Pericyte Loss Leads to Capillary Stalling Through Increased Leukocyte-Endothelial Cell Interaction in the Brain

Figures

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  FIGURE 1. Pericyte coverage is reduced in the brain of DTAi△PC mice. (A) Schematic diagram of the mouse model and experimental timeline for selective depletion of pericytes in brain capillaries by tamoxifen administration. Tamoxifen (2 mg) was treated intraperitoneally for five consecutive days in 8-week-old DTAi△PC mice. Analyses were performed 2 weeks after the initial tamoxifen administration. (B,C) Images and comparisons of CD13+-pericyte coverage over lectin+-vessels, and capillary density, capillary length, and capillary tortuosity in vehicle-treated control (n = 6) and DTAi△PC (n = 8) mice. (D) Representative image of capillary in two-photon microscopy (TPM) imaging. White arrows indicate that the line scanning directions to measure capillary diameter and RBC velocity. (E) Paired comparisons of capillary diameter, RBC velocity, and RBC volume flux in control (n = 5) and DTAi△PC (n = 4) mice. Imaging is performed twice, before tamoxifen administration (baseline) and 2 weeks after the initial tamoxifen administration (after 2 weeks), in the same mice. The measurements from at least 10 capillaries for each mouse were averaged. Error bars represent mean ± SD. ∗∗∗∗P < 0.0001 and ∗P <…

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  FIGURE 2. Capillary stalling is increased in pericyte-deficient mice. (A,B) Images and comparison of the number of capillary segments labeled with DiI-coated microspheres in control (n = 5) and DTAi△PC (n = 5) mice. The number of capillary segments labeled with DiI-coated microspheres was measured in the primary somatosensory cortex. The measurements from at least 10 capillaries for each mouse were averaged. (C) CCD and time-averaged OCTA images at different time points showing no structural changes both in control and DTAi△PC mice. Time-averaged OCTA images were obtained by averaging 10 angiograms acquired in 63 s. Imaging was performed twice, before tamoxifen administration (baseline) and 2 weeks after the initial tamoxifen administration (after 2 weeks), in the same mice. Positions of stalled segments are shaded in yellow. (D,E) Comparisons of the number of stalled segments and percentage of re-stalled segments in control (n = 6) and DTAi△PC (n = 5) mice. The number of re-stalled segments after 2 weeks was normalized to the number of stalled segments at baseline. Error bars represent median with interquartile range and mean ± SD. ∗P < 0.05 vs. control, by Mann-Whitney test for…

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  FIGURE 3. The extent of the cerebral endothelial glycocalyx is decreased in pericyte-deficient brain capillaries. (A) Representative image of isolated brain capillaries. (B) Comparisons of the transcription profiles of genes contributing to maintaining structure of glycocalyx. List of genes was selected based on previous studies and only those that showed significant difference (P < 0.05) were selected. Capillaries were isolated from the brain of control (n = 3) and DTAi△PC (n = 4) mice. (C) TPM image of WGA lectin+-glycocalyx in capillary. Dotted square in TPM image indicates the region for analysis of the cerebral endothelial glycocalyx extent. (see supplementary Figure 3 for details.) (D) Comparison of the extent of the cerebral endothelial glycocalyx in control (54 capillaries, n = 4) and DTAi△PC (56 capillaries, n = 5) mice. White circles represent the individual value of the cerebral endothelial glycocalyx extent for each capillary. Black and red circles represent the averaged value of the cerebral endothelial glycocalyx extent for each mouse. The averaged values of the cerebral endothelial glycocalyx extent for each mouse are utilized for comparison analysis. The measurements from…

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  FIGURE 4. Pericyte loss leads to increased expression of leukocyte adhesion molecules. (A,B) Images and comparisons of VCAM1+ and ICAM1+ coverage over lectin+-vessels in control (n = 5) and DTAi△PC (n = 5) mice. (C) Comparisons of VCAM1 and ICAM1 expression in the whole brain lysate in control (n = 4) and DTAi△PC (n = 4) mice. GAPDH control is shown in Supplementary Figure 6A. Error bars represent mean ± SD and median with interquartile range. ∗∗P < 0.01 and ∗P < 0.05 vs. control, by Welch’s t-test for comparison analyses of vascular surface coverage of VCAM1, vascular surface coverage of ICAM, VCAM expression in the whole brain lysate. ∗P < 0.05 vs. control, by Mann-Whitney test for comparison analysis of ICAM1 expression in the whole brain lysate. All scale bars are 100 μm. VCAM1, vascular cell adhesion molecule 1; ICAM1, intercellular adhesion molecule 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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  FIGURE 5. Cerebral hypoxia is induced in pericyte-deficient mice. (A,B) Images and comparisons of mean GLUT1 intensity in lectin+-vessels in control (n = 5) and DTAi△PC (n = 5) mice. (C) Comparisons of GLUT1 expression in the whole brain lysate in control (n = 4) and DTAi△PC (n = 4) mice. GAPDH control is shown in Supplementary Figure 6A. Error bars represent mean ± SD. ∗P < 0.05 and ∗∗P < 0.01 vs. control, by Welch’s t-test for comparison analyses. All scale bars are 100 μm. GLUT1, glucose transporter 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.