Characterization of a Novel OPA1 Mouse Model by Light and Transmission Electron Microscopy

Abstract

Purpose: Hundreds of mutations in the Opa1 gene have been identified in Dominant Optic Atrophy (DOA). This study examines the retinal nerve fiber layer and optic nerve of a novel mouse model with selective homozygous deletion of Opa1.

Methods: The conditional Opa1 mouse line was generated and previously described (Cogliati et al. Cell 2013). Genetic ablation of Opa1 in retinal ganglion cells was achieved by crossing the floxed Opa1 line with the Grik4 Cre-driver. Eyes from wildtype and heterozygous and homozygous mutants were collected and fixed at six months of age. Retinal whole mounts were dissected and immunostained with a monoclonal neurofilament antibody to visualize RGC axons. Retinas were imaged by confocal fluorescence microscopy and neurofilament staining was quantified using the radial distribution plot in ImageJ (NIH; Bethesda, MD). The densities of nerve fibers were compared between wildtype, heterozygous, and homozygous mutants. To quantify axonal counts, optic nerves were post-fixed in 2% osmium tetroxide, en bloc stained with uranyl acetate and epon embedded. Transverse semi-thin sections of optic nerves were prepared, stained with p-phenylenediamine (PPD) for myelin, and evaluated by transmission electron microscopy.

Results: When compared to wildtype samples, homozygous and heterozygous mutants of Opa1 exhibited decreased axonal density by neurofilament staining. There was no significant difference in neurofilament density between the homozygous and heterozygous mutants at six months of age. Ultra-structural analysis of the optic nerves did not show significant degeneration of axons across different genotypes.

Conclusions: This is the first study to characterize the retina and nerve in a selective homozygous knock-out mouse model of Opa1. Our analysis demonstrates decreased axonal density in heterozygous and homozygous mutants compared to wildtype at six months of age. Further characterizations of older age groups are ongoing to determine whether this is an appropriate model for DOA.

Keywords: 539 genetics • 600 mitochondria • 613 neuro-ophthalmology: optic nerve