Immunolabeling for occludin, claudin-3, and claudin-5 (magenta) was performed to assess the localization of these tight junction proteins in brain vessels (visualized with the basal lamina LAMγ1 marker, yellow) in focal NMOSD-like lesions. Immunoreactivity for occludin (A), claudin-3 (B), and claudin-5 (C) was localized at the tight junctions between adjacent endothelial cells and formed sharply defined, continuous strands in uninjected, naive controls. Loss of occludin immunoreactivity was observed 10 hours and 24 hours after lesion induction in astrocyte-depleted areas. Vascular occludin localization recovered to 68.5% ± 4.6% within 3 days after focal injection of AQP4 Ab and human complement (A). Quantification of occludin-positive vessels confirms the transient loss of occludin immunoreactivity from blood vessels in areas of GFAP loss (41–307 vessels/animal evaluated depending on astrocyte lesion size; n = 3 animals per time point, except 6 hours n = 4; D). In contrast, no loss of claudin-3 and claudin-5 immunoreactivity was detected after lesion induction, and sharply defined immunopositive strands were observed at the tight junctions of LAMγ1-positive vessels at 10 hours (B and C, respectively). Quantification of claudin-3–positive vessels (45–218 vessels/animal evaluated; ctrl n = 3, 6h: n = 4, 10h: n = 2, 24h: n = 3, 3d: n = 2, 6d: n = 3; E) and claudin-5–positive vessels (28–209 vessels/animal, n = 3 animals per time point, except 6h n = 4; F) confirms this observation. Temporal evolution of experimental NMOSD lesions (G). The y axis represents the extent of the investigated factors in arbitrary units. (D–F) Kruskal-Wallis test followed by Dunn’s multiple comparison test. *P < 0.05. Data are shown as mean ± SEM. bv, blood vessels. Scale bars: 50 μm (A–C).