A mouse ocular explant model that enables the study of living optic nerve head events after acute and chronic intraocular pressure elevation: Focusing on retinal ganglion cell axons and mitochondria

Abstract

We developed an explant model of the mouse eye and optic nerve that facilitates the study of retinal ganglion cell axons and mitochondria in the living optic nerve head (ONH) in an ex vivo environment. Two transgenic mouse strains were used, one expressing yellow fluorescent protein in selected axons and a second strain expressing cyan fluorescent protein in all mitochondria. We viewed an explanted mouse eye and optic nerve by laser scanning microscopy at and behind the ONH, the site of glaucoma injury. Explants from previously untreated mice were studied with the intraocular pressure (IOP) set artificially at normal or elevated levels for several hours. Explants were also studied from eyes that had undergone chronic IOP elevation from 14 hours to 6 weeks prior to ex vivo study. Image analysis in static images and video of individual mitochondria or axonal structure determined effects of acute and chronic IOP elevation. At normal IOP, fluorescent axonal structure was stable for up to 3 hours under ex vivo conditions. After chronic IOP elevation, axonal integrity index values indicated fragmentation of axon structure in the ONH. In mice with fluorescent mitochondria, the normal density decreased with distance behind the ONH by 45% (p=0.002, t-test). Density increased with prior chronic IOP elevation to 21,300 ± 4,176 mitochondria/mm2 compared to control 16,110 ± 3159 mitochondria/mm2 (p=0.025, t-test), but did not increase significantly after 4 hours, acute IOP elevation (1.5% decrease in density, p=0.83, t-test). Mean normal mitochondrial length of 2.3 ± 1.4 μm became 13% smaller after 4 hours of IOP elevation ex vivo compared to baseline (p=0.015, t-test, N-10). Normal mitochondrial speed of movement was significantly slower in the anterograde direction (towards the brain) than retrograde, but there were more mitochondria in motion and traveling longer lengths in anterograde direction. The percent of mitochondria in motion decreased by >50% with acute IOP increase to 30 mm Hg after 60 minutes. A new ocular explant model implemented with eyes from transgenic mice with fluorescent cellular components provided real time measurement of the early events in experimental glaucoma and quantitative outcomes for neuroprotection therapy experiments.

Pressure-Induced Changes in Astrocyte GFAP, Actin, and Nuclear Morphology in Mouse Optic Nerve

“… 7 , 39 – 41 Similarly, in larger mammalian eyes and humans, transport block in axons occurs in the connective tissue zone of the LC and myelination begins just posterior to this. 3 , 42 – 44 The accumulation of amyloid precursor protein 33 during axonal obstruction can lead to axonal swelling, 39 , 45 resulting in the increase in axonal area measured in this work.…”

The effects of age on mitochondria, axonal transport, and axonal degeneration after chronic IOP elevation using a murine ocular explant model

“…In an initial, pilot study describing this method (Kimball et. al., 2017), explants from young RGC-YFP mice were used to measure axonal integrity after acute and chronic pressure elevation.…”

“…Twenty-nine explants were from 4 month old mice of the Mito-CFP strain, 18 explants were from 14 month old Mito-CFP mice, and 31 explants were from 14–17 month old RGC-YFP mice. For comparison to the older RGC-YFP mice, we utilized comparable data on younger (4 months old) mice, published previously (Kimball et al, 2017).…”

“…To measure the degree of degeneration in the RGC-YFP explants, the axonal integrity ratio (Knöferle et al, 2010) was used as previously published (Kimball et al, 2017). Each Z-stack of ONH images from RGC-YFP mice was converted into a maximum projection image, allowing all axons from the 70–100 μm thickness to appear within a single 2-dimensional image.…”

“…These tools permit assessment of initial effects after chronic IOP exposure. We developed (Kimball et al, 2017) an ex vivo explant of the eye and optic nerve to study acute and chronic effects of IOP elevation in mice. In the present research, we studied explants from the two mouse strains, one with yellow fluorescent protein (YFP) in a select number of RGC and a second strain with cyan fluorescent protein (CFP) in all mitochondria.…”