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的主要原因 缺陷，包括脑瘫和神经发育障碍。而新生儿中风则被视为 与老年人一样常见的是，文学已经出现了大脑发育阶段的时候 中风对脑损伤的病理生理机制有重要影响。上一首 治疗的努力主要集中在敏锐的神经元保护上，但这样的策略似乎 短程飞行。我们报道了间充质干细胞(MSC)延迟鼻腔给药 保护脑白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模型。细胞外小泡(EV)现在是 被认为在健康的细胞与细胞之间没有直接接触的情况下，在细胞之间的交流中起着基本的作用 和患病的有机体，而EV是神经退化场景的一部分。根据我们的初步调查 从MSC释放的外切体(MSC-exo)保护亚急性中风后新生儿大脑的数据，在 我们推测，MSC-exo是MSC诱导急性白血病的潜在机制。 通过调节小胶质细胞信号调节对新生儿卒中后的神经保护和长期康复。 鉴于炎症是围产期脑损伤的标志，影响早期损伤和脑修复 以及小胶质细胞对新生儿的神经和血管保护作用 我们将确定非鼻腔给药的MSC-exo是如何被激活的 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 TMCAO雌雄小鼠及MSC-exo是否通过改变脑微环境 从小胶质细胞释放微囊和小EV(目标1)，并确定 MSC-exo给药对髓鞘形成、脑修复和功能结果的影响(目标2)。要理解 MSC-exo的机制作用及其治疗新生儿卒中的潜力，我们将利用状态- ART实验工具，包括临床相关的围产期局灶性动脉卒中模型，我们 发明，结合药理学方法和先进的非侵入性成像 方法学(NanoSight、超分辨流式细胞术Alexa)和大/小的表征 EV和它们的“货物”从损伤区域的小胶质细胞释放出来。这本书的意义和新颖性 建议的研究是为了促进对MSC-exo诱导的细胞类型的机制的理解 卒中后对新生儿脑的特异性影响及寻找新的治疗靶点以创造有效 以及对新生儿中风的安全治疗。

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.