Enlarged Optic Nerve Axons and Reduced Visual Function in Mice with Defective Microfibrils

Abstract

Glaucoma is a leading cause of irreversible vision loss due to retinal ganglion cell (RGC) degeneration that develops slowly with age. Elevated intraocular pressure (IOP) is a significant risk factor, although many patients develop glaucoma with IOP in the normal range. Mutations in microfibril-associated genes cause glaucoma in animal models, suggesting the hypothesis that microfibril defects contribute to glaucoma. To test this hypothesis, we investigated IOP and functional/structural correlates of RGC degeneration in mice of either sex with abnormal microfibrils due to heterozygous Tsk mutation of the fibrilin-1 gene (Fbn1Tsk /+). Although IOP was not affected, Fbn1Tsk /+ mice developed functional deficits at advanced age consistent with glaucoma, including reduced RGC responses in electroretinogram (ERG) experiments. While RGC density in the retina was not affected, the density of RGC axons in the optic nerve was significantly reduced in Fbn1Tsk /+ mice. However, reduced axon density correlated with expanded optic nerves, resulting in similar numbers of axons in Fbn1Tsk /+ and control nerves. Axons in the optic nerves of Fbn1Tsk /+ mice were significantly enlarged and axon diameter was strongly correlated with optic nerve area, as has been reported in early pathogenesis of the DBA/2J mouse model of glaucoma. Our results suggest that microfibril abnormalities can lead to phenotypes found in early-stage glaucomatous neurodegeneration. Thinning of the elastic fiber-rich pia mater was found in Fbn1Tsk /+ mice, suggesting mechanisms allowing for optic nerve expansion and a possible biomechanical contribution to determination of axon caliber.

Increased Susceptibility to Glaucomatous Damage in Microfibril Deficient Mice

“…Consistent with our previous finding of enlarged optic nerves in Fbn1 Tsk/+ mice, 16 optic nerves from uninjected eyes were significantly larger for Fbn1 Tsk/+ mice as compared with wt ( Fig. 2A).…”

“…Previously we reported several optic nerve phenotypes in Fbn1 Tsk/+ mice at normal IOP, including larger optic nerves, larger optic nerve axons and thinner pia mater at 6 and 16 months of age. 16 Although these differences did not result in fewer RGCs or RGC axons, we hypothesized that they could result in increased susceptibility to RGC degeneration if the Fbn1 Tsk/+ mice were challenged with elevated IOP. In the present study, we confirmed these alterations in optic nerve structure at normal IOP in Fbn1 Tsk/+ mice at 11 months of age (Figs.…”

“…However this was unlikely because we determined that tonometer calibrations were nearly identical in wt and Fbn1 Tsk/+ mice. 16 The Tsk mutation of aqueous humor outflow pathway and thereby contribute to IOP regulation. 56,57 Fibrillin-1 microfibrils are essential for elastic fiber formation and contribute to their mechanical properties.…”

“…Male and female mice seven months of age at the time of microbead or sham injections (see below) were used for experiments. This age was chosen because we previously found expanded optic nerves starting at six months of age 16 and to avoid a potential protective effect of advanced age that has been reported for 10-to 12-month-old C57BL/6J mice. 37,38 Four months after injection, mice were euthanized by carbon dioxide inhalation followed by thoracotomy and then cardiac perfused with 10 mL phosphate-buffered saline solution (PBS) followed by 10 mL 4% paraformaldehyde (PFA)/PBS.…”

“…Optic nerves were fixed in 2% osmium in PBS for one hour, dehydrated, and embedded in epon as described previously. 16 Thin cross-sections (1 μm) of optic nerves were cut at approximately 1.5 mm from the globe using an EM UC7 ultramicrotome (Leica, Wetzlar, Germany). Sections were stained with p-phenylene diamine (PPD), then mounted using Permount Mounting Medium (Thermo Fisher Scientific).…”