Ultrastructural Analysis of Optic Nerve Axons in Experimental Glaucoma: Evidence for Axon Shrinkage

Abstract

Abstract: : Purpose: To determine changes in the morphology of retinal ganglion cell axons in a rodent model of experimental glaucoma. Methods: Unilateral experimental ocular hypertension was induced in adult male Norwegian Brown rats by sclerosing the episcleral veins with hypertonic saline injections. Intraocular pressures were measured using a calibrated Tonopen XL (Mentor, USA) in animals maintained in a constant low light environment (40–90 lux). Animals were sacrificed at selected time points (4–8 weeks post induction of ocular hypertension). Following enucleation, optic nerves were fixed by immersion in 2.5% glutaraldehyde, post fixed in 1% osmium tetroxide and embedded in spurs resin. The nerves were examined by electron microscopy for quantitative measurements of axonal and myelin sheath perimeters and cross sectional areas using AnalySIS Software (Soft Imaging System, GmbH, Germany). Results:10 nerves (5–normal and 5–glaucoma) were examined in serial sections. Qualitative analysis of glaucomatous nerves revealed delamination of the myelin sheaths with fragmentation of the component lamina surrounded by electron dense deposits. Quantitative analysis revealed a reduction in cross sectional axon area from a mean (SEM) of 1.01µm² (+0.06, {n =5) in controls compared to a mean of 0.72µm² (+0.60, {n =5}) in glaucomatous optic nerves (p<0.015). Cross sectional axon perimeter did not differ significantly between normal and experimental nerves (p< 0.77). Delamination and fragmentation of the oligodendrocyte sheaths resulted in increased sheath perimeter in glaucomatous axons 7.24 µm (+0.33) compared to controls 6.56µm (+0.13), though the difference did not reach the level of statistical significance (p<0.083). Conclusions: The significant reduction in cross sectional axonal area with preservation of inner oligodendrocyte circumference is consistent with shrinkage and remodelling of the optic nerve axons in experimental glaucoma. Fragmentation of myelin sheaths in this model may propagate damage within the optic nerve.

Keywords: microscopy: electron microscopy • pathology: experimental