Redox Status of Wallerian Degeneration Slow (WldS) and Wild-Type (WT) Axons Following Single Cell Laser Axotomy of Cultured Retinal Ganglion Cells (RGCs)

Abstract

Purpose : Glaucoma is characterized by axonal degeneration and loss of RGCs. Understanding the mechanisms that lead to axonal degeneration is essential to delay RGC loss and preserve vision in glaucoma. The delayed axonal degeneration in WldS mutant animals can provide insight to such mechanisms. Here, we tested the hypothesis that initiation and progression of axonal degeneration involves the critical step of oxidative modification of sulfhydryls contained within the axon, and that its progression down the axon is significantly different in WldS rats compared to wild-type.

Methods : Retina suspensions from postnatal wild-type and WldS rat pups were immunopanned using anti-Thy-1 to obtain purified primary RGC cultures. RGCs cultured in laminin-coated dishes were allowed to extend neurites until a dominant process with a typical axon hillock was identified. A two-photon laser was used to induce single-cell axotomy. Axonal membrane degeneration was monitored by time-lapse fluorescent imaging.

Results : Following axonal damage, there was a significant delay in detection of the polarity-sensitive thiol-reactive fluorogenic marker annexin B12-IANBD signal in WldS axons compared to wild-type. The velocity of the spread of the fluorescent signal along the segments proximal and distal to the axotomy was significantly slower for WldS compared to wild-type. When the optimized cell-permeable disulfide reducing agent bis(3-propionic acid methyl ester) phenylphosphine borane complex was added to the medium, it significantly lowered the velocity of the spread of fluorescence in axotomized wild-type RGC axons.

Conclusions : The spread of axonal degeneration after axotomy can be detected by fluorescenct imaging of annexin B12-IANBD. The significant delay in the initiation and velocity of this spread in WldS axons, which is partly replicated by application of a cell-permeable disulfide-reducing agent, suggests a role for a redox signal in axonal degeneration after axotomy. This might provide a basis for a therapeutic strategy to delay axonal degeneration in glaucoma.

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