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E. E. Capowski, J. M. Meyer, R. L. Shearer, L. S. Wright, D. M. Gamm; Time Course of Cellular Degeneration in Adult Primate Retinal Explant Cultures. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5175. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To assess the usefulness of adult primate retinal explant cultures for testing ex vivo gene therapy strategies to promote cell survival.
Individual 2, 3 or 5 mm diameter biopsy punches from discrete regions (macula, mid-periphery and periphery) of the higher primate retina were placed in transwell inserts and cultured with or without matrigel in medium containing B27 supplement and 10% fetal calf serum for up to 14 days. At selected time points, explants were fixed in 4% paraformaldehyde, cryoprotected and sectioned to permit subsequent immunohistochemical evaluation. The in vitro time course of degeneration of three major classes of retinal neurons, photoreceptors, bipolar cells and ganglion cells, was determined using cell type-specific antibodies in conjunction with activated caspase3 immunostaining and TUNEL fluorescent detection. Cell counts from the outer and inner nuclear layer as well as the ganglion cell layer were also obtained.
Explants from each region exhibited a variable pattern of cellular degeneration over the time course studied, with loss of ganglion cells and photoreceptor outer segments occurring early in the process. Regional differences were also observed among the retinal areas sampled. Cell loss occurred at least in part via an apoptotic mechanism as determined by caspase3 activation and TUNEL staining.
Explant cultures provide a useful starting point for evaluating the efficacy of neuroprotective therapies for the primate retina. This culture system will provide the basis for future experiments addressing gene therapy approaches to treating macular degeneration.
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