Precision Identification and Targeting of Rod Microglia in Diffuse Brain-Injured Cortex

项目总结： 在实验性弥漫性脑损伤(TBI)后，我们重新发现了杆状小胶质细胞 在大脑的病理或修复中激活的小胶质细胞的变种。图中描绘的是杆状小胶质细胞。 尼塞尔、卡哈尔和德尔·里奥·霍特加在全身瘫痪的情况下，但在大约100年的时间里基本上被忽视了。 最近在人类阿尔茨海默病、自闭症谱系障碍和 脑损伤、细菌感染和癫痫的实验模型，但没有功能的报道。 我们重复地观察到视杆状小胶质细胞在邻近神经元突起的皮质层中成串排列。 弥漫性脑损伤后的神经病理灶形成和消退的时间进程与 神经病理学。然而，先进的分子工具缺乏理解ROD所必需的武器库 小胶质细胞的结构和功能。我们假设视杆状小胶质细胞具有独特的分子图谱。 对于其他小胶质细胞形态，这为证实弥漫性脑损伤中杆状小胶质细胞的机制提供了工具。 为了研究杆状小胶质细胞在弥漫性脑损伤中的意义和影响，目前的提议将提供关键的 噬菌体展示分子图谱技术区分杆状小胶质细胞与活化及分支小胶质细胞的数据 生物扫描和RNA测序。我们将通过中线液压冲击伤诱导成年男性弥漫性颅脑损伤 雌性CX3CR1-EGFP小鼠，基于形态学鉴定杆状小胶质细胞。激光捕获显微解剖将 分离皮质区域的视杆状小胶质细胞和非视杆状小胶质细胞(激活和分支)。AIM 1中的隔离细胞 将用于噬菌体展示生物扫描，以开发和验证杆状小胶质细胞特有的细胞表面标记。 AIM 2中分离的细胞将用于下一代测序(RNAseq)以揭示上调基因 视杆状小胶质细胞的表达与激活和分支形态的比较。通径分析将是 掺入以确定与杆状小胶质细胞的功能和来源相关的独特的表达结构域。这个 赞助商和指导团队提供集体经验和专业知识，为以下人员提供高级培训 申请者要达到目的。来自这项提议的数据将定义杆状小胶质细胞的分子轮廓和结果 在为杆状小胶质细胞的定量和分离方案开发的工具中。有了精致的分子工具，棒 可以跨神经疾病(例如阿尔茨海默病、自闭症谱系)研究小胶质细胞 精神障碍、癫痫)，并被激活或抑制，以推动神经症状的治疗。

PROJECT SUMMARY: After experimental diffuse traumatic brain injury (TBI), we rediscovered the rod microglia – a morphological variant of activated microglia in the pathology or repair of the brain. Rod microglia are depicted in the drawings of Nissl, Cajal, and del Rio Hortega in cases of general paresis, but were largely ignored for around 100 years. Rod microglia have recently been visualized in human Alzheimer’s disease, Autism spectrum disorder and experimental models of TBI, bacterial infection, and seizure, but no function has been reported. We reproducibly observe rod microglia that align in trains across cortical layers adjacent to neuronal processes in foci of neuropathology after diffuse TBI and they form and recede in a time course consistent with neuropathology. Yet, advanced molecular tools are absent from the arsenal necessary to understand rod microglia structure and function. We hypothesize that rod microglia have a unique molecular profile compared to other microglia morphologies, which provide tools to confirm rod microglia mechanisms in diffuse TBI. To investigate the significance and impact of rod microglia in diffuse TBI, the current proposal will provide critical data to distinguish rod microglia from activated and ramified microglia by molecular profile using phage display biopanning and RNA sequencing. We will induce diffuse TBI by midline fluid percussion injury in adult male and female CX3Cr1-eGFP mice and identify rod microglia based on morphology. Laser capture microdissection will isolate rod microglia and non-rod microglia (activated and ramified) from cortical regions. Isolated cells in aim 1 will be used for phage display biopanning to develop and validate a cell surface marker unique to rod microglia. Isolated cells in aim 2 will be used for next-generation sequencing (RNAseq) to reveal upregulated gene expression in rod microglia compared to activated and ramified morphologies. Pathway analysis will be incorporated to identify unique expression domains associated with the function and origin of rod microglia. The sponsor and mentoring team provide collective experience and expertise to provide advanced training for the applicant to achieve the aims. Data from this proposal will define the molecular profile of rod microglia and result in developed tools for quantitative and isolation protocols of rod microglia. With refined molecular tools, rod microglia can be investigated across neurological conditions (e.g. Alzheimer’s disease, Autism spectrum disorder, seizure) and activated or inhibited to drive treatment of neurological symptoms.