Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx mice
Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx mice
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DOI：
10.1002/glia.10216
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发表时间：
2003-05-01
期刊：
影响因子：
6.2
通讯作者：
Roncali, L
中科院分区：
文献类型：
--
作者：
Nico, B;Frigeri, A;Roncali, L
在这项研究中，我们研究了肌营养不良蛋白缺陷 mdx 小鼠大脑中血脑屏障 (BBB) 的参与情况，mdx 小鼠是杜氏肌营养不良症 (DMD) 的实验模型。为此，我们使用了两种紧密连接标记物，即闭合小带 (ZO-1) 和claudin-1 蛋白，以及一种神经胶质细胞标记物，即水通道蛋白-4 (AQP4) 蛋白，其表达与 BBB 分化和完整性相关。结果显示，mdx 小鼠的大多数脑微血管排列着改变的内皮细胞，这些细胞显示出开放的紧密连接，并被肿胀的神经胶质突起包围。此外，18% 的血管周围神经胶质末足含有电子致密的细胞碎片，并被退化的微血管包裹。蛋白质印迹显示，与对照大脑相比，mdx 小鼠的 ZO-1 蛋白含量减少了 60%，AQP4 含量也有类似的减少。 mdx 小鼠的 ZO-1 免疫细胞化学和claudin-1 免疫荧光显示，与对照小鼠相比，微血管有弥漫性染色，显示带状染色模式。 ZO-1 免疫金电子显微镜显示 mdx 小鼠中未标记的紧密连接和金颗粒分散在内皮细胞质中，而对照组中 ZO-1 金颗粒仅位于内皮紧密连接处。 a-肌动蛋白和 ZO-1 的双重免疫荧光染色揭示了这些蛋白质的共定位。与 ZO-1 染色一样，抗 α-肌动蛋白抗体的免疫标记模式在 mdx 血管中呈弥漫性，而在对照中呈尖状或带状。 α-肌动蛋白免疫金电子显微镜显示，mdx 小鼠的内皮细胞和周细胞的细胞质中存在金颗粒，而对照组的内皮紧密连接和周细胞的细胞骨架微丝上则显示有 α-肌动蛋白金颗粒。 mdx 小鼠的血管周围神经胶质突起似乎被抗 AQP4 抗体微弱染色，而在对照组中，在光和电子显微镜水平上检测到强烈的 AQP4 标记的神经胶质突起。通过辣根过氧化物酶 (HRP) 研究 mdx 脑微血管的血管通透性。注射 HRP 后，在 mdx 小鼠中观察到广泛的血管周围标记逃逸区域，而在对照组中，HRP 完全位于血管内。 mdx 脑血管周围基质中未发现炎症细胞、CD4、CD8、CD20 和 CD68 阳性细胞。这些发现表明，mdx 大脑中的抗肌营养不良蛋白缺乏会导致内皮细胞和神经胶质细胞严重损伤，导致 α-肌动蛋白细胞骨架、ZO-1、claudin-1 和 AQP4 组装紊乱，以及 BBB 破坏。 BBB 的改变表明血管通透性的变化参与了与 DMD 相关的神经功能障碍的发病机制。
In this study, we investigated the involvement of the blood-brain barrier (BBB) in the brain of the dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy (DMD). To this purpose, we used two tight junction markers, the Zonula occludens (ZO-1) and claudin-1 proteins, and a glial marker, the aquaporin-4 (AQP4) protein, whose expression is correlated with BBB differentiation and integrity. Results showed that most of the brain microvessels in mdx mice were lined by altered endothelial cells that showed open tight junctions and were surrounded by swollen glial processes. Moreover, 18% of the perivascular glial endfeet contained electron-dense cellular debris and were enveloped by degenerating microvessels. Western blot showed a 60% reduction in the ZO-1 protein content in mdx mice and a similar reduction in AQP4 content compared with the control brain. ZO-1 immunocytochemistry and claudin-1 immunofluorescence in mdx mice revealed a diffuse staining of microvessels as compared with the control ones, which displayed a banded staining pattern. ZO-1 immunogold electron microscopy showed unlabeled tight junctions and the presence of gold particles scattered in the endothelial cytoplasm in the mdx mice, whereas ZO-1 gold particles were exclusively located at the endothelial tight junctions in the controls. Dual immunofluorescence staining of a-actin and ZO-1 revealed colocalization of these proteins. As in ZO-1 staining, the pattern of immunolabeling with anti-alpha-actin antibody was diffuse in the mdx vessels and pointed or banded in the controls. a-actin immunogold electron microscopy showed gold particles in the cytoplasms of endothelial cells and pericytes in the mdx mice, whereas alpha-actin gold particles were revealed on the endothelial tight junctions and the cytoskeletal microfilaments of pericytes in the controls. Perivascular glial processes of the mdx mice appeared faintly stained by anti-AQP4 antibody, while in the controls a strong AQP4 labeling of glial processes was detected at light and electron microscope level. The vascular permeability of the mdx brain microvessels was investigated by means of the horseradish peroxidase (HRP). After HRP injection, extensive perivascular areas of marker escape were observed in mdx mice, whereas HRP was exclusively intravascularly localized in the controls. Inflammatory cells, CD4-, CD8-, CD20-, and CD68-positive cells, were not revealed in the perivascular stroma of the mdx brain. These findings indicate that dystrophin deficiency in the mdx brain leads to severe injury of the endothelial and glial cells with disturbance in alpha-actin cytoskeleton, ZO-1, claudin-1, and AQP4 assembly, as well as BBB breakdown. The BBB alterations suggest that changes in vascular permeability are involved in the pathogenesis of the neurological dysfunction associated with DMD.