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Lindsay Fague, Nicholas Marsh-Armstrong; Debris Accumulation Precedes Axonal Degeneration and Myeloid Cell Influx after Optic Nerve Crush in Xenopus Laevis. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3744. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Using Translating Ribosome Affinity Purification (TRAP) and RNA-seq to profile retinal ganglion cells (RGCs) regenerating after optic nerve crush (ONC) in Xenopus laevis, we previously reported large transcriptional changes within RGCs, including in many pro-regenerative pathways. Since regeneration of the optic nerve (ON) in frogs may also require efficient debris clearance, we set out to characterize axonal debris clearance in the ONC paradigm, to guide future profiling studies of ON astrocytes and myeloid cells.
ONC was performed in Tg(Isl2b:lynkGFP/Mett7bmCherry) transgenic frogs in 4-6 animals per timepoint. These transgenes enable visualization of RGC healthy and damaged membranes. At Days 0, 1, 3, 5 and 7 after ONC, ONs were dissected, fixed, and cryo-sectioned longitudinally. The transgenes for Mett7b-mCherry and lynk-GFP (under the control of RGC-specific promoter Isl2b) were visualized alongside Isolectin B4 labeling of myeloid cells (both CNS resident and infiltrating). An average of 16 images were taken per ON at 20x magnification using an Axiovision fluorescent microscope, using custom IPlab scripts.
Distal to the injury site, labeling of RGC axons by lnkGFP transgene shows a progressive decline from Day 3 onward. Accumulation of uncleared cellular debris, as reported by Mett7bmCherry, is visible starting at day 1. IB4-positive cells show an increase starting at Day 3 post-ONC, peaking at day 5; at day 5 and 7 post-ONC, they can be seen also lining the exterior of the nerve. Notably, our transgenic debris reporter, Mett7bmCherry, shows a large increase in fluorescence even at Day 1, before overt axon degradation or myeloid cell influx.
We report that myeloid cell influx into the ON in frogs peaks at Day 5 following ONC, and follows a progressive increase of uncleared axonal debris which begins at day 1. We also report that our transgenic debris reporter, Mett7bmCherry, detects extensive axonal injury before overt loss of axons or myeloid cell influx. Further studies will profile the responses of ON astrocytes to ON injury (day 1) and myeloid cells (day 5) using TRAP-RNAseq, so as to choose candidate genes for functional studies of genes involved in ON-debris clearance to complement ongoing Caspr-Cas9 studies perturbing the molecular machinery involved in axon degeneration and regeneration.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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