Department of Obstetrics and Feto-maternal Medicine, Inselspital, Bern University Hospital, University of Bern, 3010 Bern, Switzerland.
Department for BioMedical Research (DBMR), University of Bern, 3012 Bern, Switzerland.
Cells. 2019 Aug 8;8(8):855. doi: 10.3390/cells8080855.
Perinatal brain injury (PBI) in preterm birth is associated with substantial injury and dysmaturation of white and gray matter, and can lead to severe neurodevelopmental deficits. Mesenchymal stromal cells (MSC) have been suggested to have neuroprotective effects in perinatal brain injury, in part through the release of extracellular vesicles like exosomes. We aimed to evaluate the neuroprotective effects of intranasally administered MSC-derived exosomes and their potential to improve neurodevelopmental outcome after PBI. Exosomes were isolated from human Wharton's jelly MSC supernatant using ultracentrifugation. Two days old Wistar rat pups were subjected to PBI by a combination of inflammation and hypoxia-ischemia. Exosomes were intranasally administered after the induction of inflammation and prior to ischemia, which was followed by hypoxia. Infrared-labeled exosomes were intranasally administered to track their distribution with a LI-COR scanner. Acute oligodendrocyte- and neuron-specific cell death was analyzed 24 h after injury in animals with or without MSC exosome application using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunohistochemical counterstaining. Myelination, mature oligodendroglial and neuronal cell counts were assessed on postnatal day 11 using immunohistochemistry, Western blot or RT-PCR. Morris water maze assay was used to evaluate the effect of MSC exosomes on long-term neurodevelopmental outcome 4 weeks after injury. We found that intranasally administered exosomes reached the frontal part of the brain within 30 min after administration and distributed throughout the whole brain after 3 h. While PBI was not associated with oligodendrocyte-specific cell death, it induced significant neuron-specific cell death which was substantially reduced upon MSC exosome application prior to ischemia. MSC exosomes rescued normal myelination, mature oligodendroglial and neuronal cell counts which were impaired after PBI. Finally, the application of MSC exosomes significantly improved learning ability in animals with PBI. In conclusion, MSC exosomes represent a novel prevention strategy with substantial clinical potential as they can be administered intranasally, prevent gray and white matter alterations and improve long-term neurodevelopmental outcome after PBI.
围产期脑损伤（PBI）与早产儿的白质和灰质严重损伤和发育不良有关，并可导致严重的神经发育缺陷。间充质基质细胞（MSC）被认为具有围产期脑损伤的神经保护作用，部分原因是通过释放细胞外囊泡（如外泌体）。我们旨在评估鼻内给予 MSC 衍生的外泌体的神经保护作用及其改善 PBI 后神经发育结局的潜力。使用超速离心法从人 Wharton 果冻 MSC 上清液中分离出外泌体。将 2 天大的 Wistar 幼鼠通过炎症和缺氧缺血联合诱导 PBI。在诱导炎症后和缺血前鼻内给予外泌体，随后进行缺氧。用 LI-COR 扫描仪鼻内给予红外标记的外泌体以追踪其分布。在损伤后 24 小时，使用末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）测定和免疫组织化学复染分析有无 MSC 外泌体应用的动物中急性少突胶质细胞和神经元特异性细胞死亡。使用免疫组织化学、Western blot 或 RT-PCR 在出生后第 11 天评估髓鞘形成、成熟少突胶质细胞和神经元细胞计数。Morris 水迷宫试验用于评估 MSC 外泌体对损伤后 4 周长期神经发育结局的影响。我们发现，鼻内给予的外泌体在给药后 30 分钟内到达大脑前部，并在 3 小时后分布在整个大脑中。虽然 PBI 与少突胶质细胞特异性细胞死亡无关，但它诱导了明显的神经元特异性细胞死亡，而在缺血前给予 MSC 外泌体可显著减少这种细胞死亡。MSC 外泌体挽救了正常的髓鞘形成、成熟少突胶质细胞和神经元细胞计数，这些细胞计数在 PBI 后受损。最后，MSC 外泌体的应用显著改善了 PBI 动物的学习能力。总之，MSC 外泌体作为一种新的预防策略具有重要的临床潜力，因为它们可以通过鼻内给予，防止灰质和白质改变，并改善 PBI 后的长期神经发育结局。
