Quantifying blood-brain-barrier leakage using a combination of evans blue and high molecular weight FITC-Dextran

Background: 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. New Method: 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. Results: 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. Comparison with Existing Methods: 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. Conclusions: The proposed method proved powerful in quantifying BBB leakage of specific regions, which may benefit studies regarding BBB disruption.