnitude of activation than that seen in neutrophils.