December 2018
Volume 59, Issue 15
Open Access
Glaucoma  |   December 2018
Reduced Cerebrospinal Fluid Inflow to the Optic Nerve in Glaucoma
Author Affiliations & Notes
  • Emily Mathieu
    Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada
    Department of Ophthalmology and Vision Sciences, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada
    Department of Laboratory Medicine and Pathobiology, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada
  • Neeru Gupta
    Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada
    Department of Ophthalmology and Vision Sciences, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada
    Department of Laboratory Medicine and Pathobiology, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada
    Glaucoma Unit, St. Michael's Hospital, Toronto, Ontario, Canada
    Dalla Lana School of Public Health, University of Toronto, Toronto, Ontario, Canada
  • Luz A. Paczka-Giorgi
    Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada
    Department of Ophthalmology and Vision Sciences, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada
  • Xun Zhou
    Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada
    Department of Ophthalmology and Vision Sciences, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada
  • Amir Ahari
    Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada
    Department of Ophthalmology and Vision Sciences, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada
  • Rafael Lani
    Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada
  • Joseph Hanna
    Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada
    Department of Ophthalmology and Vision Sciences, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada
    Department of Laboratory Medicine and Pathobiology, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada
  • Yeni H. Yücel
    Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada
    Department of Ophthalmology and Vision Sciences, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada
    Department of Laboratory Medicine and Pathobiology, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada
    Department of Physics, Faculty of Science, Ryerson University, Toronto, Ontario, Canada
    Faculty of Engineering and Architectural Science, Ryerson University, Toronto, Ontario, Canada
    Institute of Biomedical Engineering, Science and Technology (iBEST), St. Michael's Hospital, Ryerson University, Toronto, Ontario, Canada
  • Correspondence: Yeni H. Yücel, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, 30 Bond Street, 209 LKSKI Room 409, Toronto, Ontario M5B 1W8, Canada; yucely@smh.ca
Investigative Ophthalmology & Visual Science December 2018, Vol.59, 5876-5884. doi:10.1167/iovs.18-24521
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      Emily Mathieu, Neeru Gupta, Luz A. Paczka-Giorgi, Xun Zhou, Amir Ahari, Rafael Lani, Joseph Hanna, Yeni H. Yücel; Reduced Cerebrospinal Fluid Inflow to the Optic Nerve in Glaucoma. Invest. Ophthalmol. Vis. Sci. 2018;59(15):5876-5884. doi: 10.1167/iovs.18-24521.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

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.

Glaucoma is an optic neuropathy and a leading cause of blindness worldwide.1,2 It is characterized by progressive loss of retinal ganglion cells and their axons, resulting in optic disc cupping and vision loss. Glaucoma is often associated with elevated intraocular pressure (IOP), the main risk factor for development and progression of this disease. 
Emerging evidence suggests a potential role for cerebrospinal fluid (CSF) in glaucoma. Several investigators have proposed that the pressure gradient across the lamina cribrosa between IOP and the pressure immediately behind the eye (orbital CSF pressure) may be relevant as a risk factor for the development of glaucomatous damage.36 This hypothesis is supported by clinical studies showing that lumbar CSF pressure is lower in primary open angle glaucoma3 and low-tension glaucoma4 patients than controls. Altered CSF dynamics also may contribute to optic nerve pathology in glaucoma7,8; however, experimental evidence supporting this hypothesis is lacking. 
Mice are known to have similar IOP,9 optic nerve anatomy,10 CSF drainage pathways,11 and CSF pressure12 to humans. These similarities make mice a useful model in glaucoma research. We have recently shown that CSF enters the mouse optic nerve via a “glymphatic” pathway.13 Along this pathway, CSF permeates through the optic nerve along paravascular spaces formed between blood vessels and aquaporin 4 (AQP4)-laden astrocytic endfeet.13 Similar to the brain's glymphatic system,14,15 this inflow of CSF may play crucial roles in nutrient delivery and clearance of metabolic waste in the optic nerve. This newly discovered fluid pathway may be relevant to glaucoma; however, it is not yet known whether CSF flow into the optic nerve is affected in glaucoma. 
DBA/2J (DBA) mice develop spontaneous age-related elevated IOP leading to optic nerve head excavation and retinal ganglion cell loss that closely mimic characteristics of human glaucoma.16,17 This well-characterized model presents a unique opportunity to assess whether CSF entry to the optic nerve is altered in glaucoma. Here we assess the entry of CSF to the optic nerve subarachnoid space and into the optic nerve in the DBA mouse model of glaucoma. 
Methods
Experimental Animals
Two-month-old C57Bl/6J (n = 9; 5 male/4 female) and DBA/2J (n = 10; 6 male/4 female), and 10-month-old C57 (n = 9; 3 male/6 female) and DBA mice (n = 9; 5 male/4 female) were housed on a 12-hour light-dark cycle and fed standard food ad libitum. Experimental animals were supplied by The Jackson Laboratory (Bar Harbor, ME, USA). All experimental protocols adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the institutional animal care committee. 
Fluorescent Tracer
Lysine-fixable lyophilized Alexa Fluor 647 conjugated to 10-kDa dextran (Invitrogen, Carlsbad, CA, USA) tracer was reconstituted in phosphate buffered saline (PBS) to a final concentration of 10 mg/mL and stored at −20°C until the day of injection. 
IOP Measurements
Approximately 10 minutes before tracer injection into the CSF, IOP was measured in both eyes using a rebound tonometer (Tonolab; Icare Finland Oy, Vantaa, Finland) under general isoflurane anesthesia (2% isoflurane in 100% O2).18 Each measurement was the average of six repeat measures and each eye was measured three times. These readings were then averaged to obtain the final IOP for each eye. Reported IOPs are the average of measurements from both eyes. 
Fluorescent Tracer Injection into the CSF
A detailed description of tracer injection into the CSF can be found in Mathieu et al.13 Briefly, under general isoflurane anesthesia as described above, 3 μL tracer was injected into the cisterna magna as a slow bolus through a suboccipital incision. It was previously shown that this injection technique does not increase intracranial pressure.19 Eight 2-month-old C57 (4 male/4 female), nine 2-month-old DBA (5 male/4 female), eight 10-month-old C57 (3 male/5 female), and eight 10-month-old DBA (4 male/4 female) mice were injected. After tracer injection into the CSF, mice were kept in a warm chamber under isoflurane anesthesia for the remainder of the experiment. One additional animal from each group did not undergo injection and these were used as noninjected controls. 
Euthanasia and Tissue Preparation
One hour after tracer injection, mice were killed under deep general isoflurane anesthesia (5% in 100% O2) by intracardiac perfusion with cold saline followed by 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA). Noninjected control mice (n = 1 for each group) were killed using the same procedure without tracer injection into the CSF to account for background autofluorescence in optic nerve tissue during analysis. 
Whole head specimens from each mouse were harvested and immersion-fixed overnight in 2% paraformaldehyde (Electron Microscopy Sciences) at 4°C followed by cryoprotection in 10% sucrose solution in PBS for 1 day and 20% sucrose for 2 days. Globes with attached intraorbital optic nerves were isolated and snap frozen in isopentane cooled by dry ice before embedding in optimal cutting temperature medium (Tissue-Tek O.C.T. Compound; Sakura Finetek USA, Torrance, CA, USA). Optic nerves were serially sectioned in the coronal plane using a cryostat (30 μm; CM 1900 Cryostat; Leica, Wetzlar, Germany). Optic chiasms from 10-month-old C57 and DBA mice were processed in the same way and coronally sectioned (30 μm). 
Confocal Microscopy of CSF Tracer in Optic Nerve Sections
Optic nerve sections from the mid-orbit (600–700 μm behind the posterior sclera) and from the glial lamina (at the level of the posterior sclera) were evaluated. The sections were rinsed with PBS and mounted on glass slides (Fisherbrand Superfrost Plus; Fisher Scientific, Pittsburgh, PA, USA) with antifade mounting medium (Dako Fluorescence Mounting Medium; Dako North America Inc., Carpinteria, CA, USA) and coverslipped (Fisherbrand #1.5; Fisher Scientific, Pittsburgh, PA, USA). Confocal imaging (LSM 700; Zeiss, Oberkochen, Germany) of sections was performed to identify and map tracer distribution in the optic nerve subarachnoid space and within the optic nerve. Confocal images were taken using a ×20 objective. Acquisition settings (laser intensity, pinhole size, gain) for the 647 channel (tracer imaging) were consistent across all samples. Z-stacks of 22 μm thickness were collected with 1.17 μm step size. Z-stacks were processed using ImageJ (Fiji, version 2.0.0-rc-54/1.51g; http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) image analysis software to create maximum intensity projections and color composites. All sections were imaged alongside noninjected control sections to account for background tissue autofluorescence. 
Optic chiasm sections were imaged to assess the presence of tracer in the intracranial chiasmatic cistern surrounding the chiasm and to confirm that tracer injection into the CSF was successful. Acquisition settings were consistent across all samples. 
Histochemistry and Immunofluorescence Staining
Isolectin B4 staining was used to label blood vessel endothelium in coronal optic nerve sections. Sections were rinsed (3 × 5 minutes in PBS) and stained overnight at 4°C with isolectin B4 from Griffonia simplicifolia conjugated to Alexa Fluor 488 (Invitrogen) diluted 1:50 from a 1-mg/mL stock solution in 0.5% Triton X-100 PBS solution. 
AQP4 on optic nerve astrocytes was labeled by immunofluorescence staining of coronal sections from each mouse by first rinsing (3 × 5 minutes in PBS) and blocking with goat serum (5% in PBS with 0.5% Triton X-100) for 40 minutes. Sections were incubated overnight at 4°C with rabbit anti-rat AQP4 primary antibody (1:200; Chemicon, Billerica, MA, USA), followed by a rinsing step and secondary antibody (goat anti-rabbit Alexa Fluor 555; 1:500; Invitrogen) for 2 hours at room temperature. 
Phosphorylated neurofilament (pNF) was assessed as a marker of retinal ganglion cell axons20 in optic nerve cross-sections with immunofluorescence staining against phosphorylated heavy molecular weight neurofilament.21 Sections from each mouse were selected from the glial lamina. After rinsing (3 × 5 minutes in PBS), sections were blocked with goat serum (5% in PBS with 0.5% Triton X-100; Sigma, St. Louis, MO, USA) for 1 hour at room temperature, followed by overnight primary antibody incubation (SMI-31; mouse monoclonal; 1:500 in blocking buffer; BioLegend, San Diego, CA, USA) at 4°C. Sections were then rinsed and incubated for 1 hour at room temperature in secondary antibody (Alexa Fluor 555 goat anti-mouse; 1:1000 in blocking buffer; Invitrogen) before a final rinse and mounting with fluorescence mounting medium. Negative controls were obtained by omitting primary antibody. 
pNF immunoreactivity was variable among glaucoma mice. pNF immunoreactivity decreases were determined to be mild focal (only a few axon bundles affected in a small region of the nerve), moderate decrease, or severe decrease (nearly all axon bundles affected; almost complete absence of pNF immunoreactivity in some bundles). 
Quantification of CSF Tracer in Optic Nerve Sections
Quantification of CSF tracer fluorescence in the optic nerve was conducted for all four groups at the glial lamina. Tracer fluorescence was quantified using two parameters: mean pixel intensity (mean gray value) and suprathreshold area fraction (% area), indicating the tracer intensity and the cross-sectional tracer coverage area, respectively. These measures were obtained using ImageJ by first drawing a region of interest (ROI) around the nerve that included the entirety of the optic nerve parenchyma while excluding the surrounding subarachnoid space, sclera, and dura. Mean pixel intensity was acquired on unthresholded images and background autofluorescence measured in noninjected controls was subtracted to obtain the final tracer intensity values. Images were then thresholded to eliminate background autofluorescence and suprathreshold area fraction was measured. For each mouse, one laminar optic nerve cross-section consisting of 10 consecutive confocal z planes was assessed. This corresponded to an 11-μm-thick digital stack. 
Quantification of CSF Tracer in Optic Chiasm Sections
CSF tracer at the border of optic chiasms from all 10-month-old mice was quantified by measuring mean pixel intensity in a rectangular 200 × 10-μm ROI next to the midline of the chiasm. Background autofluorescence measured in noninjected controls was subtracted to obtain the final tracer intensity values. 
Quantification of AQP4 Expression in Optic Nerve Sections
AQP4 immunoreactivity was quantified using the same protocol used to quantify CSF tracer in the optic nerve. AQP4 signal intensity and cross-sectional area coverage were measured in an ROI that included only the optic nerve parenchyma. 
Correlation Between Optic Nerve Cross-Sectional Area and CSF Tracer Intensity
Optic nerve cross-sectional area was measured in coronal sections from all 10-month-old C57 and DBA mice using ROIs drawn with ImageJ image analysis software. Linear regression analysis using GraphPad (GraphPad Prism 7.0c for Mac; La Jolla, CA, USA) software was performed to assess any correlation between optic nerve cross-sectional area and CSF tracer intensity and CSF tracer area coverage within the nerve. 
Statistical Analysis
IOP, tracer intensity, and cross-sectional distribution measurements in laminar optic nerve sections were assessed for all groups using linear mixed effects regression models that included the main effects of strain and age along with a strain × age interaction term. If the interaction between age and strain was statistically significant at P < 0.10, we compared strains within the same age category and each strain across age categories. Stata commands, including margins and contrasts, were used to interpret the comparisons. Two-sided P < 0.05 was considered to be statistically significant. Three extreme outliers were identified and excluded from statistical analysis. All analyses were completed using Stata 15.1 (2017; StataCorp LLC, College Station, TX, USA). 
All other quantification data were tested for normality using d'Agostino and Pearson tests (GraphPad). Normally distributed data sets were compared using two-tailed Student's t-tests assuming unequal variance (Welch corrected) (GraphPad). This included AQP4 distribution data and CSF tracer intensity in optic chiasms. Non-normally distributed data sets were compared using two-tailed Mann-Whitney U tests (GraphPad). This included AQP4 signal intensity data. P values less than 0.05 were considered significant. 
Results
Intraocular Pressure
Mean IOP in 2-month-old C57, 2-month-old DBA, 10-month-old C57, and 10-month-old DBA mice was 15.3 ± 0.8, 12.6 ± 1.5, 15.7 ± 0.6, and 20.3 ± 5.1 mm Hg (mean ± SD), respectively. IOP was significantly higher in 10-month-old DBA mice compared with 2-month-old DBA mice (P = 0.0000), and 10-month-old C57 controls (P = 0.0023). 
Distribution of CSF Tracer in Glaucomatous Optic Nerves
CSF-injected tracer was present in the optic nerve subarachnoid space and in the optic nerve in branching tubular formations at the laminar level in 2-month-old C57 (n = 6/8), 2-month-old DBA (n = 6/9), and all 10-month-old C57 controls (n = 8/8) (Figs. 1a, 1b). Tracer within the nerve was primarily in spaces immediately surrounding isolectin-labeled blood vessels (Fig. 2). Signal intensity in and around the optic nerve varied between individuals within each mouse group, whereas signal intensity in the subarachnoid space and in paravascular spaces of a given optic nerve appeared similar. 
Figure 1
 
(a) Optic nerve sections of 10-month-old C57 mice showed CSF tracer (green) in the subarachnoid space (arrow) and within the nerve (arrowhead). (b) At higher power, CSF tracer signal was evident in channels within the nerve. (c) Optic nerve sections in 10-month-old DBA mice showed absent CSF tracer, also at higher power (d). This was similar to controls without tracer injection (e, f). Scale bars: (a, c, e) 50 μm, (b, d, f) 20 μm.
Figure 1
 
(a) Optic nerve sections of 10-month-old C57 mice showed CSF tracer (green) in the subarachnoid space (arrow) and within the nerve (arrowhead). (b) At higher power, CSF tracer signal was evident in channels within the nerve. (c) Optic nerve sections in 10-month-old DBA mice showed absent CSF tracer, also at higher power (d). This was similar to controls without tracer injection (e, f). Scale bars: (a, c, e) 50 μm, (b, d, f) 20 μm.
Figure 2
 
CSF tracer (green) in optic nerve coronal sections from age-matched controls is primarily within paravascular spaces surrounding isolectin-labeled blood vessels (red) seen at (a) low, and (b) high power. Scale bars: (a) 50 μm, (b) 20 μm.
Figure 2
 
CSF tracer (green) in optic nerve coronal sections from age-matched controls is primarily within paravascular spaces surrounding isolectin-labeled blood vessels (red) seen at (a) low, and (b) high power. Scale bars: (a) 50 μm, (b) 20 μm.
Within the 10-month-old DBA group, tracer was absent in the subarachnoid space and in the optic nerve in five of eight mice (Figs. 1c, 1d). Remaining mice showed minimal tracer (two of eight), and no difference compared with controls (one of eight). Ten-month-old DBA mice lacking CSF tracer in and around the nerve showed background autofluorescence similar to the noninjected control (Figs. 1e, 1f). CSF tracer was absent in the laminar optic nerve in all 10-month-old DBA females (n = 4/4) and in one of four males. CSF tracer was present surrounding the optic chiasm in all 10-month-old C57 (n = 8/8) and DBA mice (n = 8/8), including those without tracer at the laminar optic nerve (Figs. 3a–d). 
Figure 3
 
(a) Abundant CSF tracer (green) lined the optic chiasm (Ch) of a 10-month-old C57 mouse. Dashed line indicates midline of chiasm. (b) In the same animal depicted in (a), there was CSF tracer in the optic nerve subarachnoid space and laminar optic nerve. (c) CSF tracer lined the subarachnoid space of a 10-month-old DBA mouse. (d) In the same animal depicted in (c), there was an absence of CSF tracer in the optic nerve subarachnoid space and laminar optic nerve. (e) Quantification of CSF tracer intensity (mean ± SD) in optic chiasms from 10-month-old C57 and DBA mice showed no significant difference between groups (P = 0.053). Scale bars: (a, c) 200 μm, (b, d) 50 μm.
Figure 3
 
(a) Abundant CSF tracer (green) lined the optic chiasm (Ch) of a 10-month-old C57 mouse. Dashed line indicates midline of chiasm. (b) In the same animal depicted in (a), there was CSF tracer in the optic nerve subarachnoid space and laminar optic nerve. (c) CSF tracer lined the subarachnoid space of a 10-month-old DBA mouse. (d) In the same animal depicted in (c), there was an absence of CSF tracer in the optic nerve subarachnoid space and laminar optic nerve. (e) Quantification of CSF tracer intensity (mean ± SD) in optic chiasms from 10-month-old C57 and DBA mice showed no significant difference between groups (P = 0.053). Scale bars: (a, c) 200 μm, (b, d) 50 μm.
CSF Tracer Quantification
CSF tracer intensity in the laminar optic nerve of 2-month-old C57 and DBA mice, and 10-month-old C57 and DBA mice, was 2109 ± 1761, 1906 ± 1485, 3477 ± 1815, and 724 ± 635 arbitrary intensity units (a.u.), respectively (mean ± SD; Fig. 4a). There was a statistically significant interaction between the effects of age and strain on CSF tracer intensity (P = 0.0202). At 2 months of age, DBA and C57 mice showed no significant differences in CSF tracer intensity (P = 0.7828). However, at 10 months of age, DBA mice showed significantly lower CSF tracer intensity compared with C57 mice (P = 0.0008). In both strains, tracer intensity in mice aged 10 months did not differ significantly from mice aged 2 months (C57, P = 0.0718; DBA, P = 0.1171). 
Figure 4
 
(a) Optic nerve CSF tracer fluorescence intensity for all groups. Optic nerve signal was significantly decreased in 10-month-old DBA mice compared with age-matched controls (P = 0.0008). (b) CSF tracer distribution (% area) as a percentage of optic nerve cross-sectional area for all groups. The overall distribution of CSF tracer was significantly less in 10-month-old DBA mice compared with age-matched controls (P = 0.0033). Bars indicate mean ± SD. **P < 0.01.
Figure 4
 
(a) Optic nerve CSF tracer fluorescence intensity for all groups. Optic nerve signal was significantly decreased in 10-month-old DBA mice compared with age-matched controls (P = 0.0008). (b) CSF tracer distribution (% area) as a percentage of optic nerve cross-sectional area for all groups. The overall distribution of CSF tracer was significantly less in 10-month-old DBA mice compared with age-matched controls (P = 0.0033). Bars indicate mean ± SD. **P < 0.01.
CSF tracer cross-sectional area coverage (tracer distribution) in the laminar optic nerves of 2-month-old C57 and DBA mice, and 10-month-old C57 and DBA mice was 20.4% ± 18.1%, 15.8% ± 17.0%, 28.1% ± 16.3%, and 4.2% ± 6.0%, respectively (mean ± SD; Fig. 4b). There was a statistically significant interaction between the effects of age and strain on tracer distribution (P = 0.0765). At 2 months of age, DBA and C57 mice showed no significant differences in mean area coverage (P = 0.5332). However, at 10 months of age, DBA mice showed significantly lower tracer distribution in comparison with age-matched C57 mice (P = 0.0033). In both strains, tracer distribution area in mice aged 10 months did not differ significantly from mice aged 2 months (C57, P = 0.3084; DBA, P = 0.13). 
There was no significant difference in CSF tracer intensity in optic chiasms of 10-month-old DBA mice compared with age-matched controls (8812 ± 6610 and 19,510 ±12,731 a.u., respectively; mean ± SD; P = 0.053) (Fig. 3e). 
Assessment of AQP4 and Axonal Phosphorylated Neurofilament
Assessment of AQP4 immunofluorescence demonstrated abundant staining across optic nerve sections in both 10-month-old control and DBA mice (Figs. 5a, 5b). Quantification of AQP4 immunoreactivity showed no significant difference in staining intensity (P = 0.38; Fig. 5c) or cross-sectional area coverage (P = 0.36; Fig. 5d) between the two groups. 
Figure 5
 
AQP4 immunofluorescence staining (green) of coronal sections showed abundant immunoreactivity throughout the nerve in both 10-month-old C57 (a) and DBA (b) mice. There was no significant difference in AQP4 signal intensity (c; P = 0.38) or area coverage (d; P = 0.36) between aged C57 and DBA mice. Bars indicate mean ± SD.
Figure 5
 
AQP4 immunofluorescence staining (green) of coronal sections showed abundant immunoreactivity throughout the nerve in both 10-month-old C57 (a) and DBA (b) mice. There was no significant difference in AQP4 signal intensity (c; P = 0.38) or area coverage (d; P = 0.36) between aged C57 and DBA mice. Bars indicate mean ± SD.
pNF immunofluorescence staining in 10-month-old C57 controls showed an even distribution of pNF immunoreactivity within individual axon bundles and among bundles throughout the nerve at the lamina (Figs. 6a, 6b). A comparison of controls and 10-month-old glaucoma mice revealed a decrease in pNF immunoreactivity in 50% of 10-month-old DBA mice (n = 4/8) (Figs. 6c, 6d). The decrease seen in pNF immunoreactivity was variable among glaucoma mice. This ranged from mild and focal decrease (only a few axon bundles affected in a small region of the nerve; n = 2/8) to severe (nearly all axon bundles affected; almost complete absence of pNF immunoreactivity in some bundles; n = 1/8). One animal showed an intermediate phenotype (n = 1/8) and was classified as moderate. All 10-month-old DBA mice with decreased pNF labeling (n = 4) had a complete absence of CSF tracer in the optic nerve subarachnoid space and in the optic nerve at the lamina. This corresponded to pNF disruptions in 80% of DBA mice that did not have CSF tracer in the optic nerve at the laminar level (n = 4/5). All 10-month-old glaucoma mice with CSF tracer in the optic nerve showed normal pNF immunoreactivity (n = 3/3). 
Figure 6
 
(a) Control optic nerve sections showed abundant CSF tracer in green and homogeneous distribution of pNF in red. (b) At higher power, CSF tracer was visible in the form of channels within the nerve and pNF immunostaining was observed in bundles. (c) Glaucoma optic nerve sections showed absent CSF tracer and patchy pNF immunostaining. (d) This was also seen at higher power, with markedly decreased pNF compared with controls. Scale bars: (a, c) 50 μm, (b, d) 20 μm.
Figure 6
 
(a) Control optic nerve sections showed abundant CSF tracer in green and homogeneous distribution of pNF in red. (b) At higher power, CSF tracer was visible in the form of channels within the nerve and pNF immunostaining was observed in bundles. (c) Glaucoma optic nerve sections showed absent CSF tracer and patchy pNF immunostaining. (d) This was also seen at higher power, with markedly decreased pNF compared with controls. Scale bars: (a, c) 50 μm, (b, d) 20 μm.
Effect of Optic Nerve Cross-Sectional Area on CSF Tracer Intensity
Linear regression analysis showed little correlation between optic nerve cross-sectional area and optic nerve CSF tracer intensity in 10-month-old C57 (r2 = 0.0001) and 10-month-old DBA mice (r2 = 0.13) (Fig. 7a). There was minimal correlation between optic nerve cross-sectional area and CSF tracer distribution area in the optic nerve for 10-month-old C57 (r2 = 0.0035) and 10-month-old DBA (r2 = 0.39) (Fig. 7b). 
Figure 7
 
(a) Scatterplot displaying optic nerve cross-sectional area and CSF tracer intensity in laminar optic nerves. Linear regression lines show minimal correlation between these variables in 10-month-old C57 (r2 = 0.0001) and DBA mice (r2 = 0.13). (b) Scatterplot showing optic nerve cross-sectional area and CSF tracer distribution area in laminar optic nerves. There was minimal correlation between these variables in 10-month-old C57 (r2 = 0.0035) and DBA mice (r2 = 0.39).
Figure 7
 
(a) Scatterplot displaying optic nerve cross-sectional area and CSF tracer intensity in laminar optic nerves. Linear regression lines show minimal correlation between these variables in 10-month-old C57 (r2 = 0.0001) and DBA mice (r2 = 0.13). (b) Scatterplot showing optic nerve cross-sectional area and CSF tracer distribution area in laminar optic nerves. There was minimal correlation between these variables in 10-month-old C57 (r2 = 0.0035) and DBA mice (r2 = 0.39).
Discussion
This study provides the first evidence that CSF entry into the optic nerve subarachnoid space and optic nerve is impeded in a mouse model of glaucoma. Impaired CSF inflow to the optic nerve in glaucoma is particularly relevant given our recent finding of CSF flow through the optic nerve via paravascular spaces formed between penetrating pial blood vessels and an ensheathing layer of astrocytic endfeet.13 This CSF flow into the optic nerve shares a number of similarities with the brain's “glymphatic” system, whereby CSF flows into and out of the brain along paravascular spaces.14 Although a clear mechanism for directional bulk flow in optic nerve paravascular spaces has yet to be elucidated, optic nerve paravascular flow may fulfill similar functions as the brain's glymphatic system. These include nutrient delivery,22 circulation of apolipoprotein E, a cholesterol carrier that supports lipid transport and central nervous system injury repair,23 astrocytic paracrine signaling,24 and toxic metabolite clearance.14 The importance of intact optic nerve CSF flow has been demonstrated in sheep with orbital CSF stasis induced by distal ligation of the optic nerve subarachnoid space, which resulted in marked axonal loss.25 Impaired CSF flow in the optic nerve may, therefore, contribute to a number of pathological conditions including glaucoma. 
Impaired CSF inflow to the optic nerve paravascular spaces in 10-month-old DBA mice appears to be secondary to blocked flow of CSF to the optic nerve subarachnoid space, as opposed to localized obstruction of paravascular inflow into the optic nerve. We did not find any instances in which CSF tracer was present in the optic nerve subarachnoid space, but absent in optic nerve paravascular spaces of the same nerve. Furthermore, when we assessed optic nerve AQP4, a cell membrane water channel heavily expressed on astrocytic endfeet that plays a crucial role in the brain's glymphatic system,14 we found no evidence of altered AQP4 immunoreactivity levels in 10-month-old DBA/2J glaucoma mice compared with age-matched controls. This is in contrast to the findings of Dibas et al.,26 who found increased AQP4 expression in optic nerve heads and proximal optic nerves in rats with sustained induced ocular hypertension. The observed difference in AQP4 expression between the study in rats and the present study may be due to species difference and the type of optic nerve injury. It is unclear how changes in optic nerve AQP4 expression may affect CSF flow through the optic nerve. Further studies to address this should be undertaken in a model in which CSF tracer can reliably be delivered to the optic nerve subarachnoid space so that localized disruption to paravascular flow into the optic nerve can be assessed. 
Possible explanations for the observed decrease in tracer-loaded CSF in the optic nerve of 10-month-old DBA mice include increased resistance to flow due to elevated IOP or expansion of DBA nerves.27 Optic nerves of DBA mice are known to increase in diameter as a result of axon expansion before overt axon loss,27 a phenomenon that could potentially cause narrowing of the subarachnoid space. We found minimal correlation between optic nerve cross-sectional area and the amount of CSF tracer in the nerve, suggesting that optic nerve expansion is likely not responsible for blocked CSF inflow to the orbital subarachnoid space. Histological analysis by electron microscopy may provide further insight into any anatomical abnormalities of the CSF spaces in this model. Our findings also may be explained by a global disruption of CSF secretion or flow in these glaucoma mice. In normal mice, CSF is formed at a rate of 0.37 μL/min, resulting in a complete turnover of the 40 μL CSF volume 12 to 13 times per day.12,28 The finding of CSF tracer surrounding all DBA optic chiasms, including those from animals lacking tracer at the lamina, indicates that CSF flow into the intracranial chiasmatic cistern in these mice was intact. Another possibility may be a reduced pressure gradient between the intracranial CSF space and the termination of the optic nerve subarachnoid space. CSF outflow at the subarachnoid space termination is thought to drain into lymphatic vessels of the dura mater29 or orbit.3033 If this CSF outflow pathway is compromised in glaucoma, the pressure gradient would no longer favor CSF flow from the intracranial space to the optic nerve and orbit. Future studies exploring outflow of CSF to the optic nerve and orbit in glaucoma are warranted. 
DBA mice in this study showed considerable variability in CSF tracer entry to the optic nerve subarachnoid and paravascular spaces. This mouse strain of spontaneously occurring glaucoma is known to display large interindividual variation in disease severity and progression, as well as a more severe phenotype in females.17 Of the 10-month-old DBA animals in this study, all females had a complete loss of CSF tracer at the laminar level, whereas CSF tracer was present (although diminished) in three of four males. pNF immunoreactivity also showed considerable interindividual variation among DBA mice. Altered expression of pNF in the optic nerve in glaucoma is associated with pathological changes to anterograde transport and can be used as an indicator of the severity of axon pathology.20 We found decreased pNF staining in laminar optic nerves only in aged DBA mice lacking CSF tracer at the glial lamina. This corresponded to axon pathology in 80% of aged DBA mice that did not have CSF tracer at the glial lamina, whereas all three animals positive for tracer showed normal pNF staining. This finding suggests an association between CSF flow obstruction and axon pathology in this glaucoma model, although a causative relationship cannot be concluded. Further studies with larger sample sizes would be valuable for an in-depth exploration of the relationship between axonal pathology and CSF entry to the optic nerve. The characteristics of DBA/2J mice, a valuable model in glaucoma, are often contrasted with those of age-matched C57Bl/6 mice.27,34 Although impaired CSF inflow into the optic nerve observed in 10-month-old DBA mice was not seen in age-matched control C57 mice, variation in vasculature and blood-brain barrier across mouse strains could not be excluded. Future investigations with DBA/2J mice would benefit from the use of the DBA/2J-Gpnmb+ control strain to better control for variation due to genetic background.35 
Evidence from glaucoma models including DBA mice indicates accumulation of amyloid precursor protein and amyloid peptides in the optic nerve.3638 It also has been reported that amyloid-beta colocalizes with apoptotic retinal ganglion cells in the retina in a rat model of ocular hypertension and that direct intravitreal injection of amyloid-beta causes retinal ganglion cell death in a dose-dependent manner.39 Whether this is related to reduced CSF flow in the optic nerve and metabolite clearance in glaucoma needs further exploration. In addition, studies investigating reduced optic nerve CSF flow in relation to elevated IOP, and optic nerve gliosis40 in glaucoma are warranted. 
Conclusions
This study provides the first evidence that CSF entry into the optic nerve13 is impaired in a glaucoma model. This finding points to a new CSF-related mechanism that may help to understand optic nerve damage in glaucoma. 
Acknowledgments
The authors thank Hyacinth Irving, MA, for her expert help with statistical analysis. 
Supported by Canada Foundation for Innovation (NG, YHY), Canadian Institutes of Health Research (MOP119432; YHY, NG), Glaucoma Research Society of Canada (NG, YHY), Lloyd and Marie Barbara (NG), Thor and Nicky Eaton Research Fund (NG), Henry Farrugia Research Fund (YHY), National Science and Engineering Research Council CGS Award (EM), Vision Science Research Program Award (EM, JH), and Ontario Graduate Scholarship Award (JH). 
Disclosure: E. Mathieu, None; N. Gupta, None; L.A. Paczka-Giorgi, None; X. Zhou, None; A. Ahari, None; R. Lani, None; J. Hanna, None; Y.H. Yücel, None 
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Figure 1
 
(a) Optic nerve sections of 10-month-old C57 mice showed CSF tracer (green) in the subarachnoid space (arrow) and within the nerve (arrowhead). (b) At higher power, CSF tracer signal was evident in channels within the nerve. (c) Optic nerve sections in 10-month-old DBA mice showed absent CSF tracer, also at higher power (d). This was similar to controls without tracer injection (e, f). Scale bars: (a, c, e) 50 μm, (b, d, f) 20 μm.
Figure 1
 
(a) Optic nerve sections of 10-month-old C57 mice showed CSF tracer (green) in the subarachnoid space (arrow) and within the nerve (arrowhead). (b) At higher power, CSF tracer signal was evident in channels within the nerve. (c) Optic nerve sections in 10-month-old DBA mice showed absent CSF tracer, also at higher power (d). This was similar to controls without tracer injection (e, f). Scale bars: (a, c, e) 50 μm, (b, d, f) 20 μm.
Figure 2
 
CSF tracer (green) in optic nerve coronal sections from age-matched controls is primarily within paravascular spaces surrounding isolectin-labeled blood vessels (red) seen at (a) low, and (b) high power. Scale bars: (a) 50 μm, (b) 20 μm.
Figure 2
 
CSF tracer (green) in optic nerve coronal sections from age-matched controls is primarily within paravascular spaces surrounding isolectin-labeled blood vessels (red) seen at (a) low, and (b) high power. Scale bars: (a) 50 μm, (b) 20 μm.
Figure 3
 
(a) Abundant CSF tracer (green) lined the optic chiasm (Ch) of a 10-month-old C57 mouse. Dashed line indicates midline of chiasm. (b) In the same animal depicted in (a), there was CSF tracer in the optic nerve subarachnoid space and laminar optic nerve. (c) CSF tracer lined the subarachnoid space of a 10-month-old DBA mouse. (d) In the same animal depicted in (c), there was an absence of CSF tracer in the optic nerve subarachnoid space and laminar optic nerve. (e) Quantification of CSF tracer intensity (mean ± SD) in optic chiasms from 10-month-old C57 and DBA mice showed no significant difference between groups (P = 0.053). Scale bars: (a, c) 200 μm, (b, d) 50 μm.
Figure 3
 
(a) Abundant CSF tracer (green) lined the optic chiasm (Ch) of a 10-month-old C57 mouse. Dashed line indicates midline of chiasm. (b) In the same animal depicted in (a), there was CSF tracer in the optic nerve subarachnoid space and laminar optic nerve. (c) CSF tracer lined the subarachnoid space of a 10-month-old DBA mouse. (d) In the same animal depicted in (c), there was an absence of CSF tracer in the optic nerve subarachnoid space and laminar optic nerve. (e) Quantification of CSF tracer intensity (mean ± SD) in optic chiasms from 10-month-old C57 and DBA mice showed no significant difference between groups (P = 0.053). Scale bars: (a, c) 200 μm, (b, d) 50 μm.
Figure 4
 
(a) Optic nerve CSF tracer fluorescence intensity for all groups. Optic nerve signal was significantly decreased in 10-month-old DBA mice compared with age-matched controls (P = 0.0008). (b) CSF tracer distribution (% area) as a percentage of optic nerve cross-sectional area for all groups. The overall distribution of CSF tracer was significantly less in 10-month-old DBA mice compared with age-matched controls (P = 0.0033). Bars indicate mean ± SD. **P < 0.01.
Figure 4
 
(a) Optic nerve CSF tracer fluorescence intensity for all groups. Optic nerve signal was significantly decreased in 10-month-old DBA mice compared with age-matched controls (P = 0.0008). (b) CSF tracer distribution (% area) as a percentage of optic nerve cross-sectional area for all groups. The overall distribution of CSF tracer was significantly less in 10-month-old DBA mice compared with age-matched controls (P = 0.0033). Bars indicate mean ± SD. **P < 0.01.
Figure 5
 
AQP4 immunofluorescence staining (green) of coronal sections showed abundant immunoreactivity throughout the nerve in both 10-month-old C57 (a) and DBA (b) mice. There was no significant difference in AQP4 signal intensity (c; P = 0.38) or area coverage (d; P = 0.36) between aged C57 and DBA mice. Bars indicate mean ± SD.
Figure 5
 
AQP4 immunofluorescence staining (green) of coronal sections showed abundant immunoreactivity throughout the nerve in both 10-month-old C57 (a) and DBA (b) mice. There was no significant difference in AQP4 signal intensity (c; P = 0.38) or area coverage (d; P = 0.36) between aged C57 and DBA mice. Bars indicate mean ± SD.
Figure 6
 
(a) Control optic nerve sections showed abundant CSF tracer in green and homogeneous distribution of pNF in red. (b) At higher power, CSF tracer was visible in the form of channels within the nerve and pNF immunostaining was observed in bundles. (c) Glaucoma optic nerve sections showed absent CSF tracer and patchy pNF immunostaining. (d) This was also seen at higher power, with markedly decreased pNF compared with controls. Scale bars: (a, c) 50 μm, (b, d) 20 μm.
Figure 6
 
(a) Control optic nerve sections showed abundant CSF tracer in green and homogeneous distribution of pNF in red. (b) At higher power, CSF tracer was visible in the form of channels within the nerve and pNF immunostaining was observed in bundles. (c) Glaucoma optic nerve sections showed absent CSF tracer and patchy pNF immunostaining. (d) This was also seen at higher power, with markedly decreased pNF compared with controls. Scale bars: (a, c) 50 μm, (b, d) 20 μm.
Figure 7
 
(a) Scatterplot displaying optic nerve cross-sectional area and CSF tracer intensity in laminar optic nerves. Linear regression lines show minimal correlation between these variables in 10-month-old C57 (r2 = 0.0001) and DBA mice (r2 = 0.13). (b) Scatterplot showing optic nerve cross-sectional area and CSF tracer distribution area in laminar optic nerves. There was minimal correlation between these variables in 10-month-old C57 (r2 = 0.0035) and DBA mice (r2 = 0.39).
Figure 7
 
(a) Scatterplot displaying optic nerve cross-sectional area and CSF tracer intensity in laminar optic nerves. Linear regression lines show minimal correlation between these variables in 10-month-old C57 (r2 = 0.0001) and DBA mice (r2 = 0.13). (b) Scatterplot showing optic nerve cross-sectional area and CSF tracer distribution area in laminar optic nerves. There was minimal correlation between these variables in 10-month-old C57 (r2 = 0.0035) and DBA mice (r2 = 0.39).
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