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.

诱导一氧化氮合酶（INOS）是参与炎症反应的NOS同工型。 iNOS可以控制损伤，保护和修复演变的关键机制，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以多种细胞类型表达。我们进行了研究，研究了TBI之后的长期结局，并观察到iNOS在两个物种中具有强大的内源性神经保护剂作用。我们的研究得到了不断扩大的文献支持，揭示了INOS对中枢神经系统内部和外部的伤害的重要有益作用。该提议的假设是iNOS表示TBI是一种强大的内源性神经保护剂。具体目的如下：1）确定在小鼠和大鼠实验性TBI后iNOS诱导的时间过程，大小和细胞定位，2）测试INOS是否是内源性神经保护剂，并在TBI后使用INOS KO MICE和INOS抑制剂来改善TBI后的组织病理学和功能性结果。 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.我们已建立的CCI模型将用于在小鼠和大鼠中生产TBI。将使用RT-PCR，酶活性和免疫组织化学研究iNOS的表达。将使用两个iNOS（氨基瓜胺和N6-（Ininoethyl1）-L-赖氨酸）和iNOS KO小鼠的抑制剂。表达人iNOS的复制缺陷腺病毒将用于在损伤前后的体内转染大脑区域。结果评估将包括运动和认知（Morris Water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化学分析）。在患有严重TBI的人类中，亚硝酸盐/硝酸盐水平将用作CSF和脑间质性液（微透析）中NO的标记。将使用免疫组织化学研究接受紧急切除或要进行紧急切除的患者的大脑样本。确认iNOS是一种神经保护剂，表明对iNOS的过表达是有益的，并且定义了这种效果所涉及的机制是迈向新治疗的关键步骤。最后，这些研究将在TBI中的这一重要机制中将基准统一为床边。