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1.
Figure 1

Figure 1. Time course of cell generation and BBB development in the rat cerebral cortex. From: Pericytes are required for blood-brain barrier integrity during embryogenesis.

ag, Sections of rat cerebral cortex at indicated ages were stained for endothelial cells with Bandeiraea simplicifolia lectin I (BSL) (green, af) and nuclei with DAPI (blue, a, f (left), g), pericytes with anti-PDGFR-β (red, b; white arrows point to pericytes), oligodendrocyte progenitors with anti-PDGFR-α (red, c), astrocytes with anti-aquaporin 4 (red, d), anti-occludin (red, e; yellow arrows indicate tight-junction strands), anti-Glut1 (red, f (right)), and anti-Pgp (red, g). Scale bars represent 100 µm (ad, f, g) and 20 µm (e). hj, Rats aged E15 (left), E21 (middle) and adults (right) were given a trans-cardiac perfusion of biotin, and liver (h), muscle (i) and brain (j) tissue sections were stained with streptavidin (green) and DAPI (blue). Scale bar represents 100 µm.

Richard Daneman, et al. Nature. ;468(7323):562-566.
2.
Figure 4

Figure 4. Vascular expression of LAMs in Pdgfrb−/− mice. From: Pericytes are required for blood-brain barrier integrity during embryogenesis.

a, b, Western blots of brain lysates from E18 Pdgfrb−/− (KO) and littermate controls, probing occludin, claudin 5, Icam1, Alcam, Lgals3, β-actin and PDGFR-β. a, Representative blots; b, quantification; *P < 0.05 by Student’s t-test. ce, Cerebral cortex of E18 Pdgfrb−/− mice (d) and littermate controls (c) were stained with anti-Icam1 (purple) and BSL (green, bottom; white arrows indicate Icam1+ vessels), and per cent Icam1+ vascular length was quantified (e). Scale bar represents 250 µm. **P < 0.005 by Student t-test. fh, Purified murine brain endothelial cells cultured alone (f) or with a feeding layer of purified brain pericytes (g) and stained for DAPI (blue) and anti-Icam1 (green), and proportion of Icam1+ cells was quantified (h). Scale bar represents 200 µm. **P < 0.005 by Student t-test. ik, Five-week-old PdgfrbF7/F7 mice (j) and littermate controls (i) were stained with anti-Gr1 (red) and BSL (green, bottom), and number of Gr1+ cells per sagittal section was counted (k). Scale bar represents 250 µm. *P < 0.05 by Students t-test. All error bars represent s.e.m.

Richard Daneman, et al. Nature. ;468(7323):562-566.
3.
Figure 2

Figure 2. Pericytes are required for BBB formation. From: Pericytes are required for blood-brain barrier integrity during embryogenesis.

a, b, E18 Pdgfrb−/− mice (b) and littermate controls (a) were given a trans-cardiac perfusion of biotin, and tissue sections were stained with streptavidin (green; white arrows indicate tracer in vessels). Scale bars represent 200 µm (upper panel) and 100 µm (lower panel). c, E18 Pdgfrb−/− mice and littermate controls were given a trans-cardiac perfusion of 3 kDa or 70 kDa biotinylated dextran, tissue sections stained with streptavidin-Alexa 488, fluorescence was quantified in ImageJ and permeability relative to control was graphed. *P < 0.05 by Student’s t-test. df, Neonatal mouse cerebral cortex from PdgfrbF7/− (f), PdgfrbF7/F7 (e) and littermate controls (d) were stained with BSL (green, df (bottom)) and for pericytes with anti-desmin (purple, df). Scale bar represents 100 µm. g, Pericyte coverage of CNS vessels in PdgfrbF7/−, PdgfrbF7/F7 and littermate control mice was quantified by analysing per cent length of BSL+ vessels opposed to desmin+ pericytes. hj, P5 PdgfrbF7− mice (h), PdgfrbF7/F7 mice (i) and littermate controls (j) were given an intraperitoneal injection of Evan’s blue dye, and their brains were dissected the following day after PBS perfusion. k, Neonatal PdgfrbF7/−, PdgfrbF7/F7 and littermate controls were given a trans-cardiac perfusion of biotin and leakage was quantified in tissue sections with streptavidin-Alexa-488 (y axis) and graphed versus pericyte coverage (x axis; values from panel g). All error bars represent s.e.m.

Richard Daneman, et al. Nature. ;468(7323):562-566.
4.
Figure 3

Figure 3. Pericytes regulate structural aspects of the BBB. From: Pericytes are required for blood-brain barrier integrity during embryogenesis.

A, B, Electron microscopy images of CNS vessels from E18 Pdgfrb−/− mice (B) and littermate controls (A) including whole endothelial cell cross-sections (a), cytoplasm (b; yellow arrows indicate cytoplasmic vessels), tight junctions (c; yellow arrows indicate altered junction alignment; yellow arrowheads indicate junctions dipping into parenchyma), and after perfusion with biotin followed by staining with streptavidin–HRP (d, e; white arrows indicate uptake of tracer). Scale bars represent 2 µm (a), 0.5 µm (b, c) and 0.2 µm (d, e). C, Quantification of the number of vesicles per endothelial cross-section for Pdgfrb−/− mice and littermates. D, Angles of tight junctions (TJs) for Pdgfrb−/− mice and littermate controls were classified as parallel to the lumen (0°), perpendicular to the lumen (90°) or in between (45°). *P < 0.05 by Student’s t-test. E, F, Cerebral cortex of E18 Pdgfrb−/− mice (F) and littermate controls (E) were stained with BSL (green) and anti-occludin (red). Scale bars represent 20 µm. G, H, Purified murine brain endothelial cells were cultured alone (G) or with a feeding layer of purified brain pericytes (H) and stained with DAPI (blue) and anti-claudin 5 (left, red) or anti-occludin (right, red; yellow arrows indicate cell borders). Scale bars represent 100 µm (left) and 50 µm (right). I, Per cent length of sealed claudin 5 and occludin junctions in endothelial cells cultured alone or with pericyte feeder layers. **P < 0.01 by Student’s t-test. J, TEER measurements for purified murine brain endothelial cells cultured alone or with a feeding layer of purified brain pericytes. *P < 0.05 by Student’s t-test. All error bars represent s.e.m.

Richard Daneman, et al. Nature. ;468(7323):562-566.

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