Figure 3.
Figure 3. The expression of the chemokine CXCL1 is increased in Cdh5-Cre;Cdk5f/f mice. (A) Effect of endothelial Cdk5 deletion on BBB leakage in mice at 4 and 16 wk. Left: Representative stitched images of immunostaining for Evans blue in the hippocampus in Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 4 wk (bars, 500 µm). The black represents areas that were not captured. Right: Representative images of immunostaining for GFAP (green) and Evans blue (red) in the hippocampus in Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 16 wk (bars, 100 µm; insets, 20 µm). DAPI staining is shown in blue. (B) Representative confocal microscopy images of Biocytin-TMR, Evans blue, 40-kD Dextran (red), and Lectin-positive microvessels (gray) in the hippocampus from Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 4 and 16 wk. Bars, 50 µm. (C) Representative confocal microscopy images of fibrinogen and IgG (red) and Lectin-positive microvessels (gray) in the hippocampus from Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 4 and 16 wk. Bars, 50 µm. (D) Transmission electron microscopy images of hippocampal endothelial tight junctions in samples from Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 4 and 16 wk. Blue arrows indicate tight junctions between ECs on vessels. Red arrows indicate gaps lacking tight junctions between ECs on vessels. Bars: main images, 200 nm; insets, 100 nm. (E) Representative immunoblots and quantification of the changes in tight junction proteins in the microvessels of Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 16 wk (n = 3 mice per group; **, P < 0.01; unpaired two-tailed Student’s t test). (F) Double immunostaining (left) for Glut1 (red) and ZO-1 (green) in the hippocampus from Cdh5-Cre;Cdk5f/f and control mice. Z-stacking through the microvessels was performed to confirm the colocalization of ZO-1 with endothelial marker Glut1. Bar, 25 µm. Quantification (right) of the ZO-1 fluorescence intensity in the vessels (n = 3 mice per group; ***, P < 0.001; unpaired two-tailed Student’s t test). (G) Heat map of all the differentially expressed genes in Cdh5-Cre;Cdk5f/f vs. control mice at 4 wk. The threshold was set to a fold change ≥ 2 and a t test P value ≤ 0.05. The data were standardized along the rows. (H) IGV genome browser view of the RNA sequencing profile from the analysis of 4-wk-old Cdh5-Cre;Cdk5f/f and Cdk5f/f mice. The Cxcl1 is shown up-regulated, and Cxcl16 is shown unchanged upon conditional KO Cdk5 in ECs. (I) The chemokine expression in primary cultured ECs from the cerebral microvessels of Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 4 wk was evaluated by qRT-PCR. Note that the Cxcl1 level was increased in Cdh5-Cre;Cdk5f/f mice, while that of another Cxcl family member, Cxcl16, was unchanged between Cdh5-Cre;Cdk5f/f and control mice. Cxcl1 and Cxcl16 mRNA levels were normalized to the corresponding Gapdh level (n = 5 mice per group; *, P < 0.05; unpaired two-tailed Student’s t test). (J) CXCL1 protein levels were detected through ELISA (n = 5 mice per group; **, P < 0.01; unpaired two-tailed Student’s t test). (K) RNAscope for Cxcl1 probes (red) and CD31 (an EC marker; green) in the hippocampal CA1 stratum pyramidale for the indicated groups. The DAPI counterstaining (blue) indicates the nuclei. The quantification of the relative Cxcl1 mRNA level is shown at the right (n = 3 mice per group; *, P < 0.05; unpaired two-tailed Student’s t test). Bar, 25 µm. The error bars represent means ± SEM. FC, fold change; n.s., not significant.

The expression of the chemokine CXCL1 is increased in Cdh5-Cre;Cdk5f/f mice. (A) Effect of endothelial Cdk5 deletion on BBB leakage in mice at 4 and 16 wk. Left: Representative stitched images of immunostaining for Evans blue in the hippocampus in Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 4 wk (bars, 500 µm). The black represents areas that were not captured. Right: Representative images of immunostaining for GFAP (green) and Evans blue (red) in the hippocampus in Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 16 wk (bars, 100 µm; insets, 20 µm). DAPI staining is shown in blue. (B) Representative confocal microscopy images of Biocytin-TMR, Evans blue, 40-kD Dextran (red), and Lectin-positive microvessels (gray) in the hippocampus from Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 4 and 16 wk. Bars, 50 µm. (C) Representative confocal microscopy images of fibrinogen and IgG (red) and Lectin-positive microvessels (gray) in the hippocampus from Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 4 and 16 wk. Bars, 50 µm. (D) Transmission electron microscopy images of hippocampal endothelial tight junctions in samples from Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 4 and 16 wk. Blue arrows indicate tight junctions between ECs on vessels. Red arrows indicate gaps lacking tight junctions between ECs on vessels. Bars: main images, 200 nm; insets, 100 nm. (E) Representative immunoblots and quantification of the changes in tight junction proteins in the microvessels of Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 16 wk (n = 3 mice per group; **, P < 0.01; unpaired two-tailed Student’s t test). (F) Double immunostaining (left) for Glut1 (red) and ZO-1 (green) in the hippocampus from Cdh5-Cre;Cdk5f/f and control mice. Z-stacking through the microvessels was performed to confirm the colocalization of ZO-1 with endothelial marker Glut1. Bar, 25 µm. Quantification (right) of the ZO-1 fluorescence intensity in the vessels (n = 3 mice per group; ***, P < 0.001; unpaired two-tailed Student’s t test). (G) Heat map of all the differentially expressed genes in Cdh5-Cre;Cdk5f/f vs. control mice at 4 wk. The threshold was set to a fold change ≥ 2 and a t test P value ≤ 0.05. The data were standardized along the rows. (H) IGV genome browser view of the RNA sequencing profile from the analysis of 4-wk-old Cdh5-Cre;Cdk5f/f and Cdk5f/f mice. The Cxcl1 is shown up-regulated, and Cxcl16 is shown unchanged upon conditional KO Cdk5 in ECs. (I) The chemokine expression in primary cultured ECs from the cerebral microvessels of Cdh5-Cre;Cdk5f/f and Cdk5f/f mice at 4 wk was evaluated by qRT-PCR. Note that the Cxcl1 level was increased in Cdh5-Cre;Cdk5f/f mice, while that of another Cxcl family member, Cxcl16, was unchanged between Cdh5-Cre;Cdk5f/f and control mice. Cxcl1 and Cxcl16 mRNA levels were normalized to the corresponding Gapdh level (n = 5 mice per group; *, P < 0.05; unpaired two-tailed Student’s t test). (J) CXCL1 protein levels were detected through ELISA (n = 5 mice per group; **, P < 0.01; unpaired two-tailed Student’s t test). (K) RNAscope for Cxcl1 probes (red) and CD31 (an EC marker; green) in the hippocampal CA1 stratum pyramidale for the indicated groups. The DAPI counterstaining (blue) indicates the nuclei. The quantification of the relative Cxcl1 mRNA level is shown at the right (n = 3 mice per group; *, P < 0.05; unpaired two-tailed Student’s t test). Bar, 25 µm. The error bars represent means ± SEM. FC, fold change; n.s., not significant.

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