Glaucoma is a neurodegenerative disease of the visual system and is the leading cause of irreversible blindness worldwide. Lowering of intraocular pressure (IOP) is the only clinically proven way to slow disease progression. However, glaucoma may continue to progress in some patients even after lowering IOP to normal levels, which suggests that other key factors may be contributing to the disease [1]. Recent studies suggest the potential of citicoline, an intermediate in the generation of phosphatidylcholine from choline, as a targeted treatment to improve visual outcomes in glaucoma patients [2, 3], but its neuroprotective effects remain unclear. In this study, we used an experimental rat model of chronic glaucoma to induce sustained IOP elevation for up to 5 weeks. A subgroup of animals received citicoline treatment via repeated oral dosing, and the effects on the structure and function of the visual system were determined with MR neuroimaging and optokinetics. Animal preparation: 20 adult Long Evans rats were intracamerally injected with 20µL of a solution containing 6% vinylsulfonated hyaluronic acid and 6% thiolated hyaluronic acid [4] using a microinjection system at 12 psi and a glass pipette. The solution solidified to an optically clear hydrogel shortly after injection. Only the right eye of each animal was injected, leaving the left eye as an internal control. Eight of the rats received daily citicoline treatment (500mg/kg, via oral gavaging) for 7 days prior to hydrogel injection, and every 48 hours for 14 days after hydrogel injection. IOP measurements: IOP was measured before and at days 3, 7, 14, and 35 after hydrogel injection using a handheld TonoLab tonometer (Icare, Finland). A total of 18 measurements were taken and averaged for each eye at each time point. Optokinetics: Visual acuity was quantified before and at 7, 14, and 35 days after IOP elevation using an OptoMotry virtual reality system (CerebralMechanics, Inc.) to assess the visuomotor behavior [5, 6]. 100% contrast and rotation speed of 0.12 degrees/s were maintained throughout, while spatial frequency ranged from 0.042 to 0.750 cycles/degree. MRI protocol: Diffusion tensor imaging (DTI) was acquired at 35 days after IOP elevation induction for both untreated and citicoline-treated groups in a 9.4 Tesla MRI scanner. DTI was acquired using 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: TR/TE= 2300/27.8ms, echo train length=8, diffusion gradient duration time (δ)/diffusion gradient separation time (Δ)=5/17ms, number of repetitions=4, field of view=2.6x2.6cm2, acquisition matrix= 192x192 (zero-filled to 256×256), and slice thickness=1mm. Slices were oriented orthogonal to the prechiasmatic optic nerves. MRI data analysis: Fractional anisotropy (FA) parametric maps were computed using DTIStudio. Regions of interest were manually drawn on the prechiasmatic optic nerve and the optic tract with reference to color-encoded FA directionality maps and the rat brain atlas. DTI parametric values were compared between visual pathways projected from the hypertensive and normotensive eyes within each group, as well as between citicoline-treated and untreated animals using ANOVA and post-hoc multiple comparisons correction tests. Results are presented as mean ± standard error of mean.Intracameral hydrogel injection to the right eye significantly elevated IOP in all animals for the duration of the experimental period, with no significant IOP difference between citicoline-treated and untreated animals (Fig. 1). The visual acuity of the left, normotensive eyes remained unchanged over time, and was comparable with the right, hypertensive eyes before IOP elevation for all animals (Fig. 2), whereas the visual acuity of the hypertensive eyes of both citicoline-treated and untreated animals became worse starting at day 7. However, the worsening of visual function in the hypertensive eye appeared significantly slower for the citicoline-treated group compared to the untreated group. The decrease in FA in the right optic nerve was significantly smaller in the citicoline-treated animals compared to the untreated animals (Figs. 3, 4). In addition, FA of the right optic nerve was positively associated with visual acuity of the right eye among all animals (r=0.57, p<0.01)(Fig. 5). Our results indicate that intracameral injection of the optically clear hydrogel induced chronic IOP elevation up to the experimental period of 5 weeks and caused significant changes in the visuomotor behavior and white matter integrity along the visual pathway, whereas oral citicoline treatment ameliorated the effects on visual pathway integrity and visual outcomes. These results appear consistent with recent literature which suggests that citicoline may act upon demyelination in white matter and improve corresponding functional outcomes in neurodegenerative diseases [7].