INOS AND TBI

伊诺斯和TBI

• 批准号: 6448241
• 负责人: PATRICK M KOCHANEK
• 金额: $ 20.73万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6448241
• 关键词: Adenoviridae; brain circulation; brain injury; clinical research; cytokine; disease /disorder model; human subject; laboratory mouse; laboratory rat; neuroprotectants; nitric oxide synthase; pathologic process; protein biosynthesis; transfection; trauma

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.

诱导型一氧化氮合酶 (iNOS) 是一种参与炎症反应的 NOS 亚型。 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 在创伤周围区域的多种细胞类型中表达。我们进行了研究，检查 TBI 后的长期结果，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方面的重要有益作用。该提议的假设是 iNOS 在 TBI 中表达，并且是一种强大的内源性神经保护剂。具体目标如下：1) 确定小鼠和大鼠实验性 TBI 后 iNOS 诱导的时间过程、强度和细胞定位，2) 测试 iNOS 是否是一种内源性神经保护剂并改善 TBI 后的组织病理学和功能结果，使用iNOS KO 小鼠和大鼠 iNOS 抑制剂。还研究了 TBI 后 iNOS 存在双相作用的可能性，具有早期有害影响，但总体上有利影响，3) 在小鼠和大鼠中测试通过基于腺病毒的载体进行基因转移的 iNOS 过度表达是否具有神经保护作用TBI，4) 在我们的小鼠和大鼠模型中确定 iNOS 如何赋予其神经保护作用，包括评估下游介质，如细胞因子、神经生长因子 (NGF) 和脑血流(CBF) 和 5) 定义患有严重 TBI 的人类的整体和局部 NO 产生，分别通过脑脊液 (CSF) 和脑间质液中的亚硝酸盐/硝酸盐水平进行评估，并确定时间进程、幅度，以及人脑挫伤中 iNOS 诱导的细胞定位。我们建立的 CCI 模型将用于在小鼠和大鼠中产生 TBI。将使用 RT-PCR、酶活性和免疫组织化学研究 iNOS 的表达。将使用两种 iNOS 抑制剂（氨基胍和 N6-（亚乙基 1）-L-赖氨酸）和 iNOS KO 小鼠。表达人类 iNOS 的复制缺陷型腺病毒将用于在损伤前后体内转染大脑区域。结果评估将包括运动和认知（莫里斯水迷宫）任务、组织病理学、CBF（灌注 NMR）、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术分析）。在患有严重 TBI 的人类中，亚硝酸盐/硝酸盐水平将用作脑脊液和脑间质液（微透析）中 NO 的标志物。将使用免疫组织化学研究接受紧急挫伤切除术的患者的脑样本。证实 iNOS 是一种神经保护剂，表明 iNOS 的过度表达是有益的，并确定这种作用所涉及的机制，是开发新疗法的关键步骤。最后，这些研究将把实验室和临床结合起来，研究 TBI 中的这一重要机制。