Figure 9. Notch3 deficiency delays glymphatic flow.

(A) Experimental design. Mice were anesthetized and injected with PECAM antibodies. A cannula inserted in the cisterna magna delivered fluorescent beads, which reached the perivascular space and were visualized by live imaging from the intact skull. (B) In vivo images of PECAM-labeled vessels in Notch3^–/– and control. Arrows indicate flow of FITC-beads; asterisks indicate aneurysms. (C) Quantification of vessel diameter (from live images). Data are shown as the mean ± SD; n = 20 vessels, 5 animals (control), and 28 vessels, 8 animals (Notch3^–/–), from mixed sexes at 6 months. Mann-Whitney test. (D) Quantification of aneurysms per vessel. n = 20 vessels, 5 animals (control), and 28 vessels, 8 animals (Notch3^–/–), from mixed sexes at 6 months. One-sample Wilcoxon’s test. (E) Quantification of mean bead velocity. (F) Quantification of beads per visual field in mixed sexes at 6 months. Data are shown as the mean ± SD; n = 4; Welch’s t test for E and F. (G) Representative inferior and lateral images of Notch3^–/– and control brains harvested 3 hours after cisternal injection. Yellow arrows indicate bead stagnation. (H) Experimental design. Mice were injected with FITC-dextran into the cisterna magna; blood was collected by cardiac puncture to assess fluorescence as a measurement of glymphatic clearance. (I) Quantification of fluorescence intensity after cisternal FITC-dextran injection across multiple time points in a mixed-sex cohort of 3-month C57BL/6J mice. n = 5–7 animals per time point and/or condition, as indicated in graph; unpaired Student’s t test. (J) Quantification of plasma fluorescence intensity 5 minutes after cisternal FITC-dextran injection in a mixed-sex cohort of 6-month Notch3^–/– and control animals. Data are shown as the mean ± SD; n = 8–10 animals per time point and/or condition, as indicated; Welch’s t test. For I and J: squares, male; inverted triangles, female.