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：缺氧缺血（HI）引起的新生儿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的神经功能缺损。我们之前在新生儿HI中的研究表明，间充质干/基质细胞（MSC）与大脑的微环境之间存在意想不到的相互作用，从而导致治疗效率的改变。MSC被认为可以通过旁分泌模式通过细胞外囊泡（EVs）介导大多数治疗作用，这可能是细胞疗法的替代方法。在本研究中，我们调查了MSC-EV对新生儿HI所致脑损伤的影响。

方法：通过结扎右颈总动脉，然后缺氧1 h（10％氧气），将9天大的C57BL / 6小鼠暴露于HI。HI后1、3和5天腹膜内注射MSC-EV。HI后一周，通过区域神经病理学评分，萎缩测量和免疫组织化学评估脑损伤，以评估其对神经元，少突胶质细胞和血管密度，增殖，少突胶质细胞成熟，髓鞘形成，星形胶质细胞和小胶质细胞活化的影响。免疫组织化学分析得到了广泛的M1 / M2-和A1 / A2相关分子和神经生长因子的mRNA表达分析的补充。

结果：虽然总神经病理学评分和组织萎缩没有改变，但MSC-EVs可以显着保护免受HI引起的纹状体组织损失，并减少微和星形胶质细胞的活化。MSC-EVs导致促炎细胞因子TNFa显着下调，同时伴随M2标志物YM-1和抗炎细胞因子TGFb显着上调。MSC-EVs显着降低了A1标记物C3的星形细胞表达，并伴有神经生长因子（即BDNF，VEGF和EGF）表达的增加。这些改变与神经元和血管密度的增加有关，与并列在纹状体的神经源性脑室下区域中增殖细胞的显着增加相一致。

结论：本研究表明，MSC-EVs介导抗炎作用，促进再生反应并改善受伤的新生儿大脑中的关键发育过程。目前的结果表明，MSC-EVs具有不同的细胞靶向机制，可预防继发性HI引起的脑损伤。MSC-EV治疗可能是新生儿脑损伤中与风险相关的细胞疗法的有前途的替代方法。