Kuhlmann Christoph R W, Zehendner Christoph M, Gerigk Marlis, Closhen Dorothea, Bender Bianca, Friedl Peter, Luhmann Heiko J
Institute of Physiology and Pathophysiology, Johannes Gutenberg University of Mainz, Duesbergweg 6, 55128 Mainz, Germany.
Neurosci Lett. 2009 Jan 16;449(3):168-72. doi: 10.1016/j.neulet.2008.10.096. Epub 2008 Nov 6.
The aim of the present study was to examine the signaling pathways of hypoxia followed by reoxygenation (H/R)-induced disruption of the blood-brain-barrier (BBB) in a co-culture of astrocytes and brain endothelial cells (BEC) in vitro. We analyzed the possible stabilizing effect of MK801, a highly selective N-methyl-d-aspartate receptor (NMDAR) antagonist, on BBB integrity. Levels of reactive oxygen species (ROS), glutamate (Glut) release and monocyte adhesion were measured under normoxia and H/R. BBB integrity was monitored measuring the trans-endothelial electrical resistance (TEER). TEER values dropped under H/R conditions which was abolished by MK801. Glut release from astrocytes, but not from endothelial cells was significantly increased under H/R, as were ROS levels and monocyte adhesion. The oxidative stress was blocked by MK801 and the NAD(P)H-oxidase inhibitor apocynin. We observed that calcium (Ca(2+)) signaling plays a crucial role during ROS generation and monocyte adhesion under H/R. ROS levels were decreased by applying ryanodine, a blocker of Ca(2+) release from the endoplasmic reticulum (ER) and by lowering the extracellular Ca(2+) concentration. Xestospongin C, which blocks IP(3) mediated Ca(2+) release from the ER did not alter ROS production under H/R conditions. These findings indicate that both extracellular Ca(2+) influx and ryanodine-mediated intracellular Ca(2+) release from the ER during H/R contribute to ROS formation at the BBB. Blocking ROS or Ca(2+) signaling prevented H/R-induced monocyte adhesion to BEC. We conclude, that the activation of NMDAR under H/R by Glut increases intracellular Ca(2+) levels, contributes to BBB disruption, ROS generation and monocyte adhesion.
本研究的目的是在体外星形胶质细胞和脑内皮细胞（BEC）共培养体系中，研究缺氧复氧（H/R）诱导血脑屏障（BBB）破坏的信号通路。我们分析了高选择性N-甲基-D-天冬氨酸受体（NMDAR）拮抗剂MK801对BBB完整性的可能稳定作用。在常氧和H/R条件下测量活性氧（ROS）水平、谷氨酸（Glut）释放和单核细胞黏附。通过测量跨内皮电阻（TEER）监测BBB完整性。H/R条件下TEER值下降，而MK801可消除这种下降。H/R条件下，星形胶质细胞而非内皮细胞的Glut释放显著增加，ROS水平和单核细胞黏附也增加。MK801和NAD（P）H氧化酶抑制剂夹竹桃麻素可阻断氧化应激。我们观察到，钙（Ca(2+)）信号在H/R条件下ROS生成和单核细胞黏附过程中起关键作用。应用内质网（ER）Ca(2+)释放阻滞剂ryanodine并降低细胞外Ca(2+)浓度可降低ROS水平。阻断IP(3)介导的ER Ca(2+)释放的西司他汀C在H/R条件下未改变ROS产生。这些发现表明，H/R期间细胞外Ca(2+)内流和ryanodine介导的ER细胞内Ca(2+)释放均有助于BBB处ROS形成。阻断ROS或Ca(2+)信号可防止H/R诱导的单核细胞黏附于BEC。我们得出结论，H/R条件下Glut激活NMDAR会增加细胞内Ca(2+)水平，导致BBB破坏、ROS生成和单核细胞黏附。
