Extended Data Fig. 11. Expansion of nasopharyngeal lymphatic plexus and CSF outflow to deep cervical lymph nodes in aged mice by intracisternal AAV-mVEGF-C.

a, Diagram of the sequence of intracisternal (i.c.) infusion of 3 × 10¹⁰ genome copies of AAV9-mCherry (mCherry) or AAV9-mVEGF-C-mCherry (mVEGF-C) in 3 μl of PBS followed 3 weeks later by i.c. infusion of TMR-Dextran at 1.0 μl/min for 3 min (or 0.5-µm FluoSpheres) and measurement of TMR-Dextran fluorescence in deep cervical lymph nodes (dcLN) 30 min later in aged (75–78 weeks old) Prox1-GFP or C57BL/6J mice. b,c, Immunofluorescence images comparing whole mounts of the nasopharyngeal lymphatic plexus after AAV delivery of mCherry or mVEGF-C. mCherry expression (red) is present in the NPLP of both groups, indicating that the AAV9-vectors were similarly transduced in these lymphatics. Scale bars, 500 μm. Each dot is the value for one mouse; Prox1⁺ lymphatic area (mCherry, n = 4; mVEGF-C, n = 6) in two independent experiments. Bars indicate mean ± s.e.m. AU, arbitrary unit. P value was calculated by two-tailed Mann-Whitney test. d, Fluorescence images showing the distribution of FluoSpheres in lymphatics of whole mounts of the posterior nasal and nasopharyngeal plexuses at 1 h after the i.c. infusion to mice that received AAV9-mVEGF-C-mCherry. Scale bar, 500 μm. Similar findings were obtained from n = 3 mice in two independent experiments. e, Fluorescence images and measurements of TMR-Dextran fluorescence in deep cervical lymph nodes (dcLN, outlined by green dashed lines) in aged mice after AAV delivery of mCherry or mVEGF-C. Scale bars, 200 μm. Each dot is the value for one mouse; TMR-Dextran intensity in dcLN (mCherry, n = 6; mVEGF-C, n = 4) in three independent experiments. Bars present mean ± s.e.m. AU, arbitrary unit. P value was calculated by two-tailed Mann-Whitney test.