Institute of Physiology and Pathophysiology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany.
PLoS One. 2013 Dec 6;8(12):e82823. doi: 10.1371/journal.pone.0082823. eCollection 2013.
Re-canalization of cerebral vessels in ischemic stroke is pivotal to rescue dysfunctional brain areas that are exposed to moderate hypoxia within the penumbra from irreversible cell death. Goal of the present study was to evaluate the effect of moderate hypoxia followed by reoxygenation (MHR) on the evolution of reactive oxygen species (ROS) and blood-brain barrier (BBB) integrity in brain endothelial cells (BEC). BBB integrity was assessed in BEC in vitro and in microvessels of the guinea pig whole brain in situ preparation. Probes were exposed to MHR (2 hours 67-70 mmHg O2, 3 hours reoxygenation, BEC) or towards occlusion of the arteria cerebri media (MCAO) with or without subsequent reperfusion in the whole brain preparation. In vitro BBB integrity was evaluated using trans-endothelial electrical resistance (TEER) and transwell permeability assays. ROS in BEC were evaluated using 2',7'-dichlorodihydrofluorescein diacetate (DCF), MitoSox and immunostaining for nitrotyrosine. Tight-junction protein (TJ) integrity in BEC, stainings for nitrotyrosine and FITC-albumin extravasation in the guinea pig brain preparation were assessed by confocal microscopy. Diphenyleneiodonium (DPI) was used to investigate NADPH oxidase dependent ROS evolution and its effect on BBB parameters in BEC. MHR impaired TJ proteins zonula occludens 1 (ZO-1) and claudin 5 (Cl5), decreased TEER, and significantly increased cytosolic ROS in BEC. These events were blocked by the NADPH oxidase inhibitor DPI. MCAO with or without subsequent reoxygenation resulted in extravasation of FITC-albumin and ROS generation in the penumbra region of the guinea pig brain preparation and confirmed BBB damage. BEC integrity may be impaired through ROS in MHR on the level of TJ and the BBB is also functionally impaired in moderate hypoxic conditions followed by reperfusion in a complex guinea pig brain preparation. These findings suggest that the BBB is susceptible towards MHR and that ROS play a key role in this process.
在缺血性中风中，重新开通血管对于拯救处于半影区的功能失调的大脑区域免受不可逆的细胞死亡至关重要。本研究的目的是评估中度缺氧后再复氧（MHR）对脑内皮细胞（BEC）中活性氧（ROS）和血脑屏障（BBB）完整性演变的影响。在体外 BEC 中和在豚鼠全脑原位准备的微血管中评估 BBB 完整性。探针暴露于 MHR（2 小时 67-70mmHg O2，3 小时复氧，BEC）或大脑中动脉闭塞（MCAO），并在全脑准备中进行随后的再灌注。体外 BBB 完整性通过跨内皮电阻（TEER）和 Transwell 通透性测定进行评估。使用 2'，7'-二氯二氢荧光素二乙酸酯（DCF）、MitoSox 和硝基酪氨酸免疫染色评估 BEC 中的 ROS。通过共聚焦显微镜评估 BEC 中的紧密连接蛋白（TJ）完整性、硝基酪氨酸染色和 FITC-白蛋白漏出。使用二苯基碘（DPI）研究 NADPH 氧化酶依赖性 ROS 演变及其对 BEC 中 BBB 参数的影响。MHR 损害了 TJ 蛋白紧密连接蛋白 1（ZO-1）和紧密连接蛋白 5（Cl5），降低了 TEER，并显著增加了 BEC 中的细胞质 ROS。这些事件被 NADPH 氧化酶抑制剂 DPI 阻断。MCAO 伴或不伴随后的再氧合导致豚鼠脑准备的半影区 FITC-白蛋白漏出和 ROS 生成，并证实了 BBB 损伤。MHR 中 ROS 可能导致 BEC 完整性受损，而在中度缺氧后再复氧的复杂豚鼠脑准备中，BBB 也存在功能障碍。这些发现表明，BBB 易受 MHR 影响，ROS 在该过程中起关键作用。
