Neonatal Stroke: The Role of Microglia-derived Extracellular Vesicles

摘要 新生儿(围产期)动脉缺血性中风与老年人一样常见，是导致脑血管疾病的主要原因 长期的神经和认知缺陷，包括脑瘫和神经发育 残疾人士。有文献表明，中风时的大脑发育阶段有一个 主要影响脑损伤的病理生理机制，但尚无有效的研究方法 治疗。炎症是围产期脑损伤的标志，影响早期损伤和脑损伤 在以后的生活中进行修复和连接。我们发现小胶质细胞作为内源性保护剂在 新生大鼠和小鼠的急性中风，包括通过清除凋亡的神经元碎片来保护， 限制神经炎症，保护血脑屏障的完整性。我们将确定是否 小胶质细胞通过释放细胞外小泡(EV)发挥神经和血管保护作用。这个 快速发展的EV场作为细胞间通讯的基本方式而不需要细胞间的直接接触 在健康和患病的生物体中，根据细胞类型的不同，EV显示出异质性 释放它们，释放的机制和神经退化的场景。 我们假设小胶质细胞部分通过释放小胶质细胞来减轻新生儿中风后的损伤。 衍生EV(MEV)。在三个目标中，我们将确定新生儿中风如何改变MEV特性和 它们与急性卒中后分离的脑片细胞和小胶质细胞的通讯(目标1)； 检测脑内脂类CD36依赖信号和EV释放受阻对脑内EV的影响 两种小鼠模型的神经炎症和损伤--新生儿中风或脑内注射IL-1β。 我们将在体内分析EV停止释放对神经元和小胶质细胞转录/蛋白质组的影响 同一损伤新生儿脑内的情况(目标2)；并确定是否在术后早期给予MEV 新生儿中风保护短期和增强长期大脑修复(目标3)。 为了了解MEV在新生儿卒中中的机制作用及其治疗潜力，我们将 利用最先进的实验工具，包括临床相关的围产期局灶性动脉卒中 我们发明的模型，结合功能丧失和功能获得遗传和 药理学方法和先进的非侵入性成像方法。我们会操纵 EV从神经元释放(清晰编辑方法)，分离和表征单个EV和MEV 分数(超灵敏ImageStreamX技术和MEV“货物”)，图像MEV通信 利用静息/激活的小胶质细胞和血管系统(实时成像)，并利用新的双转基因 RiboTag小鼠在活体内实时检测小胶质细胞-神经元分子串扰。建议进行的研究 将增进对细胞信号和通信的了解，并有助于实现我们的长期 目的寻找新的治疗靶点，为新生儿卒中创造安全有效的治疗方法。

Abstract Neonatal (perinatal) arterial ischemic stroke is as common as in the elderly and is a major cause of long-term neurological and cognitive deficits, including cerebral palsy and neurodevelopmental disabilities. Literature has emerged that the stage of brain development at the time of stroke has a major impact on the pathophysiological mechanisms of brain damage, but there are no effective treatments. Inflammation is a hallmark of perinatal brain injury and affects both early injury and brain repair and connectivity later in life. We discovered that microglia serve as endogenous protectants after acute stroke in neonatal rats and mice, including protection by removing apoptotic neuronal debris, limiting neuroinflammation and protecting blood-brain barrier integrity. We will determine whether microglial cells exert neuro- and vasoprotective effects via release of extracellular vesicles (EV). The fast-growing EV field as the fundamental way of cell-cell communication without direct cell-cell contacts in healthy and diseased organism has demonstrated heterogeneity of EV depending on the cell type releasing them, the mechanism of release and a neurodegenerative scenario. We hypothesize that microglial cells alleviate injury after neonatal stroke in part via released microglia- derived EV (MEV). In three aims, we will determine how neonatal stroke changes MEV properties and their communication with cells in brain slices and microglial cells that isolated after acute stroke (Aim 1); examine effects of disrupted lipid CD36-dependent signalling and disrupted EV release in the brain on neuroinflammation and injury in two mouse models—neonatal stroke or intracerebral IL1beta injection. We will analyse in vivo effects of halted EV release on neuronal and microglial transcriptome/proteome profiles in same injured neonatal brain (Aim 2); and determine if MEV administration early after neonatal stroke protects short-term and enhance long-term brain repair (Aim 3). To understand the mechanistic role of MEV and their therapeutic potential for neonatal stroke, we will utilize state-of-the art experimental tools, including a clinically relevant perinatal focal arterial stroke model that we invented, in conjunction with loss-of-function and gain-of function genetic and pharmacological approaches and advanced non-invasive imaging methodologies. We will manipulate EV release from neurons (CRISP editing approach), isolate and characterize individual EV and MEV fractions (super-sensitive ImageStreamX technology and MEV “cargo”), image MEV communication with resting/activated microglia and the vasculature (live imaging), and utilize novel double transgenic RiboTag mice to examine in vivo real time microglia-neuron molecular crosstalk. The proposed studies will enhance the understanding of cell signalling and communication and help achieve our long-term goal to identify novel therapeutic targets to create effective and safe therapy for neonatal stroke.