Sacral spinal cord outflow. (A and B) Macroscope imaging of the sacrococcygeal region 90 min after ICM injection of OVA-A⁵⁵⁵ tracer. (A) Tracer deposits were detected at intervertebral spaces (white arrows, S3–S4, S4-Co1, Co1–Co2) and in the sciatic LNs (siLN, green arrowheads). (B) In the peritoneal cavity, the tracer (white) was detected in collecting lumbar LNs (lbLN) and renal LNs (reLN) (green arrowheads). Co, coccygeal vertebrae; S, sacral vertebrae. (C–E) LSFM sagittal (C) and coronal (D and E) views of clarified sacral vertebrae, spinal cord, and dura mater. (C) Outflow of tracer (red) in the epidural space (white arrows) between S3–S4 and S4-Co1. Asterisks, vertebral bodies. (D and E) Pattern of LYVE1⁺ lymphatics (green) and tracer (red) in the S3–S4 region. Tracer deposits (red) were detected in the vertebral canal and in siLNs (green arrowheads in D). (E) Magnification of dotted frame in D. Tracer accumulated in the epidural space (ES), including in LYVE1⁺ lymphatics (yellow, white arrowheads in E). A, anterior; D, dorsal; L, lateral; P, posterior; V, ventral. (F) Quantifications of OVA-A⁵⁵⁵⁺ MΦ (CD45⁺/CD11b⁺/CD11c⁻) among total CD45⁺ cells FACS-sorted from the siLNs and lbLNs of noninjected mice or mice injected into the Th-Lb spine with OVA-A⁴⁸⁸. n = 6 mice/group. Data show mean + SEM; one-way ANOVA with Dunn’s multiple-comparisons test; *, P < 0.05. (G–K) Light-field microscope images of coronal paraffin sections of S3–S4 vertebrae from mice with intraspinal injection of ink tracer. (G) Low magnification of highly magnified areas shown in H–K. Tracer was detected on the ependymal layer and the pial surface of the spine (H), as well as inside phagocytic cells of the subarachnoid (I and J) and epidural (K) spaces. Note the thin monolayer of dura mater (black arrow in I) and the accumulation of tracer-containing mononuclear cells in subdural arachnoid sacs (black arrow in J). Asterisk, vertebral body. Scale bar: 2 mm (A and B); 500 μm (C–E and G); 10 μm (H–K).