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，导致神经元电路中断和慢性神经功能障碍。随后的神经炎症会加剧组织损伤，并导致神经变性，比最初的损伤严重许多倍。最近的证据表明，作为人体主要的天然免疫细胞，单核细胞来源的巨噬细胞可以加重病理或诱导组织再生，这取决于它们渗透的时间和程度，它们表现的行为表型，以及在组织中停留的时间。然而，巨噬细胞在推动颅脑损伤后继发性损伤病理和再生中所起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，本项目的目的是确定这些细胞在脑外伤病理中的作用，并同时实时跟踪单核细胞的渗透，以产生翻译的、微创的诊断策略。此外，我建议利用高保真的闭合头弥漫性脑损伤的临床前猪模型，以便使用磁共振成像来表征单核细胞在脑中的渗透，以跟踪磁性标记的细胞，并使用成像质量细胞术来表征脑中巨噬细胞的行为。具体地说，猪单核细胞将被外源性地负载磁性微粒，这些装载了磁性微粒的细胞将在脑创伤后静脉注射。此后，将使用磁共振成像(MRI)随时间的推移实时跟踪磁性标记的细胞，以表征细胞渗透到大脑中的特征。我假设，浸润性巨噬细胞将定位于具有微观病理特征的部位，用磁性微粒追踪巨噬细胞浸润可以作为一种新的诊断策略。重要的是，这种猪脑损伤模型是当今使用的最具临床相关性的脑损伤模型，因为该模型与人类头部损伤生物力学密切相关。在目标1中，我将产生、验证和实施一组猪抗体，以利用成像质量细胞术来表征存档的猪脑损伤组织中巨噬细胞的渗透、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，以便实时追踪细胞在脑外伤后的脑内渗透。从这项建议中获得的信息将为脑外伤制定一种与翻译相关的诊断战略，可以改善受影响个人的治疗和护理，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退伍军人医学中心完成，因为它拥有世界上其他地方没有的独特资源和设备。在此期间，我将接触到成像质量细胞仪，120多个猪脑的档案，核磁共振，磁性微粒制造设备，以及导致猪闭合性弥漫性脑损伤的损伤设备。总之，这些资源、实验、赞助商、合作者和机构环境将培养神经免疫相互作用的专业研究利基，这将促进我不断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.