An in vivo Assay to Test Blood Vessel Permeability
An in vivo Assay to Test Blood Vessel Permeability
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DOI：
10.3791/50062
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发表时间：
2013-03-01
影响因子：
1.2
通讯作者：
Chernoff, Jonathan
中科院分区：
文献类型：
--
作者：
Radu, Maria;Chernoff, Jonathan
本方法是以静脉注射伊文思蓝的小鼠作为试验动物模型。伊文思蓝是一种结合白蛋白的染料。在生理条件下，内皮对白蛋白是不可渗透的，因此伊文思蓝结合的白蛋白仍然限制在血管内。在促进血管渗透性增加的病理条件下，内皮细胞部分失去密切接触，内皮变得对白蛋白等小蛋白具有渗透性。这种情况允许组织中的伊文思蓝外渗。健康的内皮可防止染料外渗到邻近的血管化组织中。与内皮完整的器官相比，通透性增加的器官将显示显著增加的蓝色。血管渗透性的水平可以通过简单的可视化或通过定量测量每毫克对照组织与实验动物/组织中掺入的染料来评估。该测定的两个强有力的方面是其简单性和定量特征。通过在甲酰胺中孵育特定量的组织，可以从组织中提取伊文思蓝染料。伊文思蓝吸收最大值在620 nm处，吸收最小值在740 nm处。通过使用伊文思蓝的标准曲线，光密度测量值可以转换为每毫克组织捕获的毫克染料。应使用统计分析来评估血管通透性的显著差异。
This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.