A new ocular explant model to study living optic nerve head events in experimental mouse glaucoma

Abstract

Purpose : To solve a major drawback of glaucoma research, we developed an explant of mouse eye and optic nerve to permit study of retinal ganglion cell (RGC) axons within the optic nerve head (ONH) in living tissue.

Methods : Using transgenic mouse strains, expressing Thy-1-YFP in RGC axons (selectively) or Thy-1-CFP in mitochondria (all cells), we developed an eye and optic nerve explant studied in confocal microscopy at the ONH, the site of glaucoma injury. Explants without treatment, after chronic glaucoma induction or after therapeutic alterations are imaged, to investigate changes in RGC axons or mitochondria at the ONH and first nerve segment.

Results : Axon structure was stable in both strains for at least 3 hours after explantation. In Thy-1-YFP explants, axon integrity index was altered to 0.61 + 0.18 along 250 µm length (Figure 1) after 3 days of bead induced IOP elevation (similar to that in crushed, living rat RGC axons (Koch et al., Cell Death Dis 2015)). In Thy-1-CFP explants, axonal transport block led to mitochondrial density increase of 51.7% after 3 day IOP elevations (p=0.006, Figure 2). High resolution mitochondrial length measurements (normal mean=2.2 + 2.0 µm) permit study of fission or fusion. Mitochondria were most abundant directly at the ONH, slowly decreasing distally by 45% (p=0.002). In glaucoma tissue, this decrease was only 25% (p=0.03). By custom kymography, mean mitochondria speeds in explant axons is 0.15 µm/s anterograde and 0.13 µm/s retrograde, with twice as many moving anterograde, and 15% in motion at a time, as in cultured neurons. Further experiments include measurement of oxidative free radical content and transport obstruction of amyloid precursor protein in additional mouse fluorescent strains after periods of treatment with potential neuroprotectives.

Conclusions : The ocular explant allows real time measurement of the earliest events in experimental glaucoma and quantitative outcomes for development of neuroprotection.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

 

Figure 1. A: Selective Thy1-YFP expression in control axons. B: Axons after 3 days of prior IOP elevation have swelling indicating transport obstruction and early degeneration (bar = 25µm).

 

Figure 2. A: Thy1-CFP expressing mitochondria in control explant. B: After 3 days of prior IOP elevation, mitochondria exhibit transport block with clumping (arrow head, bar = 10µm).