Boveri M, Kinsner A, Berezowski V, Lenfant A-M, Draing C, Cecchelli R, Dehouck M-P, Hartung T, Prieto P, Bal-Price A
European Centre for the Validation of Alternative Methods (ECVAM), Institute of Health and Consumer Protection, European Commission Joint Research Centre, Via E. Fermi 1, 21020 Ispra (VA), Italy.
Neuroscience. 2006;137(4):1193-209. doi: 10.1016/j.neuroscience.2005.10.011. Epub 2005 Dec 15.
The co-culture of bovine brain capillary endothelial cells and rat primary glial cells was established as an in vitro blood-brain barrier model to investigate the mechanisms by which the Gram-positive bacterial cell wall components lipoteichoic acid and muramyl dipeptide induced injury of blood-brain barrier structure and function. We found that highly purified lipoteichoic acid disrupted blood-brain barrier integrity in a concentration- and time-dependent manner indirectly, through glia activation. Low trans-endothelial electrical resistance and high permeability to fluorescein isothiocyanate-inulin observed in the presence of lipoteichoic acid-activated glial cells were potentiated by muramyl dipeptide and could be reversed only when glial cells were activated by lipoteichoic acid at 10 microg/ml but not with a higher lipoteichoic acid concentration (30 microg/ml). Immunocytochemistry analysis revealed no evident changes in the distribution of the cytoskeleton protein F-actin and tight junction proteins occludin and claudin after lipoteichoic acid treatment. However, the tight junction associated protein AHNAK clearly revealed the morphological alteration of the endothelial cells induced by lipoteichoic acid. Lipoteichoic acid-activated glial cells produced nitric oxide and pro-inflammatory cytokines (tumor necrosis factor-alpha and interleukin-1beta) that contributed to lipoteichoic acid-induced blood-brain barrier disruption, since the direct treatment of the endothelial monolayer with tumor necrosis factor-alpha or interleukin-1beta increased blood-brain barrier permeability, whereas the pre-treatment of lipoteichoic acid-activated glial cells with antibodies against these two cytokines blocked lipoteichoic acid effects. Additionally, nitric oxide was also involved in blood-brain barrier damage, since the nitric oxide donor itself (diethylenetriamine-nitric oxide adduct) increased blood-brain barrier permeability and inducible nitric oxide synthase inhibitor (1400W) partially reversed lipoteichoic acid-induced trans-endothelial electrical resistance decrease.
建立牛脑微血管内皮细胞与大鼠原代神经胶质细胞的共培养体系作为体外血脑屏障模型，以研究革兰氏阳性菌细胞壁成分脂磷壁酸和胞壁酰二肽诱导血脑屏障结构和功能损伤的机制。我们发现，高度纯化的脂磷壁酸通过激活神经胶质细胞，以浓度和时间依赖性方式间接破坏血脑屏障的完整性。在脂磷壁酸激活的神经胶质细胞存在的情况下观察到的低跨内皮电阻和对异硫氰酸荧光素 - 菊粉的高通透性，被胞壁酰二肽增强，并且只有当神经胶质细胞被10微克/毫升的脂磷壁酸激活时才能逆转，而在较高的脂磷壁酸浓度（30微克/毫升）下则不能。免疫细胞化学分析显示，脂磷壁酸处理后细胞骨架蛋白F - 肌动蛋白以及紧密连接蛋白闭合蛋白和Claudin的分布没有明显变化。然而，紧密连接相关蛋白AHNAK清楚地揭示了脂磷壁酸诱导的内皮细胞形态改变。脂磷壁酸激活的神经胶质细胞产生一氧化氮和促炎细胞因子（肿瘤坏死因子 - α和白细胞介素 - 1β），这导致了脂磷壁酸诱导的血脑屏障破坏，因为用肿瘤坏死因子 - α或白细胞介素 - 1β直接处理内皮单层会增加血脑屏障通透性，而用针对这两种细胞因子的抗体预处理脂磷壁酸激活的神经胶质细胞可阻断脂磷壁酸的作用。此外，一氧化氮也参与了血脑屏障损伤，因为一氧化氮供体本身（二乙三胺 - 一氧化氮加合物）增加了血脑屏障通透性，而诱导型一氧化氮合酶抑制剂（1400W）部分逆转了脂磷壁酸诱导的跨内皮电阻降低。
