iDISCO-LSFM imaging of CSF tracer drainage. (A) Schematic of the experimental workflow. Arrows indicate sites of tracer injection, ICM, Th-Lb, or Lb-Sa. (B) LSFM imaging of OVA-A555 (white) in the pia mater of the spinal cord (SC) 90 min after Th-Lb injection. Asterisk, vertebral body. (C) Coronal section of cervical SC 45 min after Th-Lb injection shows OVA-A555 in the SC and in the dcLNs (green arrowheads). (D) LSFM view of a coronal section of the forebrain shows OVA-A⁵⁵⁵ in phagocytic cells (inset) along the glymphatic perivascular spaces. Note that brain size is reduced by the iDISCO⁺ protocol, but meningeal layers are preserved. The dura and superior sagittal sinus (SSS) are in contact with the skull (Sk), and tracer drainage can be followed through the intact calvaria. OVA-A⁵⁵⁵⁺ cells are also found along the SSS and the rostral confluence of sinuses (RCS). Cx, cortex. (E) 3D schematic of the lateral view of the head. Cb, cerebellum; CP, cribriform plate; ica, internal carotid artery; ijgv, internal jugular vein; NP, nasopharynx; OB, olfactory bulb; OC, oral cavity; OE, olfactory epithelium; ON, optic nerve. Green arrowheads, scLN. (F–H) Lateral views of OVA-A⁵⁵⁵ in mice sacrificed 15 min (F) or 45 min (G and H) after Lb-Sa injection. OVA-A⁵⁵⁵–labeled perivascular glymphatic spaces (F and G). OVA-A⁵⁵⁵ labeled the dcLN but not the scLN at 15 min (F, green arrowheads), while both dcLN and scLN are labeled at 45 min (G). Lymphatic afferent vessels extend from the NP toward the LNs (G). (H) The same pattern in another mouse with a zoom on the anterior part of the CP and OE. Note OVA-A⁵⁵⁵⁺ vessels in the OE and around the NP (G and H). (I) Summary of tracer injections and number of tracer-positive cervical LNs per mouse. (J and K) Quantification of OVA-A⁵⁵⁵⁺ DC1s (CD45⁺/CD11b⁺/CD11c⁺), DC2s (CD45⁺/CD11b⁻/CD11c⁺), and MΦ (CD45⁺/CD11b⁺/CD11c⁻) among total CD45⁺ cells FACS-sorted from the dcLN, mandibular LN (mLN), accessory mandibular LN (amLN), and superficial parotid LN (spLN) of non-injected mice or mice injected into the Th-Lb spine with OVA-A⁴⁸⁸. n = 6 mice/group. Data show mean + SEM; Mann–Whitney U test (J) and one-way ANOVA with Dunn’s multiple-comparisons test (K); *, P < 0.05; **, P < 0.01. A, anterior; D, dorsal; L, lateral; P, posterior; V, ventral. Scale bar: 500 μm (B–D and H); 800 μm (F and G).