Introduction

Glaucoma is a neurodegenerative disease that causes irreversible vison over time, making it the second leading cause of blindness worldwide. Elevated intraocular pressure (IOP) is a risk factor associated with glaucoma, and while lowering IOP is a clinically accepted method to slow the progression of the disease, degeneration of the visual system may still occur after lowering the IOP1. Citicoline is an endogenous compound that acts in the biosynthetic pathway of cellular membrane synthesis, and recent studies suggest citicoline may be improve visual function outcome in glaucoma patients2,3. In this study, we used an experimental glaucoma model to elevate IOP for up to 5 weeks and treated two groups of animals with varying doses of citicoline. The effects of citicoline on visual behavior response and white matter integrity were analyzed with optokinetics and MR neuroimaging.

Methods

Animal preparation: 18 adult Long Evans received an intracameral injection of a crosslinking hydrogel4 that solidified to an optically clear hydrogel shortly after injection. The right eyes of all animals were injected and the left eye was uninjured and used as internal control. The 18 animals were divided into three groups: 1. Gel injection only (untreated, n=6), 2. low-dose citicoline (n=6), and 3. high-dose citicoline (n=6). The 12 animals in groups 2 and 3 received daily oral citicoline treatment (500mg/kg and 2500mg/kg, respectively) for 7 days prior to hydrogel injection, and every 48 hours for 14 days after hydrogel injection. IOP measurements: IOP was measured for 35 days after hydrogel injection with a handheld tonometer (ICare, Finland). A total of 18 measurements were averaged for each eye. Optokinetics: Visual behavior response was measured with an OptoMotry virtual reality system (CerebralMechanics) for 35 days after hydrogel injection5,6. MRI protocol: Diffusion tensor imaging was done at 35 days after hydrogel injection using a 9.4T MRI scanner. DTI was acquired with a fast-spin echo sequence with 12 diffusion gradient directions at b=1.0ms/μm2 and 2 non-diffusion-weighted images at b=0ms/μm2 (b0). Other imaging parameters included: slice thickness=1mm, acquisition matrix= 192x192 (zero-filled to 256×256), field of view=2.6x2.6cm2, number of repetitions=4, diffusion gradient duration time (δ)/diffusion gradient separation time (Δ)=5/17ms, ETL=8, and TR/TE= 2300/27.8ms. Slices were oriented orthogonal to the prechiasmatic optic nerves. MRI data analysis: Fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) were computed using DSIStudio. Regions of interest were manually drawn on the prechiasmatic optic nerve and the optic tract with reference to the rat brain atlas. DTI values were calculated as ratios between visual pathways projected from the IOP elevated eyes and the contralateral control eyes. Values were compared between untreated, low-dose citicoline, and high-dose citicoline groups using ANOVA and post-hoc multiple comparisons correction tests. Results are presented as average ± standard error of mean.

Results

Intracameral injection of the cross-linking hydrogel to the right eyes significantly elevated the IOP in all animals for up to 35 days, with no statistical difference between the untreated, 500mg/kg citicoline treated, or 2500mg/kg citicoline treated animals (Fig. 1). The IOP of the left uninjured eyes remained unchanged over time. The visual acuity (VA) of the right untreated, low-dose citicoline, and high-dose citicoline animals decreased as early as at day 7. The VA of the untreated animals decreased further to day 14 and 35, while the VA of the citicoline treated animals stabilized at day 14, with the high dose animals having a significantly higher VA than the untreated animals (Fig. 2a). At the end experimental time point, untreated animals had 45% difference in VA between left and right eyes, whereas low and high dose citicoline treatment resulted in 25% and 10% VA difference, respectively (Figure 2b). The decreases in FA in the optic tract and AD in the optic nerve were significantly smaller in the 2500mg/kg animals compared to untreated animals (Fig. 3). Interestingly, both low and high citicoline dose resulted in less than 5% difference in FA between left and right optic nerves at day 35, as compared to 25% difference in the untreated group.

Discussion and conclusions

Our results indicate oral citicoline administration ameliorates changes in the visual behavior response and white matter integrity after 5 weeks of IOP elevation in an experimental glaucoma model under both low and high citicoline doses. These results appear consistent with recent literature which suggests citicoline can slow down neurodegeneration and improve functional outcomes7. The fact that VA continued to improve at increasing citicoline dose while FA differences between injured and uninjured optic nerves remained similar at both doses, suggests that citicoline may act both on the optic nerve axons and beyond8.

Acknowledgements

This work was supported by the National Institutes of Health P30-EY008098 and R01-EY028125 (Bethesda, Maryland); BrightFocus Foundation G2013077 and G2016030 (Clarksburg, Maryland), Eye and Ear Foundation (Pittsburgh, Pennsylvania); and Research to Prevent Blindness (New York, New York).

References

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