Reduced Cerebrospinal Fluid Inflow to the Optic Nerve in Glaucoma.

Purpose: To determine whether cerebrospinal fluid (CSF) entry into the optic nerve is altered in glaucoma. Methods: Fluorescent 10-kDa dextran tracer was injected into the CSF of 2-month-old (n = 9) and 10-month-old DBA/2J glaucoma mice (n = 8) and age-matched controls (C57Bl/6; n = 8 each group). Intraocular pressure (IOP) was measured in all mice before tracer injection into CSF. Tracer distribution was assessed using confocal microscopy of optic nerve cross-sections of mice killed 1 hour after injection. Paravascular tracer distribution in the optic nerve was studied in relation to isolectin-stained blood vessels. Tracer intensity and cross-sectional area in the laminar optic nerve were quantitatively assessed in all four groups and statistically compared. Aquaporin 4 (AQP4) and retinal ganglion cell axonal phosphorylated neurofilament (pNF) were evaluated using immunofluorescence and confocal microscopy. Results: IOP was elevated in 10-month-old glaucoma mice compared with age-matched controls. One hour after tracer injection, controls showed abundant CSF tracer in the optic nerve subarachnoid space and within the nerve in paravascular spaces surrounding isolectin-labeled blood vessels. CSF tracer intensity and signal distribution in the optic nerve were significantly decreased in 10-month-old glaucoma mice compared with age-matched controls (P = 0.0008 and P = 0.0033, respectively). AQP4 immunoreactivity was similar in 10-month-old DBA and age-matched control mice. Half of the 10-month-old DBA mice (n = 4/8) showed a decrease in pNF immunoreactivity compared to controls. Altered pNF staining was seen only in DBA mice lacking CSF tracer at the laminar optic nerve (n = 4/5). Conclusions: This study provides the first evidence that CSF entry into the optic nerve is impaired in glaucoma. This finding points to a novel CSF-related mechanism that may help to understand optic nerve damage in glaucoma.

Progressive loss of retinal blood vessels in a live model of retinitis pigmentosa.

OBJECTIVE: To assess retinal blood vessels in a live retinitis pigmentosa (RP) model with rd1 mutation and green fluorescent protein (GFP) expressed in vascular endothelium. METHODS: Homozygous (hm) Tie2-GFP mice with rd1 mutation and known retinal degeneration were crossed with wild-type CD1 mice to generate control heterozygous (ht) Tie2-GFP mice. The retinas of 16 live hm mice were evaluated at 2 weeks and 3, 5, and 8 months of age, and compared with age-matched control ht and CD1 mice by optical coherence tomography (OCT) and confocal scanning laser ophthalmoscopy (cSLO). Fluorescence intensity was measured and compared between strains at 3, 5, and 8 months. In vivo findings were validated by immunostaining with collagen IV and isolectin histopathology. RESULTS: All hm Tie2-GFP mice showed progressive outer retinal degeneration by OCT. Loss of small branches of blood vessels and then larger main vessels was seen by cSLO. Retinal tissue and vessels were preserved in control ht mice. At all ages, measurements of fluorescence intensity were reduced in hm compared with ht mice (p < 0.001). In all strains, intensity at 8 months was reduced compared with 3 months (p < 0.001) and 5 months (p = 0.021). Histopathological studies confirmed in vivo findings and revealed a pattern of blood vessel regression in the deep plexus, followed by intermediate and superficial retinal plexuses. CONCLUSIONS: This is the first evidence of progressive loss of retinal blood vessels in a live mouse model of RP. These findings may be highly relevant to understanding retinal degeneration in RP to prevent blindness.

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.

In vivo imaging of lymphatic drainage of cerebrospinal fluid in mouse.

BACKGROUND: Mouse models are commonly used to study central nervous system disorders, in which cerebrospinal fluid (CSF) drainage may be disturbed. However, mouse CSF drainage into lymphatics has not been thoroughly characterized. We aimed to image this using an in vivo approach that combined quantum dot fluorescent nanoparticles with hyperspectral imaging. FINDINGS: Quantum dot 655 was injected into the CSF of the cisterna magna in seven mice and visualized by in vivo hyperspectral imaging at time points 20 and 40 min, 1, 2, and 6 h after injection. In controls (n = 4), quantum dots were applied directly onto intact dura mater covering the cisterna magna. After imaging, lymph nodes in the neck were harvested and processed post-mortem for histological analysis. After injection into the CSF, quantum dot signal was detected in vivo in submandibular lymph nodes of all mice studied as early as 20 min, but not in controls. Post-mortem gross and histological examination of lymph nodes confirmed in vivo observations. CONCLUSIONS: Non-invasive in vivo hyperspectral imaging is a useful tool to study CSF lymphatic drainage and is relevant to understanding this pathway in CNS disease models.