Astro KO and Endo KO mice display differences in 3D distribution profiles of luminal and perivascular cells. Isosurface-rendered images were generated from confocal z-stacks of 60-μm thick cryosections from WT, Astro KO, and Endo KO mice at d16 EAE. The BM is highlighted by staining of Lam1 (red). (Top row) DRAQ5+ nuclei in luminal and perivascular compartments were optically isolated using 3D contour based segmentation (as described in Materials and Methods), and pseudo-colored blue (luminal) and turquoise (perivascular), respectively. Using Imaris® spot creation module, each of these nuclei is shown in the 3D dataset (volume) as a “spot object,” designating its luminal or perivascular location. Scale = 10 μm. (Bottom row) Imaris® vantage plots showing the 3D distributions of luminal and perivascular cells along microvascular x, y, and z-axes in the corresponding vessels from the top row. Scale = 20 μm. (a) Representative WT vessel showing an empty lumen (*). (b) The lumen in the Astro KO vessel also appears empty (*) but partially collapsed, possibly owing to accumulation of perivascular cells that are missing guidance cues from deleted astrocyte-derived CCL2. (c) In contrast, Endo KO vessel shows evidence of congregation of cells in the lumen (blue), possibly reflecting stalled leukocyte transmigration in absence of endothelial-derived CCL2. Box-and-whisker plots are shown indicating the maximum and minimum spread from the median, in μm, of luminal or perivascular nuclei along the x, y, and z-axes.