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Kevin Joseph Donaldson, Henry M Skelton, Jana T Sellers, Hans Grossniklaus, J M Nickerson; Novel ex and in vivo methods for non-invasive longitudinal tracking of RPE dysmorphology following subretinal injections. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4982. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal diseases and surgically-induced physical damage are associated with abnormal RPE morphology. Subretinal injections (SRIs) used in gene-delivery therapies are thought to be self-resolving and relatively innocuous. Recovery is assumed to occur shortly after reattachment of the retina. However, the time course and extent of RPE recovery have not been systematically described in the mouse with either histological or non-invasive techniques. We tested the hypothesis that the RPE remains in an abnormal state for much longer than previously expected.
Adult C57BL/6J mice received either SRI of Dulbecco’s PBS (n=12) or mock injection (n=10) in one eye, with the contralateral eye remaining naive. SD-OCT and fundoscopy was performed at multiple time points (0–60 days) post-procedure on all eyes. RPE flatmounts were prepared from each eye after the final imaging time-point. Confocal imaging of IHC labeling of alpha-catenin and ZO-1 enabled quantitative single-cell metrics to be obtained. We generated two distinct transgenic mouse lines with RPE-specific fluorescent protein expression (Best1-Confetti, Best1-ZsGREEN1) for in vivo longitudinal imaging. To verify morphology and condition of the RPE sheet at the level of individual cells, in vivo cSLO and fundoscopic images were then co-registered with post-mortem flatmounts.
Retinal detachments resolved 8 days after SRI in DPBS and mock injection eyes, however abnormal RPE cell morphology (enlarged cell perimeter and area) persisted for up to 60 days post-procedure. Visible discoloration patterns in fundus images corresponded with regions of damage in flatmounts. It was possible to identify unique, individual, RPE cells in vivo with color fundoscopy and cSLO imaging for up to 4 weeks.
Here, we highlight the success of a novel, non-invasive technique that allows for simple, efficient, and repeated in vivo visualization of RPE over multiple weeks. Individual cells identified in live imaging can be subsequently co-registered in histological samples. Studies tracking in vivo RPE cell dynamics in disease models and after injury are now possible. Even after retinal detachments resolve, the underlying RPE remains abnormal for up to two months, much longer than previously thought. Therapeutic approaches employing SRIs may be negatively affected as evidenced by prolonged disruption of the RPE.
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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