The roles and the kinetics of gp91 phox after traumatic brain injury in mice

前言：我们假设NADPH氧化酶的一个亚基gp91Phox(NOX2)产生超氧阴离子(O2-)，在创伤性脑损伤(TBI)中起主要作用。为了评估gp91Phox和活性氧物种(ROS)在脑损伤中的作用，我们在gp91Phox基因敲除小鼠(gp91Phox-/-)中进行了受控皮质撞击。我们还使用小胶质细胞系来确定参与gp91Phox生成的激活细胞表型。方法：在gp91Phox-/-和野生型(Wt)小鼠(C57/B6J)(25-30g)诱导单侧脑损伤。应用免疫印迹和染色技术研究gp91Phox在脑外伤后的表达及作用。在小鼠脑内原位测定O2-和过氧亚硝酸根的水平。小胶质细胞系BV-2在不同浓度的干扰素和IL-4作用下，检测小胶质细胞中表达gp91Phox的活化表型。结果：创伤后Wt小鼠同侧大脑半球小胶质细胞中gp91Phox的表达增加，主要是阿米巴样小胶质细胞。Gp91Phox-/-小鼠的挫伤面积、TUNEL阳性细胞数、O2-和过氧亚硝酸根代谢产物的产生量均小于Wt小鼠。在干扰素γ作用下，BV-2细胞诱导型一氧化氮合酶和一氧化氮水平升高，gp91phx表达显著增加，与经典激活表型一致。结论：经典激活的小胶质细胞通过gp91Phox促进ROS的形成，在脑损伤中起重要作用。调节gp91Phox及其衍生的ROS可能为治疗创伤后脑损伤提供一种新的治疗策略。

INTRODUCTION :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.METHODS :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.RESULTS :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.CONCLUSIONS :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.