Dengue virus NS1 cytokine-independent vascular leak is dependent on endothelial glycocalyx components
(A-D) Hair was removed from the dorsal dermis of mice, and mice were allowed to recover for 3 days. On the day of the assay, retro-orbital injections of (A-B) Evans Blue dye (EBD) or (C-D) Alexa Fluor 680-conjugated dextran were administered, followed by intradermal injections of PBS (black circles), 200 ng VEGF (purple squares), 15 μg DENV2 NS1 (blue triangles), 7.5 μg DENV2 NS1 (green triangles), and 15 μg WNV NS1 (orange diamonds). The dermis from each mouse was collected and processed two hours post-injection. (A) Representative image of mouse dorsal dermis following Evans Blue assay. (B) EBD was extracted from tissue in formamide for 24 hours at 56°C and quantified using a standard curve of EBD (30.5 to 500,000 ng/ml) using linear regression analysis. Data represent quantified EBD from 8 animals. (C) Representative image of mouse dorsal dermis following fluorescent dextran assay. (D) Dermises were scanned using a fluorescent detection system (LI-COR Odyssey CLx Imaging System) at a wavelength of 700 nm, and extravasated fluorescent dextran was quantified in tissue using Image Studio software (LI-COR Biosciences). Data represent the quantification of mean fluorescent intensity from mice in (C): PBS (n = 9); VEGF (n = 7); DENV2 NS1–15 μg (n = 9); DENV2 NS1–7.5 μg (n = 9); WNV NS1 (n = 4). Data in (B) and (D) represent mean +/- SEM and were collected from 3 independent experiments. An ordinary one-way ANOVA with multiple comparisons to the PBS group using Dunnett’s multiple comparison test was used to determine significance of VEGF, DENV2 NS1, and WNV NS1. An unpaired, parametric, two-tailed t-test was used to determine significance between 15 μg of DENV2 NS1 and 7.5 μg of DENV2 NS1. ns = not significant, *P < 0.05, **P < 0.01, ****P < 0.0001.