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，导致神经回路中断和慢性神经功能障碍。继发的神经炎症可加重组织损伤并诱发神经退行性变，其程度比原始损伤严重许多倍。最近的证据表明，单核细胞来源的巨噬细胞，机体的主要先天免疫细胞，可以加剧病理或诱导组织再生，这取决于它们浸润的时间和程度，它们表达的行为表型，以及在组织中停留的时间。然而，由于研究结果不完整或相互矛盾，巨噬细胞在TBI后继发性损伤病理和再生中的作用在很大程度上存在争议。因此，该项目的目的是表征这些细胞对TBI病理的贡献，同时实时跟踪单核细胞浸润，以产生可翻译的微创诊断策略。此外，我建议利用一个高保真的闭合性头部弥漫性脑损伤猪临床前模型，利用磁共振成像跟踪磁标记细胞来表征单核细胞浸润到大脑中的情况，并利用成像细胞计数技术表征巨噬细胞在大脑中的行为。具体来说，猪单核细胞将外源性装载磁性微粒，这些微粒装载的细胞将在TBI后静脉注射。此后，磁性标记的细胞将随着时间的推移被磁共振成像（MRI）实时跟踪，以表征细胞对大脑的浸润。我假设浸润的巨噬细胞会以微观病理特征定位，并且用磁微粒跟踪巨噬细胞浸润可以作为一种新的诊断策略。重要的是，这个猪脑外伤模型是目前使用的最具临床相关性的脑外伤模型，因为这个模型非常接近地复制了人类头部损伤的生物力学。在目标1中，我将生成、验证并实施一组猪抗体，用成像细胞计数技术表征存档的猪TBI组织中巨噬细胞的浸润、分布和表型。在目标2中，我将合成、表征和静脉注射磁性标记的单核细胞给猪，以便在TBI后实时跟踪细胞浸润到大脑中。从该提案中获得的信息将为TBI开发一种翻译相关的诊断策略，可以改善受影响个体的治疗和护理，并为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，因为他们拥有世界上其他地方无法获得的独特资源和设备。在此期间，我将接触到成像细胞仪，120多个猪脑的档案，核磁共振成像，磁微粒制造设备，以及诱导猪闭头弥漫性脑损伤的损伤装置。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研究神经免疫相互作用的利基，这将促进我正在发展的研究事业的发展。

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.