Astro KO and Endo KO mice display differences in perivascular cellularity associated with spinal venules durin g EAE. Isosurface-rendered images were generated from confocal z-stacks of 60 μm cryosections at d16 EAE. Staining of BM Lam 1 (red), CLN-5 (green), and nuclei/DRAQ5 (blue) is shown. (a, b, c) Longitudinal sections reveal the extent of vessel-associated leukocytes. CLN-5 staining is presented to highlight the endothelial boundary. Insets represent enlarged view of areas highlighted in white hatched boxes, while double-headed arrows denote the space between the endothelial and parenchymal BMs. All extravasated leukocytes within this space are considered “perivascular”. In WT mice, a dense accumulation of DRAQ5+ perivascular cells (representing leukocytes) is seen, a few apparently penetrating the fragmented parenchymal BM (arrowhead). In Astro KO mice, a similar dense perivascular cellularity is observed, with visibly intact parenchymal BM and lack of parenchymal leukocyte migration. In Endo KO mice, the BM is also apparently intact, with minimal perivascular cellularity. Scale = 20 μm. (d, e, f) Cross-sections highlight the spatial distribution of vessel-associated leukocytes. In WT mice, the vessel lumen (demarked by white dashes) appears empty and cells are seen in the perivascular space. A few cells are visibly penetrating the parenchymal BM (arrowheads), alongwith dense parenchymal cellularity (brackets). In Astro KO mice, the lumen again appears empty; congregated cells are evident in the perivascular space, with a few parenchymal clusters. In Endo KO mice, cells are clearly present in the lumen, with apparently fewer cells in the perivascular space as compared to WT and Astro KO mice. Parenchymal clustering is seemingly absent. The diffuse DRAQ5+ cells are likely parenchymal neural cells. (g-h) The red arrow designates the same Endo KO image subject to contour-based 3D segmentation (see Materials and Methods) to further resolve luminal (blue) from perivascular (turquoise) cells. Results are representative of 5–6 microvessels sampled from three mice in each group and two independent experiments. Scale = 10 μm.