Background The blood–brain barrier (BBB) is altered in several diseases of the central nervous system. For example, the breakdown of the BBB during cerebral ischemia in stroke or traumatic brain injury is a hallmark of the diseases’ progression. This functional damage is one key event which is attempted to be mimicked in in vitro models. Recent studies showed the pivotal role of micro-environmental cells such as astrocytes for this barrier damage in mouse stroke in vitro models. The aim of this study was to evaluate the role of micro-environmental cells for the functional, paracellular breakdown in a human BBB cerebral ischemia in vitro model accompanied by a transcriptional analysis. Methods Transwell models with human brain endothelial cell line hCMEC/D3 in mono-culture or co-culture with human primary astrocytes and pericytes or rat glioma cell line C6 were subjected to oxygen/glucose deprivation (OGD). Changes of transendothelial electrical resistance (TEER) and FITC-dextran 4000 permeability were recorded as measures for paracellular tightness. In addition, qPCR and high-throughput qPCR Barrier chips were applied to investigate the changes of the mRNA expression of 38 relevant, expressed barrier targets (tight junctions, ABC-transporters) by different treatments. Results In contrast to the mono-culture, the co-cultivation with human primary astrocytes/pericytes or glioma C6 cells resulted in a significantly increased paracellular permeability after 5 h OGD. This indicated the pivotal role of micro-environmental cells for BBB breakdown in the human model. Hierarchical cluster analysis of qPCR data revealed differently, but also commonly regulated clustered targets dependent on medium exchange, serum reduction, hydrocortisone addition and co-cultivations. Conclusions The co-cultivation with micro-environmental cells is necessary to achieve a functional breakdown of the BBB in the cerebral ischemia model within an in vivo relevant time window. Comprehensive studies by qPCR revealed that distinct expression clusters of barrier markers exist and that these are regulated by different treatments (even by growth medium change) indicating that controls for single cell culture manipulation steps are crucial to understand the observed effects properly.

中文翻译：

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微环境细胞在人血脑屏障体外脑缺血模型中的关键作用：功能和转录组学分析

背景 血脑屏障 (BBB) 在几种中枢神经系统疾病中发生改变。例如，在中风或创伤性脑损伤的脑缺血期间 BBB 的破坏是疾病进展的标志。这种功能性损伤是试图在体外模型中模拟的一个关键事件。最近的研究表明，微环境细胞（如星形胶质细胞）在小鼠中风体外模型中对这种屏障损伤的关键作用。本研究的目的是评估微环境细胞在人类 BBB 脑缺血体外模型中的功能性、细胞旁分解中的作用，并伴有转录分析。方法 将人脑内皮细胞系 hCMEC/D3 与人原代星形胶质细胞和周细胞或大鼠神经胶质瘤细胞系 C6 单培养或共培养的 Transwell 模型进行氧/葡萄糖剥夺（OGD）。将跨内皮电阻 (TEER) 和 FITC-葡聚糖 4000 渗透性的变化记录为细胞旁紧密度的量度。此外，应用 qPCR 和高通量 qPCR Barrier 芯片来研究不同处理下 38 个相关表达的屏障靶标（紧密连接，ABC 转运蛋白）的 mRNA 表达变化。结果与单培养相反，与人原代星形胶质细胞/周细胞或神经胶质瘤 C6 细胞的共培养导致 5 小时 OGD 后细胞旁通透性显着增加。这表明微环境细胞对人体模型中 BBB 分解的关键作用。qPCR 数据的分层聚类分析揭示了不同的，但也普遍受调控的聚类目标，这些目标取决于培养基交换、血清减少、氢化可的松添加和共培养。结论 与微环境细胞的共培养对于在体内相关时间窗口内实现脑缺血模型中 BBB 的功能分解是必要的。qPCR 的综合研究表明，存在不同的屏障标记表达簇，并且这些表达簇受不同处理（甚至生长培养基变化）的调节，表明单细胞培养操作步骤的控制对于正确理解观察到的效果至关重要。qPCR 数据的分层聚类分析揭示了不同的，但也普遍受调控的聚类目标，这些目标取决于培养基交换、血清减少、氢化可的松添加和共培养。结论 与微环境细胞的共培养对于在体内相关时间窗口内实现脑缺血模型中 BBB 的功能分解是必要的。qPCR 的综合研究表明，存在不同的屏障标记表达簇，并且这些表达簇受不同处理（甚至生长培养基变化）的调节，表明单细胞培养操作步骤的控制对于正确理解观察到的效果至关重要。qPCR 数据的分层聚类分析揭示了不同的，但也普遍受调控的聚类目标，这些目标取决于培养基交换、血清减少、氢化可的松添加和共培养。结论 与微环境细胞的共培养对于在体内相关时间窗口内实现脑缺血模型中 BBB 的功能分解是必要的。qPCR 的综合研究表明，存在不同的屏障标记表达簇，并且这些表达簇受不同处理（甚至生长培养基变化）的调节，表明单细胞培养操作步骤的控制对于正确理解观察到的效果至关重要。添加氢化可的松和共培养。结论 与微环境细胞的共培养对于在体内相关时间窗口内实现脑缺血模型中 BBB 的功能分解是必要的。qPCR 的综合研究表明，存在不同的屏障标记表达簇，并且这些表达簇受不同处理（甚至生长培养基变化）的调节，表明单细胞培养操作步骤的控制对于正确理解观察到的效果至关重要。添加氢化可的松和共培养。结论 与微环境细胞的共培养对于在体内相关时间窗口内实现脑缺血模型中 BBB 的功能分解是必要的。qPCR 的综合研究表明，存在不同的屏障标记表达簇，并且这些表达簇受不同处理（甚至生长培养基变化）的调节，表明单细胞培养操作步骤的控制对于正确理解观察到的效果至关重要。