Astro KO and Endo KO mice show differential loss of CLN-5 staining in spinal venules during EAE. (a) Isosurface-rendered images were generated from confocal z-stacks of 60 μm thick thoraco-lumbar spinal cord cryosections at d9 and d16 EAE, as described in Materials and Methods. Staining of CLN-5 (green isosurface) and nuclei/DRAQ5 (blue) is shown. Larger images displaying 3D perspective projection views of confocal reconstructions show CLN-5 channel only, to emphasize the fragmented pattern of TJ protein staining. Inserts depict both CLN-5 and nuclei, highlighting the close association of altered CLN-5 staining with dense perivascular cellularity representing infiltrating leukocytes. Arrows demark overt gaps in CLN-5 staining, where the TJ pattern is clearly disrupted. Notably, CLN-5 staining pattern during EAE appears most intact in Astro KO mice, least so in WT mice, and intermediate in Endo KO mice. (b) Quantification of CLN-5 staining as intensity per unit surface area of the endothelium. CLN-5 density in naïve WT mice is included as a reference for the normal state, wherein the pattern of CLN-5 junctional staining is continuous . Statistical comparisons are between groups and within days. (c) Summary of CLN-5 changes. Statistical comparisons are within groups and between days. A total of 12 venules were analyzed in each group sampled from three mice. Data reflect mean value ± standard error. Scale = 20 μm.