Gp91phox (NOX2) in classically activated microglia exacerbates traumatic brain injury. J Neuroinflammation 7:41

Department of Emergency and Critical Care Medicine, Showa University School of Medicine, Shinagawa-Ku, Tokyo 142-8555, Japan.
Journal of Neuroinflammation (Impact Factor: 4.9). 07/2010; 7:41. DOI: 10.1186/1742-2094-7-41
Source: PubMed

ABSTRACT We hypothesized that gp91phox (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 gp91phox and reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91phox knockout mice (gp91phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91phox generation.
Unilateral TBI was induced in gp91phox-/- and wild-type (Wt) mice (C57/B6J) (25-30 g). The expression and roles of gp91phox 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 gp91phox was determined in a microglial cell line, BV-2, in the presence of IFNgamma or IL-4.
Gp91phox 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 gp91phox-/- mice than in Wt. In the presence of IFNgamma, BV-2 cells had increased inducible nitric oxide synthase and nitric oxide levels, consistent with a classical activated phenotype, and drastically increased expression of gp91phox.
Classical activated microglia promote ROS formation through gp91phox and have an important role in brain damage following TBI. Modulating gp91phox and gp91phox -derived ROS may provide a new therapeutic strategy in combating post-traumatic brain injury.

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    • "NADPH oxidase is a multiunit enzyme composed of several subunits that include several isoforms of NOX (NOX 1–5) (Bedard and Krause, 2007). Although few studies have evaluated the role of gp91 phox (catalytic subunit of the enzyme, NOX 2 ) in TBI, experimental evidences suggest that use of specific NADPH oxidase inhibitors may have significant efficacy in the treatment of TBI (Dohi et al., 2010; Lo et al., 2007). A candidate for this is Apocynin, a natural organic compound isolated from the Himalayan medicinal herb Picrohiza kurroa used as an efficient inhibitor of the NADPH–oxidase complex (Hayashi et al., 2005; Stolk et al., 1994; Van den Worm et al., 2001). "
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    ABSTRACT: Traumatic brain injury (TBI) is a devastating disease that commonly causes persistent mental disturbances and cognitive deficits. Although studies have indicated that overproduction of free radicals, especially superoxide (O2(-)) derived from Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a common underlying mechanism of pathophysiology of TBI, little information is available regarding the role of apocynin, an NADPH oxidase inhibitor, in neurological consequences of TBI. Therefore, the present study evaluated the therapeutic potential of apocynin for treatment of inflammatory and oxidative damage, in addition to determining its action on neuromotor and memory impairments caused by moderate fluid percussion injury in mice (mLFPI). Statistical analysis revealed that apocynin (5 mg/kg), when injected subcutaneously (s.c.) 30 min and 24 h after injury, had no effect on neuromotor deficit and brain edema, however it provided protection against mLFPI-induced object recognition memory impairment 7 days after neuronal injury. The same treatment protected against mLFPI-induced IL-1β, TNF-α, nitric oxide metabolite content (NOx) 3 and 24 hours after neuronal injury. Moreover, apocynin treatment reduced oxidative damage (protein carbonyl, lipoperoxidation) and was effective against mLFPI-induced Na(+)K(+)-ATPase activity inhibition. The present results were accompanied by effective reduction in lesion volume when analyzed 7 days after neuronal injury. These data suggest that superoxide (O2(-)) derived from NADPH oxidase can contribute significantly to cognitive impairment, and that the post injury treatment with specific NADPH oxidase inhibitors, such as apocynin, may provide a new therapeutic approach to the control of neurological disabilities induced by TBI.
    Neurochemistry International 09/2013; 63(6). DOI:10.1016/j.neuint.2013.09.012 · 2.65 Impact Factor
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    • ", which in turn react with proteins, lipids, sugars, and nucleotides and impair the normal physiological function of cells. Although it is considered that O 2 @BULLET− does not have strong oxidative potential and that edaravone does not scavenge O 2 @BULLET− in vitro [6], we previously reported that mice deficient in Gp91 phox (NOX2), a subunit of NADPH oxidase and a generator of O 2 @BULLET− , exhibited reduced lesion size and oxidative stress following TBI [19]. Moreover, knockout mice lacking interleukin-1, a proinflammatory cytokine, were less susceptible to neuronal cell death than their wild-type littermates and displayed less inducible nitric oxide synthase gene expression and reduced O 2 @BULLET− and ONOO − production during ischemia [20] [21]. "
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    ABSTRACT: Traumatic brain injury (TBI) is a major cause of death and disability in young people. No effective therapy is available to ameliorate its damaging effects. Our aim was to investigate the optimal therapeutic time window of edaravone, a free radical scavenger which is currently used in Japan. We also determined the temporal profile of reactive oxygen species (ROS) production, oxidative stress, and neuronal death. Male C57Bl/6 mice were subjected to a controlled cortical impact (CCI). Edaravone (3.0 mg/kg), or vehicle, was administered intravenously at 0, 3, or 6 hours following CCI. The production of superoxide radicals (O2 (∙-)) as a marker of ROS, of nitrotyrosine (NT) as an indicator of oxidative stress, and neuronal death were measured for 24 hours following CCI. Superoxide radical production was clearly evident 3 hours after CCI, with oxidative stress and neuronal cell death becoming apparent after 6 hours. Edaravone administration after CCI resulted in a significant reduction in the injury volume and oxidative stress, particularly at the 3-hour time point. Moreover, the greatest decrease in O2 (∙-) levels was observed when edaravone was administered 3 hours following CCI. These findings suggest that edaravone could prove clinically useful to ameliorate the devastating effects of TBI.
    04/2013; 2013(3):379206. DOI:10.1155/2013/379206
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    • "humidified atmosphere. The dose of each cytokine used in this study was chosen based on the available literature (Pietr et al. 2009; Dohi et al. 2010; Liu et al. 2010; Soria et al. 2011). "
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    ABSTRACT: The cellular prion protein (PrP(C) ) is a glycoprotein anchored by glycosylphosphatidylinositol (GPI) to the cell surface and is abundantly expressed in the central nervous system. Numerous studies have suggested a protective function for PrP(C) , including protection from ischemic and excitotoxic lesions and several apoptotic insults, and recent reports have shown that PrP(C) has a context-dependent neuro-protective function. In the present study, we investigated the effect of PPNP downregulation on various forms of microglial activation. We first examined the mRNA expression of PRNP upon exposure to IFN-γ, IL-4, or IL-10 in BV2 microglia. We then analyzed the effect of si-RNA-mediated disruption of PRNP on different parameters of microglial activation in IFN-γ-, IL-4-, or IL-10-stimulated microglia. The results showed that PRNP mRNA expression was invariably downregulated in microglia upon exposure to IFN-γ, IL-4, or IL-10. PRNP silencing prior to cytokines treatment reduced the responsiveness of microglia to INF-γ treatment, significantly altered IL-4-induced microglial activation phenotype, and had no effect on IL-10-induced microglial activation. Together, these results support a role of PrP(C) in the modulation of the shift of microglia from a quiescent state to an activated phenotype and in the regulation of the microglial response during classical and alternative activation. © 2012 International Society for Neurochemistry, J. Neurochem. (2012) 10.1111/jnc.12053.
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