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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. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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
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