Extended Data Fig. 4: VCAM1 is not expressed in CNS cell types other than BECs in the hippocampus, is increased during neurodegeneration and is deleted in brain endothelium for Slco1c1-Cre^ERT2+/–; Vcam1^fl/fl transgenic mice.

a, Representative 2D and 3D z-stacked high-magnification confocal images (51 slices with an interval of 0.4 µm) of VCAM1 in the granular layer of the DG of the hippocampus of a young (3-month-old) NSG mouse acutely treated with plasma from aged humans. Brain sections were co-stained for DCX and NeuN to label immature and mature granule neurons, respectively. VCAM1 is not expressed in these cell types. Light blue lines outline the granule layer. Experiment repeated three times independently with similar results. 2D scale bar, 50 µm. Two 3D renderings of the 2D images are displayed. 3D scale bar, 20 µm. b, Quantification of VCAM1, AQP4 and lectin, with Hoechst labeling of cell nuclei, in the hippocampus and cerebellum of EAE (multiple sclerosis), Npc1^–/– (Niemann Pick disease type C) and Grn^–/– (frontotemporal dementia) disease models. EAE: n = 4 naive and 8 EAE induced mice, *P = 0.006, **P = 0.0125; Npc1^–/–: n = 6 mice per group, ***P = 0.0274, ****P < 0.0001; Grn^–/–: n = 4 mice per group, *****P = 0.0004. P values were determined by unpaired two-tailed Student’s t test. Data are shown as the mean ± s.e.m. c, Representative 2D and 3D z-stacked high-magnification confocal images (51 slices with an interval of 0.4 µm) of VCAM1 in the granular layer of the DG of the hippocampus co-stained with SOX2 and GFAP to label neural stem and progenitor cells (SOX2⁺GFAP⁺) and hilus GFAP⁺ astrocytes. VCAM1 is not expressed in these cell types in the DG. Light blue lines outline the granule layer. Experiment repeated three times independently with similar results. 2D scale bar, 50 µm. Two 3D renderings of the 2D images are displayed. 3D scale bar, 20 µm. d, Cre⁺ or Cre^– littermates (3 months old) were treated daily with tamoxifen (i.p., 150 mg per kg) for 5 d followed by 4 d of rest. Mice received three LPS injections (0.5 mg per kg i.p.) at 28, 22 and 2 h before perfusion. Mice also received a retro-orbital injection of fluorescently conjugated mouse anti-VCAM1 monoclonal antibody (100 µg) 2 h before perfusion. FACS gating strategy to analyze single BECs. PI⁺ dead cells were excluded. Cells negative for CD11a, CD11b, CD45 and Ter-119 were gated to exclude erythrocytes, monocytes/macrophages and microglia. CD13 and ACSA-2 staining were applied to exclude pericytes and astrocytes, respectively. CD31⁺MECA-99⁺ cells were defined as the BEC population. e,f, Quantification (e) of flow cytometry (f) that was performed on primary BECs isolated from Cre⁺ or Cre^– mice treated as described in d. n = 3 Cre⁺ or Cre^– mice received LPS, while one Cre^– mouse was given PBS vehicle control instead. The VCAM1 gate was set on the basis of a Cre^– mouse injected with fluorescently conjugated IgG. **P = 0.0011, unpaired two-tailed Student’s t test. Data are shown as the mean ± s.e.m. g, Representative confocal images of cortex and DG for VCAM1 and Hoechst (to label cell nuclei) in LPS-stimulated mice as described in d. Loss of Vcam1 in Cre⁺ mice, but not Cre^– mice, in BBB endothelium but not meninges is shown. Experiment repeated three times independently with similar results. Scale bar, 100 µm.