a, Representative two-photon images depicting ruffles on the lower superior sagittal sinus wall (white arrows) of a naïve Cdh5cre/+STOPfl/+-tdTomato mouse pre and post local application of azaindole 1 to the sinus wall via a thinned skull window (n = 4). b, Quantification of the average number of total ruffles for the duration of the pre- (0 to 30 min) and post-treatment (30 to 60 min) intravital imaging periods (n = 4 mice). Statistical analysis was performed using a paired two-tailed Student’s t-test. Three independent experiments. Data are ± s.d. c, Left graph shows quantification of lower sinus diameters from two-photon time-lapse videos of Cdh5cre/+STOPfl/+-tdTomato mice 30 min prior to (pre) and 45 min following (post) transient application of AM 22-52 through a thinned skull window (n = 3 mice). Statistical analysis was performed by obtaining the coefficient and intercept for the pre- and post-treatment fits along with the corresponding significance values as described in Extended Data Fig. 6a. Sinus diameter measurements were normalized by dividing each value in the dataset by the mean sinus diameter prior to treatment. Data are represented as a fold change ± s.d. Data represent three independent experiments. Right graph shows the total number of openings present on the lower sagittal sinus before and after local application of AM 22-52 through a thinned skull window (n = 5 mice). Each data point represents the average number of openings present on the sinus wall for the pre- and post-treatment periods. Statistical analysis was performed using a paired two-tailed Student’s t-test. Five independent experiments. d, Representative two-photon images depicting ruffles identified by ML (white) before and after local CGRP administration through the thinned skull window over the lower sagittal sinus. Mice were treated with CGRP after 30 min of image acquisition (n = 3 mice). e, Total number of ruffles present on the lower sagittal sinus before and after local application of CGRP through the thinned skull window. Each data point represents the average number of openings present on the sinus wall for the pre- and post-treatment periods. Data are normalized by dividing each value in the dataset by the mean pre-treatment velocity measurement and are ± s.d. Statistical analysis was performed using a paired two-tailed Student’s t-test. Three independent experiments. f, Representative two-photon images depicting ruffles identified by ML (white) before and after local administration of BIBN 4096 through a thinned skull window over the lower sagittal sinus. Mice were treated with BIBN 4096 after 30 min of image acquisition (n = 3 mice). g, Total number of ruffles present on the lower sagittal sinus before and after local application of BIBN 4096 through a thinned skull window. Each data point represents the average number of openings present on the sinus wall for the pre- and post-treatment period. Data are normalized by dividing each value in the dataset by the mean pre-treatment velocity measurement and are ± s.d. Statistical analysis was performed using a paired two-tailed Student’s t-test. Three independent experiments. h, Representative two-photon image captured in a Cdh5cre/+STOPfl/+-tdTomato X LysmGFP/+ mouse depicts an increase in ruffles on the VE-cadherin+ (red) sinus wall 9 days-post LCMV infection. The ruffles are predominantly occupied by LysM-GFP+ myelomonocytic cells (green) (n = 2). i, Zoomed in area of the sinus wall. Representative ruffles are denoted by white arrows (n = 2).
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