INOS AND TBI

诱导型一氧化氮合酶（iNOS）是一氧化氮合酶的一种亚型，参与炎症反应。iNOS可能控制损伤、保护和修复进化的关键机制，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（TBI）中，iNOS在创伤周围的多种细胞类型中表达。我们对创伤性脑损伤后的长期预后进行了研究，并在两个物种中观察到iNOS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对中枢神经系统内外损伤的重要有益作用。本研究假设iNOS在脑外伤中表达，是一种强大的内源性神经保护剂。具体目的如下：1)确定小鼠和大鼠实验性脑外伤后iNOS诱导的时间过程、强度和细胞定位；2)使用iNOS KO小鼠和iNOS抑制剂测试iNOS是否为内源性神经保护剂，并改善脑外伤后的组织病理学和功能结局。也调查的可能性有两相的作用伊诺的创伤性脑损伤后早期的不利影响,但是总体有利影响,3)测试在小鼠和大鼠如果伊诺的过表达基因转移与adenovirus-based向量是创伤性脑损伤后神经保护,4)决定在我们的小鼠和大鼠模型如何伊诺授予其神经保护作用,包括评估下游介质如细胞因子、神经生长因子(神经生长因子)和脑血流量(CBF)、5)通过脑脊液（CSF）和脑间质液中亚硝酸盐/硝酸盐水平分别评估严重TBI患者体内一氧化氮（NO）的整体和局部生成，并确定人类脑挫伤中iNOS诱导的时间过程、强度和细胞定位。我们建立的CCI模型将用于小鼠和大鼠的TBI。iNOS的表达将通过RT-PCR、酶活性和免疫组织化学进行研究。将使用两种iNOS抑制剂（氨基胍和N6-（氨基乙基1）- l -赖氨酸）和iNOS KO小鼠。一种表达人类iNOS的复制缺陷腺病毒将被用于在损伤前后转染体内脑区域。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）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分析）。在严重脑外伤患者中，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的标记物。采用免疫组织化学方法研究紧急切除挫伤患者的脑样本。确认iNOS是一种神经保护剂，表明iNOS的过度表达是有益的，并确定这种作用的机制是开发新治疗方法的关键步骤。最后，这些研究将把这一重要的脑外伤机制从实验到临床结合起来。

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.