Exosomes as the mechanism of mesenchymal stem cell brain repair in neonatal stroke

摘要 新生儿（围产期）动脉缺血性卒中是长期神经和认知功能障碍的主要原因 缺陷，包括脑瘫和神经发育障碍。虽然新生儿中风 在老年人中很常见，文献已经出现了大脑发育的阶段， 中风对脑损伤的病理生理学机制具有重大影响。先前 治疗努力主要集中在急性保护神经元，但这些策略似乎 短距离。我们报道了延迟鼻内注射间充质干细胞（MSC） 保护白色物质并改善长期功能结果的实验模型， 新生大鼠短暂性大脑中动脉闭塞（tMCAO）。细胞外囊泡（EV）现在 被认为在细胞间通讯中起着重要作用，而在健康的人中没有直接的细胞间接触。 和患病的生物体，EV是神经退行性疾病的一部分。根据我们初步的 从MSC释放的外泌体（MSC-exo）保护亚急性中风后新生儿大脑的数据， 我们假设MSC-exo是MSC诱导急性心肌梗死的潜在机制。 通过调节小胶质细胞信号传导，在新生儿中风后提供神经保护和长期恢复。 鉴于炎症是围产期脑损伤的标志，影响早期损伤和脑修复， 以及小胶质细胞有助于新生儿的神经和血管保护。 中风，我们将确定如何摄取非经鼻管理MSC-exo的激活 缺血再灌注区小胶质细胞/巨噬细胞对新生儿神经炎症和损伤的影响 两种性别的小鼠进行tMCAO和MSC-exo是否改变脑微环境， 从小胶质细胞中释放微泡和小EV（目的1），并确定 MSC-exo给药对髓鞘形成、脑修复和功能结局的影响（目的2）。了解 MSC-exo的机制作用及其对新生儿卒中的治疗潜力，我们将利用国家- 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模型， 与药理学方法和先进的非侵入性成像技术相结合， 方法（NanoSight，超分辨率流式细胞术Alexa）和大/小细胞的表征 EV和它们的“货物”从受伤区域的小胶质细胞释放出来。的重要性和新奇， 提出的研究是在推进机制的理解，MSC外诱导的细胞类型， 在新生儿脑卒中后的具体影响，并确定新的治疗目标，以创造有效的 安全的治疗方法。

Abstract Neonatal (perinatal) arterial ischemic stroke is a major cause of long-term neurological and cognitive deficits, including cerebral palsy and neurodevelopmental disabilities. While neonatal stroke is as common as in the elderly, 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. Previous therapeutic efforts were mostly focused on protecting neurons acutely, but such strategies appeared to be short-range. We reported that delayed intranasal administration of mesenchymal stem cells (MSC) protects the white matter and improves long-term functional outcomes in an experimental model of a transient middle cerebral artery occlusion (tMCAO) in neonatal rats. Extracellular vesicles (EV) are now believed to play fundamental role in cell-cell communication without direct cell-cell contacts in healthy and diseased organism and that EV is a part of neurodegenerative scenarios. Based on our preliminary data that exosomes released from MSC (MSC-exo) protect neonatal brain following subacute stroke, in this proposal we hypothesize that MSC-exo is the underlying mechanism of MSC-induced acute neuroprotection and long-term recovery after neonatal stroke via modulation of microglial cell signaling. Given that inflammation is a hallmark of perinatal brain injury, affecting both early injury and brain repair and connectivity later in life, and that microglial cells contribute to neuro- and vasoprotection in neonatal stroke, we will determine how uptake of untranasally administered MSC-exo by activated microglia/macrophages in ischemic-reperfused regions affects neuroinflammation and injury in neonatal mice of both sexes subjected to tMCAO and whether MSC-exo alter brain microenvironment via release of microvesicles and small EV from microglia (Aim 1), and determine the long-term effects of MSC-exo administration on myelination, brain repair and functional outcomes (Aim 2). To understand the mechanistic role of MSC-exo 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 pharmacological approaches and advanced non-invasive imaging methodologies (NanoSight, super resolution flow cytometry Alexa) and characterization of large/small EV and their “cargo” released from microglia from injured regions. The significance and novelty of the proposed studies are in advancing the mechanistic understanding of MSC-exo-induced cell-type specific effects in neonatal brain after stroke and identifying novel therapeutic targets to create effective and safe therapy for neonatal stroke.