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Christina Lee Rettinger, Heuy-Ching Hetty Wang; Quantitative Assessment of Retina Explant Viability in a Porcine Ex Vivo Neuroretina Model. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3121.
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© ARVO (1962-2015); The Authors (2016-present)
Ex vivo neuroretina explant cultures have been used to study an array of biological processes such as retinal development, neurodegeneration and neuroprotection. However, it is often difficult to interpret findings from these studies since most assessments used to evaluate retinal tissue behavior have largely been based on non-objective morphological measures. This prompted us to develop a technique that can assess the viability of adult retina explants quantitatively, in addition to qualitatively.
Neuroretina explants 8mm in diameter were harvested from adult swine and cultured on porous cell culture inserts with adjustable heights under serum free conditions. Retina explant viability was evaluated at 1, 4, 7, 11, 14 days of culture using a resazurin-based metabolic assay that was adapted to measure cell viability in tissue. The explants were also analyzed morphologically via immunohistochemistry for glial cell markers such as glial fibrillary acidic protein (GFAP), and terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) for apoptotic cells. Moreover, neuroretina thickness was determined at each time point using hematoxylin and eosin (H&E) images. Freshly isolated, non-cultured porcine retinas were used as baseline controls.
Porcine retina explants remained viable throughout the entire duration of the experiment, although the viability gradually decreased over time in culture. The laminar structure of the neuroretina was well-preserved during the first 7 days. However, by D14, most explants in culture showed significant loss of cells in each of the nuclear layers. Furthermore, there was obvious thinning of the retina explant especially in the second week of culture. Overall, the progressive loss of retinal lamination and thickness, increase in TUNEL+ nuclei with activated, hypertrophic Müller cells as evidenced by upregulated GFAP expression, were all well-correlated with the metabolic activity of the ex vivo neuroretina explants over time.
This study was the first report to describe the use of a high-throughput and quantitative method for monitoring retina explant viability in real-time. Even though there are limitations with these ex vivo culture systems, neuroretina cultures closely mimic the functional dynamics of the organ and provide a powerful tool that can be used to develop novel neuroprotective therapies for retinal neurodegenerative disease.
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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