Evidence for Cerebrospinal Fluid Entry Into the Optic Nerve via a Glymphatic Pathway

PURPOSE. The purpose of this study was to determine whether cerebrospinal fluid (CSF) enters the optic nerve via a glymphatic pathway and whether this entry is size-dependent. METHODS. Fluorescent dextran tracers (fluorescein isothiocyanate [FITC]) of four different sizes (10, 40, 70, and 500 kDa) and FITC-ovalbumin (45 kDa) were injected into the CSF of 15 adult mice. Tracer distribution in the orbital optic nerve at 1 hour after injection was assessed in tissue sections with confocal microscopy. Tracer distribution within the optic nerve was studied in relation to blood vessels and astrocytes identified by isolectin histochemistry and glial fibrillary acidic protein (GFAP) immunofluorescence, respectively. Aquaporin 4 (AQP4) immunostaining was performed to assess astrocytic endfeet in relation to CSF tracer. RESULTS. One hour following tracer injection into CSF, all tracer sizes (10-500 kDa) were noted in the subarachnoid space surrounding the orbital optic nerve. In all cases, 10 kDa (n = 4/4) and 40 kDa (n = 3/3) tracers were noted within the optic nerve, while 70-kDa tracer was occasionally noted (n = 1/4). Tracer found within the nerve was specifically localized between isolectin-labeled blood vessels and GFAP-positive astrocytes or AQP4-labeled astrocytic endfeet. The 500-kDa tracer was not detected within the optic nerve. CONCLUSIONS. To our knowledge, this is the first evidence of a glymphatic pathway in the optic nerve. CSF enters the optic nerve via spaces surrounding blood vessels, bordered by astrocytic endfeet. CSF entry into paravascular spaces of the optic nerve is size-dependent, and this pathway may be highly relevant to optic nerve diseases, including glaucoma.