Understanding and Employing Innate Immune Cell Infiltration into the Brain Following Trauma

创伤性脑损伤（TBI）每年影响数百万人，导致神经元回路中断和慢性神经功能缺损。随后的神经炎症可加剧组织损伤并诱导比原始损伤严重许多倍的神经变性。最近的证据表明，单核细胞衍生的巨噬细胞（身体的主要先天免疫细胞）可以加剧病理或可以诱导组织再生，这取决于它们的浸润的时间和程度、它们表达的行为表型以及在组织中花费的时间持续时间。然而，巨噬细胞在TBI后驱动继发性损伤病理和再生中的作用在很大程度上是有争议的，因为研究结果不完整或相互矛盾。因此，该项目的目的是表征这些细胞对TBI病理学的贡献，并同时在真实的时间内跟踪单核细胞浸润，以产生转化的微创诊断策略。此外，我建议利用闭合头弥漫性TBI的高保真临床前猪模型，以便使用磁共振成像来表征单核细胞浸润到脑中，以跟踪磁标记的细胞，并使用成像质谱细胞术来表征脑中的巨噬细胞行为。具体地，猪单核细胞将外源性负载磁性微粒，并且这些负载微粒的细胞将在TBI后静脉内施用。此后，将用磁共振成像（MRI）随时间真实的跟踪磁性标记的细胞，以表征细胞浸润到脑中。我推测，浸润巨噬细胞将本地化的微观病理特征和跟踪巨噬细胞浸润与磁性微粒可以被用作一种新的诊断策略。重要的是，这种TBI的猪模型是目前使用的最具临床相关性的TBI模型，因为该模型密切复制了人类头部损伤的生物力学。在目标1中，我将生成、验证和实施一组猪抗体，以利用成像质谱细胞术表征存档的猪TBI组织中的巨噬细胞浸润、分布和表型。在目标2中，我将合成、表征和静脉内给予猪磁性标记的单核细胞，以便在TBI后真实的时间跟踪细胞浸润到脑中。从该提案中获得的信息将为TBI制定一种与诊断相关的诊断策略，可以改善受影响个体的治疗和护理，并为有关神经免疫相互作用的基础科学问题提供信息。重要的是，这项研究只能在宾夕法尼亚大学和VA医学中心完成，因为它拥有世界其他任何地方都无法获得的独特资源和设备。在这次研究期间，我将有机会使用成像质量细胞计数器，超过120个猪脑的档案，MRI，磁性微粒制造设备，以及诱导猪闭合头弥漫性TBI的损伤装置。总之，这些资源，实验，赞助商，合作者和机构环境将培养一个专门的研究利基神经免疫相互作用，这将促进我的发展研究生涯的增长。

Traumatic brain injury (TBI) affects millions of individuals annually resulting in disrupted neuronal circuitry and chronic neurological deficits. Subsequent neuroinflammation can exacerbate tissue damage and induce neurodegeneration that is many times worse than the original injury. Recent evidence suggests that monocyte- derived macrophages, the primary innate immune cells of the body, can exacerbate pathology or can induce tissue regeneration depending on the timing and extent of their infiltration, the behavioral phenotype they express, and the duration of time spent in the tissue. However, the role that macrophages play in driving secondary injury pathology and regeneration after TBI is largely contested because of incomplete or contradictory findings. Therefore, the purpose of this project is to characterize the contribution of these cells to TBI pathology and to simultaneously track monocyte infiltration in real time to generate a translational, minimally- invasive diagnostic strategy. Additionally, I propose to utilize a high-fidelity preclinical porcine model of closed- head diffuse TBI in order to characterize monocyte infiltration into the brain using magnetic resonance imaging to track magnetically-labeled cells and characterize macrophage behavior in the brain using imaging mass cytometry. Specifically, porcine monocytes, will be exogenously loaded with magnetic microparticles and these microparticle-loaded cells will be administered intravenously after TBI. Thereafter, magnetically labeled cells will be tracked in real time with magnetic resonance imaging (MRI) over time to characterize cell infiltration into the brain. I hypothesize that infiltrating macrophages will localize with micro-pathological features and that tracking macrophage infiltration with magnetic microparticles can be utilized as a novel diagnostic strategy. Importantly, this porcine model of TBI is the most clinically relevant model of TBI in use today, as this model closely replicates human head injury biomechanics. In Aim 1, I will generate, validate, and implement a panel of porcine antibodies to characterize macrophage infiltration, distribution, and phenotype in archived porcine TBI tissue with imaging mass cytometry. In Aim 2, I will synthesize, characterize, and administer magnetically labeled monocytes intravenously to swine in order to track cell infiltration into the brain in real time after TBI. Information gained from this proposal will develop a translationally relevant diagnostic strategy for TBI that could improve treatment and care in affected individuals and inform basic science questions about neuro-immune interactions. Importantly, this research can only be completed at the University of Pennsylvania and VA Medical Center because of unique resources and equipment that is not available anywhere else in the world. During this fellowship, I will have access to an imaging mass cytometer, an archive of over 120 porcine brains, an MRI, equipment for magnetic microparticle fabrication, and the injury device that induces the porcine closed-head diffuse TBI. Together, these resources, experiments, sponsor, collaborators, and institutional environment will cultivate a specialized research niche on neuro-immune interactions that will foster the growth of my developing research career.