Redox Status in Axonal Degeneration: Application of Disulfide-Reducing Agents Following Single Cell Laser Axotomy of Cultured Retinal Ganglion Cells (RGCs)

Abstract

Purpose: The pathophysiology of glaucoma involves damage to the RGC axons and consequent death of RGC somas. We recently showed that there is a robust increase in production of superoxide after optic nerve injury. We hypothesized that initiation and progression of axonal degeneration involves a critical step of oxidative modification of sulfhydryls on key proteins that can be inhibited by delivering cell-permeable disulfide-reducing drugs.

Methods: Purified primary RGC cultures were produced by immunopanning dissociated retinas from postnatal day 6 pups with sequential anti-macrophage and anti-Thy-1 panning plates, followed by culture in laminin-coated dishes. This allowed the extension of neurites until a dominant process with a typical axon hillock was identified. Two-photon laser injury was used to induce single-cell axotomy and oxidative stress-associated biomarkers were monitored by time-lapse imaging.

Results: The polarity-sensitive thiol-reactive fluorogenic marker annexin B12-IANBD showed progressive axonal fluorescence along the segments proximal and distal to the axotomy. The signal for the distal segment appeared earlier and progressed further and more prominently than the distal segment. The proximal segment developed a swollen stump that was significantly stained by mitochondrial tracers. Addition of optimized cell-permeable disulfide reducing agents (phosphine-borane complexes) and pegylated superoxide dismutase significantly reduced and limited the activity of B12-IANBD in the distal segment of axotomized RGC axons.

Conclusions: Our study supports significant superoxide production and oxidative axonal changes after axotomy. Application of disulfide-reducing compounds preserves the proximal segment and may be therapeutically useful for glaucoma and other optic neuropathies. Finally, RGC primary cultures with laser axotomy can be used to study early events in Wallerian and retrograde axonal degeneration.