Department of Ophthalmology, National Defense Medical College, Namiki 3-2, Tokorozawa, Saitama, 359-0042, Japan; Wellcome-Wolfson Institute for Experimental Medicine, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, Northern Ireland, United Kingdom.
Wellcome-Wolfson Institute for Experimental Medicine, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, Northern Ireland, United Kingdom.
Exp Eye Res. 2021 Apr;205:108490. doi: 10.1016/j.exer.2021.108490. Epub 2021 Feb 16.
Microglia and Müller cells (MCs) are believed to be critically involved in hypoxia-induced blood-retinal barrier (BRB) disruption, which is a major pathogenic factor of various retinopathies. However, the underlying mechanism remains poorly defined. The inner BRB (iBRB) is primarily formed of microvascular endothelial cells (ECs) with tight junction (TJ), which are surrounded and supported by retinal glial cells. We developed a novel in vitro iBRB model sheet by sandwiching Transwell membrane with layered mouse brain microvascular ECs (bEnd.3) and mouse retinal MCs (QMMuC-1) on each side of the membrane. Using this model, we tested the hypothesis that under hypoxic condition, activated microglia produce inflammatory cytokines such as interleukin (IL)-1β, which may promote vascular endothelial growth factor (VEGF) production from MCs, leading to TJ disruption. The iBRB model cell sheets were exposed to 1% oxygen for 6 h with or without mouse brain microglia (BV2) or IL-1β. TJ structure and function were examined by zonula occludens (ZO)-1 immunostaining and fluorescein isothiocyanate permeability assay, respectively. Relative gene expression of IL-1β in BV2 under normoxic and hypoxic conditions was examined by real-time reverse transcription-polymerase chain reaction. VEGF protein concentration in QMMuC-1 supernatants was measured by enzyme-linked immunosorbent assay. The bEnd.3 cell sheet incubated with BV2 in hypoxic condition or with IL-1β in normoxic condition showed abnormal localization of ZO-1 and aberrated barrier function. Under normoxic condition, EC-MC iBRB model cell sheet showed lower permeability than bEnd.3 cell sheet. Under hypoxic conditions, the barrier function of EC-MC iBRB model cell sheet was more deteriorated compared to bEnd.3 cell sheet. Under hypoxic condition, incubation of EC-MC iBRB model cell sheet with BV2 cells or IL-1β significantly increased barrier permeability, and hypoxia-treated BV2 cells expressed significantly higher levels of IL-1β mRNA. Incubation of QMMuC-1 with IL-1β increased VEGF production. These results suggest that under hypoxic condition, microglia are activated to release proinflammatory cytokines such as IL-1β that promote VEGF production from MCs, leading to disruption of iBRB function. Modulating microglia and MCs function may be a novel approach to treat hypoxia-induced retinal BRB dysfunction.
小胶质细胞和 Müller 细胞（MCs）被认为在缺氧诱导的血视网膜屏障（BRB）破坏中起关键作用，这是各种视网膜病变的主要致病因素。然而，其潜在机制仍未明了。内 BRB（iBRB）主要由具有紧密连接（TJ）的微血管内皮细胞（ECs）组成，这些细胞被视网膜神经胶质细胞包围和支持。我们通过在膜的两侧夹入分层的小鼠脑微血管内皮细胞（bEnd.3）和小鼠视网膜 MCs（QMMuC-1），开发了一种新型的体外 iBRB 模型片。使用该模型，我们测试了以下假设：在缺氧条件下，激活的小胶质细胞产生炎症细胞因子，如白细胞介素（IL）-1β，其可能促进 MC 产生血管内皮生长因子（VEGF），导致 TJ 破坏。iBRB 模型细胞片在 1%氧气下孵育 6 小时，有或没有小鼠脑小胶质细胞（BV2）或 IL-1β。通过封闭蛋白（ZO）-1免疫染色和荧光素异硫氰酸酯通透性测定分别检测 TJ 结构和功能。通过实时逆转录聚合酶链反应检测正常氧和缺氧条件下 BV2 中 IL-1β的相对基因表达。通过酶联免疫吸附试验测量 QMMuC-1 上清液中的 VEGF 蛋白浓度。在缺氧条件下与 BV2 共孵育或在常氧条件下与 IL-1β 共孵育的 bEnd.3 细胞片显示 ZO-1 异常定位和异常的屏障功能。在常氧条件下，EC-MC iBRB 模型细胞片的通透性低于 bEnd.3 细胞片。在缺氧条件下，与 bEnd.3 细胞片相比，EC-MC iBRB 模型细胞片的屏障功能更差。在缺氧条件下，与 BV2 细胞或 IL-1β 孵育的 EC-MC iBRB 模型细胞片显著增加了屏障通透性，并且缺氧处理的 BV2 细胞表达的 IL-1β mRNA 水平显著升高。与 IL-1β 共孵育的 QMMuC-1 增加了 VEGF 的产生。这些结果表明，在缺氧条件下，小胶质细胞被激活释放促炎细胞因子，如 IL-1β，促进 MC 产生 VEGF，导致 iBRB 功能障碍。调节小胶质细胞和 MCs 的功能可能是治疗缺氧诱导的视网膜 BRB 功能障碍的一种新方法。
