INOS AND TBI

诱导型一氧化氮合酶(INOS)是一种参与炎症反应的一氧化氮合酶亚型。INOS可能控制着损伤、保护和修复过程中的关键机制，包括血管调节、炎症、细胞保护、细胞毒性和再生。在创伤性脑损伤中，诱导型一氧化氮合酶在创伤周围区的多种细胞中表达。我们对脑外伤后的长期结果进行了研究，并观察到iNOS在两个物种中具有强大的内源性神经保护作用。我们的研究得到了越来越多的文献的支持，这些文献揭示了iNOS在应对中枢神经系统内外损伤时的重要有益作用。这一提议的假设是，iNOS表达为脑损伤，是一种强大的内源性神经保护剂。具体目标如下：1)测定实验性脑损伤后大鼠和小鼠诱导型一氧化氮合酶的时间进程、诱导型一氧化氮合酶的大小和细胞定位；2)利用大鼠诱导型一氧化氮合酶抑制剂和诱导型一氧化氮合酶KO小鼠，检测诱导型一氧化氮合酶是否为内源性神经保护剂，并改善脑创伤后的组织病理学和功能预后。3)在小鼠和大鼠中测试iNOS的过度表达在脑损伤后是否具有神经保护作用，4)在我们的小鼠和大鼠模型中确定iNOS是如何赋予其神经保护作用的，包括评估下游介质如细胞因子、神经生长因子(NGF)和脑血流量(CBF)，以及5)确定在严重脑损伤患者中，NO的全局和局部产生，分别通过脑脊液(CSF)和脑间质液中的亚硝酸盐/硝酸盐水平来评估并确定诱导型一氧化氮合酶在人脑挫伤中的诱导时程、大小和细胞定位。我们建立的CCI模型将用于在小鼠和大鼠身上产生脑损伤。用RT-PCR、酶活性和免疫组织化学方法研究iNOS的表达。将使用两种诱导型一氧化氮合酶抑制剂(氨基胍和N6-(氨基乙基1)-L-赖氨酸)和诱导型一氧化氮合酶KO小鼠。表达人诱导型一氧化氮合酶的复制缺陷腺病毒将用于体内脑区的转基因，包括损伤前和损伤后。结果评估将包括运动和认知(Morris水迷宫)任务、组织病理学、CBF(灌注核磁共振)、细胞因子和NGF(ELISA法)以及巨噬细胞/淋巴细胞浸润(通过免疫组织化学和流式细胞仪分析)。在患有严重脑外伤的人中，亚硝酸盐/硝酸盐水平将被用作脑脊液和脑间质液(微透析)中NO的标志。采用免疫组织化学方法对急诊切除挫伤患者的脑组织样本进行研究。确认iNOS是一种神经保护剂，表明iNOS的过度表达是有益的，并确定这一效应所涉及的机制是走向新治疗的关键步骤。最后，这些研究将为脑外伤的这一重要机制统一起来。

Inducible nitric oxide synthase (iNOS) is a NOS isoform that is involved in the inflammatory response. iNOS may govern key mechanisms in the evolution of injury, protection and repair, including vascular regulation, inflammation, cytoprotection, cytotoxicity, and regeneration. In the traumatic brain injury (TBI), iNOS is expressed in a variety of cell types in the peri-trauma region. We performed studies examining long-term outcome after TBI and observed a powerful endogenous neuroprotectant effect of iNOS in two species. Our studies are supported by an expanding body of literature revealing important beneficial effects of iNOS in response to injury inside and outside of the CNS. The hypothesis of this proposal is that iNOS is expressed that TBI and is a powerful endogenous neuroprotectant. Specific aims are as follows: 1) Determine the time course, magnitude, and cellular localization of iNOS induction after experimental TBI in both mice and rats, 2) Test whether iNOS is an endogenous neuroprotectant and improves both histopathological and functional outcome after TBI, using both iNOS KO mice and iNOS inhibitors in rats. Also investigate the possibility that there is a biphasic role of iNOS after TBI, with early detrimental effects, but beneficial effects overall, 3) Test in both mice and rats if over-expression of iNOS by gene transfer with adenovirus-based vector is neuroprotective after TBI, 4) Determine in our mouse and rat models how iNOS confers its neuroprotective effects, including evaluation of downstream mediators such as cytokines, nerve growth factor (NGF), and cerebral blood flow (CBF), and 5) Define, in humans with severe TBI, the global and local production of NO, as assessed by nitrite/nitrate levels in cerebrospinal fluid (CSF) and brain interstitial fluid, respectively, and determine the time course, magnitude, and cellular localization of iNOS induction in human cerebral contusions. Our established CCI models will be used to produce TBI in mice and rats. Expression of iNOS will be studied using RT-PCR, enzyme activity, and immunohistochemistry. Two inhibitors of iNOS (aminoguanidine and N6-(ininoethyl1)-L-lysine) and iNOS KO mice will be used. A replication deficient adenovirus that expresses human iNOS will be used to transfect brain regions in vivo, both before and after injury. Outcome evaluation will include motor and cognitive (Morris water maze) tasks, histopathology, CBF (perfusion NMR), cytokines and NGF (ELISA), and macrophage/lymphocyte infiltration (by immunohistochemistry and flow cytometric analysis). In humans with severe TBI, nitrite/nitrate levels will be used as a marker of NO in both CSF and brain interstitial fluid (microdialysis). Brain samples from patients undergoing emergency resection of contusion, will be studied using immunohistochemistry. Confirming that iNOS is a neuroprotectant, showing that over-expression of iNOS as beneficial, and defining the mechanisms involved in this effect are key steps toward the of a novel treatment. Finally, these studies will unite bench to bedside for this important mechanism in TBI.