Fig. 4: L6-F4-2 treatment promotes endothelial blood-brain and blood-retina barrier function.

a WT (control) and Ndp^KO male and female mice were treated with PBS or L6-F4-2 by i.p. injection (2.5 mg/kg) at P0, P7 and P14 and Sulfo-NHS-biotin injected 30 min prior to tissue harvest at P21. Sulfo-NHS-biotin staining from a representative mouse reveals BBB/BRB defects in cerebellum and retina, but not cerebral cortex in Ndp^KO mice, while these defects can be rescued by L6-F4-2 treatment. Biotin labeling of liver and kidney parenchyma serve as a positive control. Scale bar, 500 μm. b Quantification of (a) for BBB/BRB leakage in Ndp^KO cerebellum and retina with rescue by L6-F4-2 treatment. c Mice were treated at P21, P24 and P27 (2.5 mg/kg, i.p.) and Sulfo-NHS-biotin injected 30 min prior to tissue harvest at P30. Sulfo-NHS-biotin staining from a representative mouse reveals BBB/BRB defects in cerebellum and retina but not cerebral cortex in Ndp^KO mice with rescue by L6-F4-2. Scale bar, 200 μm. d Quantification of (c) with BBB/BRB leakage in Ndp^KO cerebellum and retina and reversal by L6-F4-2. e L6-F4-2 rescues barrier phenotypes in P30 Ndp^KO mice with increased expression of the tight junction component CLDN5, P30 cerebellum IF, overlay of CD31 IF and DAPI. f Quantification of (e). g L6-F4-2 decreases expression of the EC fenestration component PLVAP in P30 Ndp^KO mice, with overlay of CD31 IF and DAPI. h Quantitation of (g). For (e) and (g), scale bars represent 100 μm. To quantify CLDN5 and PLVAP in (f) and (h) the density was measured with ImageJ and normalized to vessel area (CD31). Error bars in (b), (d), (f), and (h) represent mean ± SEM, n = 5 mice, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; one-way ANOVA. Source data are provided as a Source Data file. For box plots, whiskers indicate the minimum and maximum values in the dataset. The center is the median number. Boundaries of boxes are the first quartile and third quartile.