Data_Sheet_1_Mesenchymal Stromal Cell-Derived Extracellular Vesicles Reduce Neuroinflammation, Promote Neural Cell Proliferation and Improve Oligodendrocyte Maturation in Neonatal Hypoxic-Ischemic Brain Injury.pdf

Background: Neonatal encephalopathy caused by hypoxia-ischemia (HI) is a major cause of childhood mortality and disability. Stem cell-based regenerative therapies seem promising to prevent long-term neurological deficits. Our previous work in neonatal HI revealed an unexpected interaction between mesenchymal stem/stromal cells (MSCs) and the brains' microenvironment leading to an altered therapeutic efficiency. MSCs are supposed to mediate most of their therapeutic effects in a paracrine mode via extracellular vesicles (EVs), which might be an alternative to cell therapy. In the present study, we investigated the impact of MSC-EVs on neonatal HI-induced brain injury.Methods: Nine-day-old C57BL/6 mice were exposed to HI through ligation of the right common carotid artery followed by 1 h hypoxia (10% oxygen). MSC-EVs were injected intraperitoneally 1, 3, and 5 days after HI. One week after HI, brain injury was evaluated by regional neuropathological scoring, atrophy measurements and immunohistochemistry to assess effects on neuronal, oligodendrocyte and vessel densities, proliferation, oligodendrocyte maturation, myelination, astro-, and microglia activation. Immunohistochemistry analyses were complemented by mRNA expression analyses for a broad set of M1/M2- and A1/A2-associated molecules and neural growth factors.Results: While total neuropathological scores and tissue atrophy were not changed, MSC-EVs significantly protected from HI-induced striatal tissue loss and decreased micro- and astroglia activation. MSC-EVs lead to a significant downregulation of the pro-inflammatory cytokine TNFa, accompanied by a significant upregulation of the M2 marker YM-1 and the anti-inflammatory cytokine TGFb. MSC-EVs significantly decreased astrocytic expression of the A1 marker C3, concomitant with an increased expression of neural growth factors (i.e., BDNF, VEGF, and EGF). These alterations were associated with an increased neuronal and vessel density, coinciding with a significant increase of proliferating cells in the neurogenic sub-ventricular zone juxtaposed to the striatum. MSC-EV-mediated neuroprotection went along with a significant improvement of oligodendrocyte maturation and myelination.Conclusion: The present study demonstrates that MSC-EVs mediate anti-inflammatory effects, promote regenerative responses and improve key developmental processes in the injured neonatal brain. The present results suggest different cellular target mechanisms of MSC-EVs, preventing secondary HI-induced brain injury. MSC-EV treatment may be a promising alternative to risk-associated cell therapies in neonatal brain injury.

背景：由缺氧缺血（HI）引起的围产期脑病是儿童死亡和残疾的主要原因。基于干细胞的再生疗法似乎有望预防长期神经功能障碍。我们先前在围产期HI研究工作中，揭示了间充质干细胞/基质细胞（MSCs）与大脑微环境之间意外交互作用，导致治疗效率的改变。MSCs被认为主要通过旁分泌途径通过细胞外囊泡（EVs）介导其大部分治疗效应，这可能是细胞疗法的替代方案。在本研究中，我们探讨了MSC-EVs对围产期HI诱导的脑损伤的影响。方法：九日龄的C57BL/6小鼠通过结扎右侧颈总动脉后进行1小时缺氧（10%氧气）处理以暴露于HI。在HI后第1、3和5天，将MSC-EVs腹腔注射。在HI后一周，通过区域神经病理评分、萎缩测量和免疫组化评估对神经元、少突胶质细胞和血管密度、增殖、少突胶质细胞成熟、髓鞘化、星形胶质细胞和微胶质细胞活化的影响来评估脑损伤。免疫组化分析辅以一系列M1/M2-和A1/A2相关分子以及神经生长因子的mRNA表达分析。结果：虽然总的神经病理评分和组织萎缩没有变化，但MSC-EVs显著保护了HI诱导的纹状体组织损失，并减少了微胶质和星形胶质细胞的活化。MSC-EVs导致促炎细胞因子TNFa的显著下调，同时M2标记物YM-1和抗炎细胞因子TGFb的显著上调。MSC-EVs显著降低了A1标记物C3的星形胶质细胞表达，同时神经生长因子（例如，BDNF、VEGF和EGF）的表达增加。这些变化与神经元和血管密度的增加相关，同时与神经发生区临近纹状体的增殖细胞的显著增加相一致。MSC-EV介导的神经保护伴随着少突胶质细胞成熟和髓鞘化的显著改善。结论：本研究表明，MSC-EVs介导抗炎效应，促进再生反应，并改善受损新生儿大脑的关键发育过程。现有结果提示MSC-EVs具有不同的细胞靶机制，预防继发性HI诱导的脑损伤。MSC-EV治疗可能是新生儿脑损伤中风险相关细胞疗法的有希望替代方案。