Department of Neurosurgery, West China Hospital of Sichuan University, Chengdu, 610041, China.
Department of Neonatology, West China Second University Hospital, Sichuan University, Chengdu, 610041, China.
J Neurosci Methods. 2019 Sep 1;325:108349. doi: 10.1016/j.jneumeth.2019.108349. Epub 2019 Jul 5.
Evans blue (EB) is the most widely used tracer to assess BBB leakage. However, a well-established method to obtain visualized and quantitative results of EB extravasation is presently unavailable.
We reported a novel method to quantify BBB leakage by combining EB and high molecular weight FITC-Dextran (2000 kDa). EB was used for a long circulation duration (60 min) to detect BBB leakage. FITC-Dextran was used for a short circulation duration (10 min) to outline vascular contours. Confocal microscope imaging was used to obtain visualized images of BBB leakage. The result of dividing integrated optical density of EB by vascular areas outlined by FITC-Dextran was treated as the quantification of BBB leakage.
This method proved workable in quantifying BBB leakage of specific regions in lipopolysaccharide-induced BBB disruption mice and apoE mice. Sections processed with this method enabled further immunofluorescence staining. Through combining the results of EB extravasation and immunofluorescence staining, the colocalization of specific proteins and BBB disruption was achieved.
Colorimetric and spectrophotometric methods provide us with quantitative results of EB extravasation but fail to locate the specific regions. Fluorescence microscopy imaging can locate specific regions of EB extravasation but a well-established quantitative method is presently unavailable. Our method combines advantages of above two classic methods, providing us with visualized and quantitative information of BBB leakage based on EB extravasation in specific cerebral regions.
The proposed method proved powerful in quantifying BBB leakage of specific regions, which may benefit studies regarding BBB disruption.
伊文思蓝（EB）是最常用于评估血脑屏障（BBB）渗漏的示踪剂。然而，目前还没有一种成熟的方法可以获得 EB 外渗的可视化和定量结果。
我们报道了一种通过结合 EB 和高分子量 FITC-葡聚糖（2000 kDa）来定量 BBB 渗漏的新方法。EB 用于长循环时间（60 分钟）以检测 BBB 渗漏。FITC-葡聚糖用于短循环时间（10 分钟）以勾勒血管轮廓。使用共聚焦显微镜成像获得 BBB 渗漏的可视化图像。将 EB 的积分光密度除以 FITC-葡聚糖勾勒的血管面积的结果被视为 BBB 渗漏的定量。
该方法在定量脂多糖诱导的 BBB 破坏小鼠和 apoE 小鼠特定区域的 BBB 渗漏方面证明是可行的。用该方法处理的切片可进一步进行免疫荧光染色。通过结合 EB 外渗和免疫荧光染色的结果，实现了特定蛋白质与 BBB 破坏的共定位。
比色法和分光光度法为我们提供了 EB 外渗的定量结果，但无法定位特定区域。荧光显微镜成像可以定位 EB 外渗的特定区域，但目前还没有成熟的定量方法。我们的方法结合了上述两种经典方法的优点，基于 EB 在特定脑区的外渗，为我们提供了 BBB 渗漏的可视化和定量信息。
所提出的方法在定量特定区域的 BBB 渗漏方面证明是有效的，这可能有助于研究 BBB 破坏。
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