A combination of extrinsic forces and contractile pumping drives lymphatic outflow of CSF in scLN and dcLN pathways. (A) Schematic of cranial lymphatic anatomy showing: (a) initial lymphatics in the nasal epithelium, (b) collecting lymphatics leading to the scLN, (c) initial intracranial meningeal lymphatics leading to the (f) dcLN via the (e) nasopharyngeal lymphatic plexus and (d) along the jugular vein. Boxes showing the location of (g) scLN- and (h) dcLN-afferent cLV imaging. (i) Inset of a single lymphangion showing contractile center (arrowhead) and flanking lymphatic valves (asterisks). (B) Representative frame captures of collecting vessel contractility afferent to the scLN and dcLN indicating the contractile center of the lymphangion (solid arrows) and flanking lymphatic valves (asterisks). Scale = 100 µm. (C) Representative traces from individual animals of cLV diameter afferent to the scLN and dcLN. (D) Peak amplitude in lymphatic pumping in the typical range of 2–20 min−1 (percent mean diameter, Lomb-Scargle periodogram, n = 10, 8, Student’s t test, ***, P = 0.00035). (E) Amplitude of mean max-min vessel diameter in lymphatic contractility of scLN and dcLN afferents (percent mean diameter, n = 10, 8, Student’s t test, ***, P = 0.00025). (F) Frequency of spectral amplitude peaks in lymphatic contractility in the typical range of 2–20 min−1 (Lomb-Scargle periodogram, n = 10, 8, Student’s t test, ***, P = 0.00012). (G) Representative frame of fluorescent microsphere drainage into the dcLN used for particle velocimetry with the dcLN (dashed line) and dcLN-afferent cLV (solid lines) indicated. Scale = 200 µm. (H) Representative traces from individual animals of mean particle velocity over time in dcLN afferents showing rapid oscillatory flow. (I) Representative frames of collecting lymphatic valve opening and closing in dcLN and scLN afferents (red: raw image signal, green: filtered image signal). Scale = 50 µm. (J) Representative traces of relative lymphatic valve movement in dcLN and scLN afferents with 0 indicating the mean inter-leaflet distance. (K) Frequency of peak amplitude in lymphatic valve opening in the range of 2–200 min−1 (Lomb-Scargle periodogram, n = 4, 3, Student’s t test, *, P = 0.011). (L) Representative frame captures of GCaMP6f calcium indicator fluorescence in LMCs in scLN- and dcLN-afferent lymphatic vessels. Scale = 100 µm. (M) Kymographs of GCaMP6f calcium indicator fluorescence in LMCs along scLN- and dcLN-afferent lymphatic vessels. (N) Representative traces from individual animals of vessel diameter and GCaMP6f ∆F/F0 in scLN- and dcLN-afferent lymphatic vessels (n = 3, 3).
Figure 1.

A combination of extrinsic forces and contractile pumping drives lymphatic outflow of CSF in scLN and dcLN pathways. (A) Schematic of cranial lymphatic anatomy showing: (a) initial lymphatics in the nasal epithelium, (b) collecting lymphatics leading to the scLN, (c) initial intracranial meningeal lymphatics leading to the (f) dcLN via the (e) nasopharyngeal lymphatic plexus and (d) along the jugular vein. Boxes showing the location of (g) scLN- and (h) dcLN-afferent cLV imaging. (i) Inset of a single lymphangion showing contractile center (arrowhead) and flanking lymphatic valves (asterisks). (B) Representative frame captures of collecting vessel contractility afferent to the scLN and dcLN indicating the contractile center of the lymphangion (solid arrows) and flanking lymphatic valves (asterisks). Scale = 100 µm. (C) Representative traces from individual animals of cLV diameter afferent to the scLN and dcLN. (D) Peak amplitude in lymphatic pumping in the typical range of 2–20 min−1 (percent mean diameter, Lomb-Scargle periodogram, n = 10, 8, Student’s t test, ***, P = 0.00035). (E) Amplitude of mean max-min vessel diameter in lymphatic contractility of scLN and dcLN afferents (percent mean diameter, n = 10, 8, Student’s t test, ***, P = 0.00025). (F) Frequency of spectral amplitude peaks in lymphatic contractility in the typical range of 2–20 min−1 (Lomb-Scargle periodogram, n = 10, 8, Student’s t test, ***, P = 0.00012). (G) Representative frame of fluorescent microsphere drainage into the dcLN used for particle velocimetry with the dcLN (dashed line) and dcLN-afferent cLV (solid lines) indicated. Scale = 200 µm. (H) Representative traces from individual animals of mean particle velocity over time in dcLN afferents showing rapid oscillatory flow. (I) Representative frames of collecting lymphatic valve opening and closing in dcLN and scLN afferents (red: raw image signal, green: filtered image signal). Scale = 50 µm. (J) Representative traces of relative lymphatic valve movement in dcLN and scLN afferents with 0 indicating the mean inter-leaflet distance. (K) Frequency of peak amplitude in lymphatic valve opening in the range of 2–200 min−1 (Lomb-Scargle periodogram, n = 4, 3, Student’s t test, *, P = 0.011). (L) Representative frame captures of GCaMP6f calcium indicator fluorescence in LMCs in scLN- and dcLN-afferent lymphatic vessels. Scale = 100 µm. (M) Kymographs of GCaMP6f calcium indicator fluorescence in LMCs along scLN- and dcLN-afferent lymphatic vessels. (N) Representative traces from individual animals of vessel diameter and GCaMP6f ∆F/F0 in scLN- and dcLN-afferent lymphatic vessels (n = 3, 3).

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