LSFM imaging of known dorsal and laterobasal MLV drainage pathways. (A) Lateral view of OVA-A⁵⁵⁵ (magenta) in the posterior head 45 min after Lb-Sa injection labeled with anti-LYVE1 (green). OVA-A⁵⁵⁵ is present in perivascular spaces along the spinal cord (SC), cerebellum (Cb), and cortex (Cx) and in the dcLN (green arrowhead). Roman numbers indicate known MLV circuits: I (SSS), II (transverse sinus TS), III (sigmoid sinus SS and jugular foramen), IV (petrosquamous sinus PSS). NP, nasopharynx (solid line); dashed line, ventral skull border; dashed rectangles, regions magnified in panels I–IV. Inset: Venous sinuses and emissary veins imaged in B–K (light blue) and other venous circuits (dark blue). ijv, internal jugular vein; iptgv, interpterygoid emissary vein; pfv, posterior facial vein; rgv, retroglenoid vein. (B–E) Horizontal (B and C) and sagittal (D and E) views labeled with anti-vWF (B and C) and anti-LYVE1 (D and E) antibodies. The SSS (B, blue arrowheads) connects the rostral confluence of sinuses (RCS) with the caudal confluence of sinuses (COS) and the TS (C and E, blue arrowheads). Note discontinuous OVA-A⁵⁵⁵ labeling in the perisinusal spaces (B and C). LYVE1⁺ MLVs along the SSS (D) contain OVA-A⁵⁵⁵ at the TS (white in E). (F–H) Coronal views at the level of the jugular foramen (jf) labeled with the indicated antibodies and OVA-A⁵⁵⁵. vWF stained the SS and the jugular vein (ijv, blue arrowhead in F). OVA-A⁵⁵⁵ labels the jf and the dcLN (green arrowheads in F–H). (G) TUJ1⁺ cranial nerves exiting the skull through the jf were not colabeled with OVA-A⁵⁵⁵. (H) LYVE1⁺ MLVs follow the SS (blue arrowhead) and exit the skull through the jf toward the dcLN. IX, cranial nerve 9 (glossopharyngeal); X, cranial nerve 10 (vagus); X, cranial nerve 11 (spinal accessory); BS, brainstem; dotted line, skull border. (I–K) Coronal views at the PSS exit through the skull. (I) vWF stains the PSS passing through the petrosquamous fissure (interrupted dashed line) to join the pfv via the rgv. (J and K) LYVE1⁺ MLVs follow the PSS, rgv, and pfv. OVA-A⁵⁵⁵ accumulated at the petrosquamous fissure level (J). (K) Magnification of the dashed frame in J showing a blind-ended MLV and other OVA-A⁵⁵⁵⁺/LYVE1⁺ lymphatics (white, green arrowhead). Br, brain. Scale bars: 1,000 μm (A–D); 500 μm (E–J); 250 μm (K).