June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Alpha-catenin is a novel marker for identifying abnormal morphology following surgical damage of the RPE
Author Affiliations & Notes
  • Kevin Joseph Donaldson
    Ophthalmology, Emory University, Atlanta, Georgia, United States
  • Jana T Sellers
    Ophthalmology, Emory University, Atlanta, Georgia, United States
  • Jeffrey H Boatright
    Ophthalmology, Emory University, Atlanta, Georgia, United States
    Atlanta VA Center of Excellence in Vision and Neurocognitive Rehabilitation, Atlanta, Georgia, United States
  • J M Nickerson
    Ophthalmology, Emory University, Atlanta, Georgia, United States
  • Footnotes
    Commercial Relationships   Kevin Donaldson, None; Jana Sellers, None; Jeffrey Boatright, None; J Nickerson, None
  • Footnotes
    Support  Abraham J. & Phyllis Katz Foundation, NIH R01EY014026, R01EY021592, R01EY016470, VA RR&D C1924P I21RX001924, VA RR&D C9246C (Atlanta VA Center of Excellence in Vision and Neurocognitive Rehabilitation), P30EY006360, and an unrestricted grant to the Department of Ophthalmology at Emory University from Research to Prevent Blindness, Inc.
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 624. doi:
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    • Get Citation

      Kevin Joseph Donaldson, Jana T Sellers, Jeffrey H Boatright, J M Nickerson; Alpha-catenin is a novel marker for identifying abnormal morphology following surgical damage of the RPE. Invest. Ophthalmol. Vis. Sci. 2017;58(8):624.

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

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Abstract

Purpose : Abnormal cell morphology in RPE is a hallmark of damage in retinal diseases such as rd10 and also appears following physical damage. Some recent gene and cell-mediated therapies have delivered directly to the subretinal space via injection. It is thought that this surgical injection is self-healing and relatively benign. However, upon closer analysis, there may be more surgical damage than previously thought. Typically, the tight junction protein ZO-1 is used to label the outline of individual RPE cell borders allowing for morphometric analysis to indicate damage. However, other junctional protein patterns, such as alpha-catenin, have not been adequately explored following subretinal injections to detect surgical damage.

Methods : RPE flatmounts from adult C57BL/6J mice were prepared 8 days after either subretinal injection of Dulbecco’s PBS (n=5), or mock injection (n=5). Immunohistochemical staining was performed using antibodies against ZO-1 and Alpha-Catenin. Using ZO-1 labeling to define cell borders, alpha-catenin surface area staining in confocal images was quantified with Cell Profiler. Correlation coefficients between multiple morphometric measurements and alpha-catenin staining area, as well as digitized RPE reconstructions, were performed using MATLAB.

Results : Abnormal RPE cell morphology persists for at least 8 days following subretinal injection of an inert vehicle (PBS). The number of cells with widespread, cloud-like staining of alpha-catenin is greatly increased in injected eyes compared to non-injected. Additionally, alpha-catenin appearance is increased near injection sites vs away in injected eyes. In “undamaged” areas, ZO-1 and alpha-catenin are similar in labeling of cell borders in a collar-like fashion. RPE cell perimeter (ρ=0.86) and area (ρ=0.89) measurements are most positively correlated (Pearson correlation) with alpha catenin surface staining.

Conclusions : Damage occurs to RPE from subretinal injection and persists for at least 8 days following injection. This is longer than expected and could negatively contribute to therapeutic approaches using subretinal injections. Alpha-catenin is a novel marker for abnormal cell morphology following damage to the RPE. Without insult, alpha-catenin staining patterns mimic more traditional markers such as ZO-1, but greatly diverge in eyes with RPE damage.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

Alpha-catenin staining (red) of RPE cell near injection site

Alpha-catenin staining (red) of RPE cell near injection site

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