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 h 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 h 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 ± 4176 mitochondria/mm² compared to control 16,110 ± 3159 mitochondria/mm² (p = 0.025, t-test), but did not increase significantly after 4 h, 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 h 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 min. 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.

Keywords: Axons; Glaucoma; Mitochondria; Mouse; Retinal ganglion cell; Sclera; Transport block.

Figure 1

(A) Explant of eye and optic nerve fixed to a plastic block with agarose covering the tissue (arrow = the optic nerve head region, bar = 400 μm). Imaging of the optic nerve is performed perpendicular to nerve along axis. The attached optic nerve is ∼3 mm long. (B) Confocal image of RGC-YFP explant with selectively YFP-expressing axons. (C) Mito-CFP explant showing mitochondria (blue) and collagen in red (second harmonic generation imaging). Labels: D- distal to the eye, P-proximal to the eye (at the ONH) are used to indicate orientation (B, C: bars = 25 μm).

Figure 2

One RGC-YFP control explant imaged at 4 time points; (A) baseline, (B) 1 hour, (C) 2 hours, and (D) 3 hours from start of imaging shows selectively fluorescent axon structure stability. Appearance changes minimally from baseline IOP of 10 mm Hg. Labels: D- distal to the eye, P-proximal to the eye (at the ONH) are used to indicate orientation (bar = 10 μm).

Figure 3

RGC-YFP axons show progressive fragmenation, swelling, and loss after IOP elevation in the bead-induced model at (A) 14 hours, (B) 1 day, (C) 4 days, and (D) 1 week post chronic IOP elevation. Labels: D-distal to the eye, P-proximal to the eye (at the ONH) are used to indicate orientation (bar = 30 μm).

Figure 4

Mitochondrial Density. (A) Mitochondria in control explant. (B) Three day glaucoma explant showing mitochondrial transport block with clumping. Labels: D- distal to the eye, P-proximal to the eye (at the ONH) are used to indicate orientation (arrow head; bar = 10 μm). (C) Mitochondrial density (percent area occupied by mitochondria) in serial zones from ONH to 1 mm behind eye, showing significantly increased density after chronic IOP elevation (* = p < 0.05, locations #4 and #5 have no error bars due to limited sample size).

Figure 5

Anterograde and retrograde speed and number of mitochondria in motion in (A) Control explants and (B) 3 day chronic glaucoma explants (1 and 10 minutes after initiation of imaging). (C) Anterograde speed after acute IOP elevation from 10 mm Hg to 30mm Hg for 1 minute and 60 minutes (curves represent polynomial fits to data).