The draining route for CSF tracers from the brain to the eye. (A) Timeline of CM injection of Evans blue tracer. (B and C) Multitime point images of Evans blue spreading from the brain to the eye after injection and the corresponding statistical line chart of dye staining degree (n = 6). (D and E) 30-min and 60-min cross-sectional images of the optic nerve, along with a line graph representing the dye staining intensity along the yellow line. In the schematic diagram, the dye was visible at the sheath location at 30 min (black arrowhead), and at 60 min, the dye was within the optic nerve (yellow arrowhead). (F) Representative fluorescent microscopic image of Evans blue transportation along the optic nerve. (G) Image showing Evans blue transportation within the optic nerve sheath 10 min after injection, with the corresponding 3D image of IB4 staining (green) indicating blood vessels. (H and I) Representative images of Evans blue transportation within the optic nerve 20 and 30 min after injection along the blood vessels and nerve bundles. NS stands for the control group receiving brain injections of normal saline. (J and K) Representative images of Evans blue transportation within the optic nerve and retina 30 min after injection, with blood vessels stained by Laminin (green). (L) Line chart of fluorescence intensity showing the location relationship between Evans blue and blood vessels (n = 7). (M) Optic nerve sections demonstrated the distribution of Evans blue dye along myelinated axons along the optic nerve 30 min after injection. (N) Retinal flat mounts showing Evans blue transportation along retinal blood vessels 60 min after injection.