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Arnold Szabo, Maria Anna Hudak, Akos Kusnyerik, Akos Lukats, Zoltan Zsolt Nagy, Janos Nemeth, Agoston Szel; Gliotic remodeling in long-term organotypic culture of the human retina. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4192.
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© ARVO (1962-2015); The Authors (2016-present)
Previously we have shown that in appropriate culture system the human retina can be kept alive with near normal morphology at least for 10 weeks. The overall architecture remains intact in culture and every major cell type survives, however, over time a gliotic reaction develops. In this study we examine this gliotic reaction in detail and describe the temporal changes of Müller cells, astrocytes and microglia.
Adult human eyes with very short (0-4 h) post mortem intervals were used in this study. Approximately 5x5 mm pieces of freshly isolated retina were placed on polycarbonate membranes and were cultured in serum-free medium for up to 10 weeks. The cultures were fixed in different time points and were analyzed by immunohistochemistry using glia-specific markers. The expression levels of glia-specific proteins were determined by Western Blot.
The overall morphology was well preserved. All retinal layers were maintained even after ten weeks, but as a sign of retinal edema the retinal thickness was increased. The Müller cells became hypertrophic with increased expression of vimentin and GFAP. At the outer surface the endfeet of Müller cells developed tiny processes extending beyond the outer limiting membrane. Using antibody against S100 beta we were able to distinguish between astrocytes and Müller cells in gliotic samples. In the central retina S100 beta stained exclusively astrocytes among glial cells, and its expression remained constant during culturing. Reactive astrocytes with swollen cytoplasm were found in the inner retina. In the inner plexiform layer long astrocyte processes formed several horizontal bundles. In non-cultured controls the microglial cells were restricted to the inner retina, while in cultures a fraction of microglial cells invaded the outer retina as well. In some cases close morphological relation between microglia and degenerating photoreceptors was visible.
To our knowledge our model provides the first experimental tool which allows the long-term investigation of retinal gliosis in three dimensional human retina with preserved cytoarchitecture. Our results underline differences in the involvement of glial cell subtypes in gliotic reaction and could be a basis of further experiments with clinical significance.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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