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Nicholas Hanovice, Ross F Collery, Brian A Link, Jeffrey M Gross; Regeneration of the retinal pigment epithelium in a novel zebrafish model of macular degeneration. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5915. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Geographic Age-Related Macular Degeneration results from progressive atrophy of the retinal pigment epithelium (RPE), which leads to photoreceptor (PR) degeneration and blindness. While much has been learned about stem cell-based RPE transplant therapies, our understanding of RPE regeneration is incomplete. Zebrafish display a robust regenerative response to injury; thus we hypothesized that the RPE would successfully regenerate. Furthermore, in mammals, proliferation of neighboring RPE cells has been associated with limited endogenous repair. We therefore hypothesized that unaffected RPE proliferates to repair RPE injury.
We utilized a novel zebrafish transgenic that enables nitroreductase-mediated ablation of central RPE cells in larval zebrafish. To characterize the dynamics of cell death following the ablation of RPE, we performed immunohistochemistry to detect TUNEL-positive nuclei. To characterize the recovery of RPE cells, we performed BrdU incorporation assays and immunohistochemistry using markers of RPE and PR cells at varying time points following ablation.
Ablation of the RPE leads to apoptosis and degeneration of cells within the PR layer as early as 6 hours post injury, and the outer nuclear layer is significantly depopulated by 5 days post injury (dpi). By 7dpi, the PR layer becomes more organized, although limited RPE recovery is detectible. By 14dpi, a morphologically normal RPE and PR layer is detectable in the eye periphery. By 21dpi, regenerated RPE is detectible throughout the eye and the PR layer is completely recovered. BrdU incorporation assays reveal the presence of proliferative cells in the RPE layer following ablation. Proliferative cells are concentrated in the periphery soon after ablation and but appear more centrally as regeneration proceeds, mirroring the pattern of regenerating RPE cells.
These results establish a novel zebrafish paradigm through which the RPE can be specifically ablated, and are the first to demonstrate that the zebrafish RPE has the capacity to regenerate. Our data also suggest that proliferation of uninjured RPE cells in the periphery contributes to the regenerative response, and this is being tested by genetic lineage tracing analyses. This model enables the study of the molecular and cellular mechanisms underlying the regenerative response of the RPE in zebrafish.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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