Novel 3D analysis of Claudin-5 reveals significant endothelial heterogeneity among CNS microvessels.
Novel 3D analysis of Claudin-5 reveals significant endothelial heterogeneity among CNS microvessels.
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DOI：
10.1016/j.mvr.2012.12.001
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发表时间：
2013-03
影响因子：
3.1
通讯作者：
Pachter, Joel S.
中科院分区：
文献类型：
--
作者：
Paul, Debayon;Cowan, Ann E.;Ge, Shujun;Pachter, Joel S.
紧密连接（TJ）在维持血脑屏障（BBB）的完整性方面具有关键作用，并且在神经炎症性疾病期间经历显著破坏。因此，CLN-5的表达和分布，一个突出的TJ蛋白在中枢神经系统（CNS）微血管和血脑屏障决定因素，已被证明是平行的生理和病理生理变化的微血管功能。然而，通过使用常规的薄切片的二维免疫组织化学分析来原位定量CNS微血管系统内的CLN-5的努力受到毛细血管的曲折性和发炎小静脉的扭曲直径的阻碍。在此，我们描述了一种新的基于轮廓的3D图像可视化和定量方法，采用高分辨率共聚焦Z-堆栈厚免疫荧光染色胸腰段脊髓冷冻切片，分析CLN-5沿着不同大小的CNS微血管段的交界区。对健康小鼠和经历实验性自身免疫性脑脊髓炎（EAE）（神经炎性疾病多发性硬化症的动物模型）的小鼠的脊髓进行分析。结果表明，在正常条件下，CLN-5染色密度（CLN-5强度/内皮表面积）在毛细血管和小静脉中最大，在大静脉中最小。在EAE期间，交界处CLN-5染色的这种异质性加剧，因为脊髓小静脉显示交界处CLN-5染色显着丧失，这与局灶性白细胞渗出相关，而邻近毛细血管既没有表现出CLN-5丧失，也没有表现出浸润的白细胞。然而，尽管只有小静脉显示这些行为，毛细血管和小静脉都证明了疾病期间IgG的渗漏，进一步强调了CNS微血管中炎症反应的异质性。该方法应易于适用于分析CNS和外周微血管系统的其他连接蛋白，并用于突出它们在健康和疾病中的作用。
Tight junctions (TJs) feature critically in maintaining the integrity of the blood-brain barrier (BBB), and undergo significant disruption during neuroinflammatory diseases. Accordingly, the expression and distribution of CLN-5, a prominent TJ protein in central nervous system (CNS) microvessels and BBB determinant, has been shown to parallel physiological and pathophysiological changes in microvascular function. However, efforts to quantify CLN-5 within the CNS microvasculature in situ, by using conventional two-dimensional immunohistochemical analysis of thin sections, are encumbered by the tortuosity of capillaries and distorted diameters of inflamed venules. Herein, we describe a novel contour-based 3D image visualization and quantification method, employing high-resolution confocal z-stacks from thick immunofluorescently-stained thoracolumbar spinal cord cryosections, to analyze CLN-5 along the junctional regions of different-sized CNS microvascular segments. Analysis was performed on spinal cords of both healthy mice, and mice experiencing experimental autoimmune encephalomyelitis (EAE), an animal model of the neuroinflammatory disease multiple sclerosis. Results indicated that, under normal conditions, the density of CLN-5 staining (CLN-5 intensity/ endothelial surface area) was greatest in the capillaries and smaller venules, and least in the larger venules. This heterogeneity in junctional CLN-5 staining was exacerbated during EAE, as spinal venules revealed a significant loss of junctional CLN-5 staining that was associated with focal leukocyte extravasation, while adjacent capillaries exhibited neither CLN-5 loss nor infiltrating leukocytes. However, despite only venules displaying these behaviors, both capillaries and venules evidenced leakage of IgG during disease, further underscoring the heterogeneity of the inflammatory response in CNS microvessels. This method should be readily adaptable to analyzing other junctional proteins of the CNS and peripheral microvasculature, and serve to highlight their role(s) in health and disease.
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DOI：
10.1073/pnas.0808698106
发表时间：
2009-02-10
影响因子：
11.1
作者：
Argaw, Azeb Tadesse;Gurfein, Blake T.;John, Gareth R.
通讯作者：
John, Gareth R.
影响因子：
3.2
作者：
Dobrogowska, DH;Vorbrodt, AW
通讯作者：
Vorbrodt, AW
影响因子：
3.9
作者：
Garrido-Urbani, Sarah;Bradfield, Paul F.;Imhof, Beat A.
通讯作者：
Imhof, Beat A.
影响因子：
2.9
作者：
Alvarez, Jorge I.;Teale, Judy M.
通讯作者：
Teale, Judy M.
影响因子：
3.3
作者：
Bennett, Jami;Basivireddy, Jayasree;McQuaid, Stephen
通讯作者：
McQuaid, Stephen