July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Elucidating the mechanisms underlying zebrafish retinal pigment epithelium regeneration
Author Affiliations & Notes
  • Lyndsay L. Leach
    Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Nicholas Jay Hanovice
    Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Ana E. Gabriel
    Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Jeffrey M Gross
    Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
    Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Lyndsay Leach, 9458428 (P); Nicholas Hanovice, None; Ana Gabriel, None; Jeffrey Gross, None
  • Footnotes
    Support  NIH Grant T32 EY-17271-09; BrightFocus Foundation Grant M2016067
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3995. doi:
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    • Get Citation

      Lyndsay L. Leach, Nicholas Jay Hanovice, Ana E. Gabriel, Jeffrey M Gross; Elucidating the mechanisms underlying zebrafish retinal pigment epithelium regeneration. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3995.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Our lab has created a novel retinal pigment epithelium (RPE) ablation paradigm using zebrafish and has shown that RPE are capable of regenerating post-injury in this in vivo model system. However, the mechanisms underlying RPE regeneration remain unknown. Previous studies implicate that the innate immune system is a critical modulator of the regenerative response in damaged tissues, including the retina. This study aims to test the hypothesis that innate immune cells (e.g. macrophages and/or neutrophils) contribute to RPE regeneration and to identify the molecular underpinnings of the regenerative response.

Methods : Transgenic rpe65a:nfsB-GFP zebrafish larvae expressing the metronidazole (MTZ) converting enzyme, nitroreductase, fused to GFP in mature RPE were used as the in vivo injury model. Larvae were pre-treated with the immunosuppressive glucocorticoid, dexamethasone, or dimethyl sulfoxide (DMSO; control) for 24 hours prior to RPE ablation. Ablation of mature RPE in 5 days post-fertilization (dpf) larvae was achieved by adding 10mM MTZ to fish water for 24 hours. To assess cell proliferation in the retina and RPE post-ablation, bromodeoxyuridine (BrdU) incorporation assays were performed and dexamethasone- and DMSO-treated larvae were fixed for immunohistochemistry. Larval eyes from untreated and MTZ-treated zebrafish were enucleated and GFP-positive cells were isolated using fluorescence-activated cell sorting on days 2, 4, and 7 post-injury (dpi). Sorted cells were subsequently processed for RNA-sequencing.

Results : In control larvae, proliferation in the RPE and pigment recovery occurred by 4dpi and in a peripheral to central manner. In dexamethasone-treated larvae, the proliferative response and the overall rate of regeneration was reduced. Analysis of RNA-sequencing results revealed potential candidate genes involved in RPE regeneration.

Conclusions : Preliminary assessments support a model in which innate immune system activation facilitates RPE regeneration. Candidate factors involved in RPE regeneration have been identified and these are being validated and further analyzed.

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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