VEGF-C effects in CSF-draining lymphatics. (A) VEGF-C protein expression measured by ELISA of the CSF (n = 4–6 mice/group, **P < 0.01, Mann–Whitney test). (B and C) MLV immunolabeling. (B) Confocal imaging of MLVs labeled with the indicated antibody in the confluence of sinuses (COS) and quantification of MLV surface and diameter (n = 5 mice/group, *P < 0.05, Mann–Whitney test). (C) LSFM imaging of mice treated as indicated for 4 wk, injected with OVA-A⁶⁴⁷ 30 min before sacrifice, then stained with anti-LYVE-1 antibody (n = 3 mice/group). Posterior cavernous sinus (pCAV), ophthalmic emissary vein (ophev), olfactory emissary vein (olfev), rostral cavernous sinus (rCAV), and cribriform plate (CrPl). Insets show higher magnifications of OVA-A⁶⁴⁷ uptake into the rCAV (white). (D) dCLN immunolabeling of LYVE-1⁺ and KI67⁺ cells in mice treated for 4 wk with either AAV-mVEGF-C or -CTRL. Quantification of KI67⁺LYVE-1⁺/LYVE-1⁺ pixel area. Scale bar: 100 μm (left panel) and 20 μm (right panels). (E–G) Bulk RNA-seq analysis of FACS-sorted dural LECs. Volcano plot of DEGs between CTRL and VEGF-C–treated mice in mRNA extracted from dural LECs. Downregulated genes (blue) and upregulated genes (red). (F) HALLMARK GSEA dot-plot depicting the most upregulated and downregulated pathways in the AAV-mVEGF-C group compared to control. N = number of DEGs/pathway. NES, normalized enrichment score. (G) qPCR analysis of dura mater mRNAs. Expression of indicated genes in the AAV-mVEGF-C group compared with controls (n = 3 mice/group, **P < 0.01, ***P < 0.005 Mann–Whitney test). (H) Volcano plot of DEGs between CTRL- and VEGF-C–treated mice in mRNA from FACS-isolated dural CD45⁺ leukocytes. Downregulated genes (blue) and upregulated genes (red). (I) GSEA dot plot based on the GO biological process (GOBP) illustrating the most upregulated and downregulated pathways in the AAV-mVEGF-C group compared with control. Scale bar: 600 μm (A); 1,000 μm (C); 100 μm (D, left panel); 20 μm (D, right panels).