Inhibition of nitric-oxide synthase 2 by aminoguanidine provides neuroprotection of retinal ganglion cells in a rat model of chronic glaucoma

Abstract

Glaucoma is an optic neuropathy with cupping of the optic disk, degeneration of retinal ganglion cells, and characteristic visual field loss. Because elevated intraocular pressure (IOP) is a major risk factor for progression of glaucoma, treatment has been based on lowering IOP. We previously demonstrated inducible nitric-oxide synthase (NOS-2) in the optic nerve heads from human glaucomatous eyes and from rat eyes with chronic, moderately elevated IOP. Using this rat model of unilateral glaucoma, we treated a group of animals for 6 months with aminoguanidine, a relatively specific inhibitor of NOS-2, and compared them with an untreated group. At 6 months, untreated animals had pallor and cupping of the optic disks in the eyes with elevated IOP. Eyes of aminoguanidine-treated animals with similar elevations of IOP appeared normal. We quantitated retinal ganglion cell loss by retrograde labeling with Fluoro-Gold. When compared with their contralateral control eyes with normal IOP, eyes with elevated IOP in the untreated group lost 36% of their retinal ganglion cells; the eyes with similarly elevated IOP in the aminoguanidine-treated group lost less than 10% of their retinal ganglion cells. Pharmacological neuroprotection by inhibition of NOS-2 may prove useful for the treatment of patients with glaucoma.

Figure 1

Chronic, moderately elevated IOP in rat eyes that had three episcleral vessels cauterized, unilaterally (Operated), vs. the opposite eye (Contralateral). IOP was measured on anesthetized animals with a Mentor Classic 30 Pneumotonometer. In one group (n = 8), aminoguanidine (AG) was added to the drinking water; in the control group (n = 8), nothing was added to the drinking water (none). Throughout the 6-month follow-up, IOP was elevated in all Operated eyes compared with Contralateral eyes (P < 0.01). Elevated IOP was not different in the group treated with aminoguanidine vs. the group not treated pharmacologically (P > 0.1). All values are mean ± SD.

Figure 2

Fundus photographs and histological cross-sections of optic nerves from eyes with normal IOP and chronic, moderately elevated IOP, with and without treating the animals with aminoguanidine for 6 months. Color fundus photographs (A–C) and red-free photographs for relief imaging (D–F) were taken on anesthetized animals. The highlights in B and C are reflections of light from the camera. Optic nerve cross-sections (G–I) were stained for myelin profiles (magnification, ×1,250). The peripheral edge of the optic nerve is to the right side of each photomicrograph, opposite the label (G–I). Arrows in H point to the approximate boundary between normal-appearing optic nerve tissue and degenerated (deg) tissue. Pictures from the same eyes are as follows: A, D, and G are from an eye with normal IOP and contralateral to B, E, and H, which are from an eye with chronic, moderately elevated IOP for 6 months from an animal not pharmacologically treated. C, F, and I are from an eye with chronic, moderately elevated IOP for 6 months from an animal treated with aminoguanidine.

Figure 3

Percentage of retinal ganglion cells (RGC) lost at 6 months in rat eyes with chronic, moderately elevated IOP. One week after Fluoro-Gold labeling, retinal ganglion cells were counted in both eyes of each animal (n = 6 for the aminoguanidine-treated group; n = 6 for the untreated group). RGCs were counted in the peripheral retina (A), approximately 4.0 mm from the optic disk, and in the central retina (B), approximately 1.0 mm from the optic disk. To demonstrate uniform bilateral distribution of Fluoro-Gold in retinal ganglion cells after bilateral injection into the superior colliculus, a separate group of normal animals (n = 6) was studied. These animals had normal IOP bilaterally. The mean ± SD of the ratios of retinal ganglion cell counts for left and right eyes were 0.99 ± 0.16 in the peripheral retina and 1.02 ± 0.05 in the central retina. These values were used for the determination of percent RGC lost at “0 Month” (CONT); % RGC lost = 1 − [RGC density in eye with elevated IOP/RGC density in eye with normal IOP] × 100%. In peripheral retina at 6 months, percent RGC lost in eyes with elevated IOP from animals that were not treated pharmacologically (NT) is statistically significantly different than control (P < 0.01), whereas percent RGC lost in eyes with elevated IOP from animals that were treated with aminoguanidine (AG) is not statistically significantly different than control (P > 0.1). All values are mean ± SD.