Department of Anesthesiology and Center for Shock, Trauma and Anesthesiology Research (STAR), University of Maryland School of Medicine, Baltimore, MD 21201, USA; Medical Scientist Training Program, University of Maryland School of Medicine, Baltimore, MD 21201, USA; Program in Neuroscience, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Department of Anesthesiology and Center for Shock, Trauma and Anesthesiology Research (STAR), University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Brain Behav Immun. 2021 Feb;92:165-183. doi: 10.1016/j.bbi.2020.12.007. Epub 2020 Dec 9.
Extracellular vesicles (EVs) have been implicated mechanistically in the pathobiology of neurodegenerative disorders, including central nervous system injury. However, the role of EVs in spinal cord injury (SCI) has received limited attention to date. Moreover, technical limitations related to EV isolation and characterization methods can lead to misleading or contradictory findings. Here, we examined changes in plasma EVs after mouse SCI at multiple timepoints (1d, 3d, 7d, 14d) using complementary measurement techniques. Plasma EVs isolated by ultracentrifugation (UC) were decreased at 1d post-injury, as shown by nanoparticle tracking analysis (NTA), and paralleled an overall reduction in total plasma extracellular nanoparticles. Western blot (WB) analysis of UC-derived plasma EVs revealed increased expression of the tetraspanin exosome marker, CD81, between 1d and 7d post-injury. To substantiate these findings, we performed interferometric and fluorescence imaging of single, tetraspanin EVs captured directly from plasma with ExoView®. Consistent with WB, we observed significantly increased plasma CD81+ EV count and cargo at 1d post-injury. The majority of these tetraspanin EVs were smaller than 50 nm based on interferometry and were insufficiently resolved by flow cytometry-based detection. At the injury site, there was enhanced expression of EV biogenesis proteins that were also detected in EVs directly isolated from spinal cord tissue by WB. Surface expression of tetraspanins CD9 and CD63 increased in multiple cell types at the injury site; however, astrocyte CD81 expression uniquely decreased, as demonstrated by flow cytometry. UC-isolated plasma EV microRNA cargo was also significantly altered at 1d post-injury with changes similar to that reported in EVs released by astrocytes after inflammatory stimulation. When injected into the lateral ventricle, plasma EVs from SCI mice increased both pro- and anti-inflammatory gene as well as reactive astrocyte gene expression in the brain cortex. These studies provide the first detailed characterization of plasma EV dynamics after SCI and suggest that plasma EVs may be involved in posttraumatic brain inflammation.
细胞外囊泡 (EVs) 在神经退行性疾病的病理生物学中发挥作用，包括中枢神经系统损伤。然而，EVs 在脊髓损伤 (SCI) 中的作用迄今为止受到的关注有限。此外，与 EV 分离和表征方法相关的技术限制可能导致误导或矛盾的发现。在这里，我们使用互补的测量技术在多个时间点 (1d、3d、7d、14d) 检查了 SCI 后小鼠血浆 EV 的变化。通过纳米颗粒跟踪分析 (NTA) 显示，超速离心 (UC) 分离的血浆 EV 在损伤后 1d 减少，并且与总血浆细胞外纳米颗粒的总体减少相平行。UC 衍生的血浆 EV 的 Western blot (WB) 分析显示，损伤后 1d 和 7d 之间四跨膜蛋白外泌体标志物 CD81 的表达增加。为了证实这些发现，我们使用 ExoView®直接从血浆中捕获单个四跨膜蛋白 EV 进行干涉和荧光成像。与 WB 一致，我们观察到损伤后 1d 时血浆 CD81+EV 计数和货物明显增加。根据干涉测量法，这些四跨膜蛋白 EV 中的大多数小于 50nm，并且基于流式细胞术的检测无法充分解析。在损伤部位，EV 生物发生蛋白的表达增强，WB 也直接从脊髓组织中分离的 EV 中检测到这些蛋白。在损伤部位，多种细胞类型的四跨膜蛋白 CD9 和 CD63 的表面表达增加；然而，流式细胞术显示星形胶质细胞 CD81 的表达独特地降低。UC 分离的血浆 EV 微 RNA 货物在损伤后 1d 也发生了显著变化，变化与炎症刺激后星形胶质细胞释放的 EV 报告的变化相似。当注射到侧脑室时，SCI 小鼠的血浆 EV 增加了大脑皮质中促炎和抗炎基因以及反应性星形胶质细胞基因的表达。这些研究首次详细描述了 SCI 后血浆 EV 动力学的特征，并表明血浆 EV 可能参与创伤后脑炎症。
