Gp91^phox (NOX2) in classically activated microglia exacerbates traumatic brain injury

Background: We hypothesized that gp91^phox (NOX2), a subunit of NADPH oxidase, generates superoxide anion (O₂^-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91^phox and reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91^phox knockout mice (gp91^phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91^phox generation.Methods: Unilateral TBI was induced in gp91^phox-/- and wild-type (Wt) mice (C57/B6J) (25-30 g). The expression and roles of gp91^phox after TBI were investigated using immunoblotting and staining techniques. Levels of O₂^- and peroxynitrite were determined in situ in the mouse brain. The activated phenotype in microglia that expressed gp91^phox was determined in a microglial cell line, BV-2, in the presence of IFNγ or IL-4.Results: Gp91^phox expression increased mainly in amoeboid-shaped microglial cells of the ipsilateral hemisphere of Wt mice after TBI. The contusion area, number of TUNEL-positive cells, and amount of O₂^- and peroxynitrite metabolites produced were less in gp91^phox-/- mice than in Wt. In the presence of IFNγ, BV-2 cells had increased inducible nitric oxide synthase and nitric oxide levels, consistent with a classical activated phenotype, and drastically increased expression of gp91^phox.Conclusions: Classical activated microglia promote ROS formation through gp91^phox and have an important role in brain damage following TBI. Modulating gp91^phox and gp91^phox -derived ROS may provide a new therapeutic strategy in combating post-traumatic brain injury.