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K. M. Arner, K. Engelsberg, F. Ghosh; Isolation of Photoreceptors in Full-Thickness Retinal Cultures. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1279. doi: https://doi.org/.
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To create a retina consisting mainly of photoreceptors for future use as a graft in the transplantation paradigm.
Full-thickness neuroretinas from embryonic Sprague Dawley rats were placed in culture 17 (E17) and 20 (E20) days post conception. The explants were cultured for 7 days (E17: n=9 and E20: n=13) and 14 days (E7: n=10 and E20: n=13). Eyes from corresponding ages (E17, E20, postnatal day (P) 3, 6, 10 and 13) were used as controls.The explants and controls were examined after preparation for light microscopy and immuno-histochemistry using antibodies against calbindin (horizontal cells), Protein Kinase C (bipolar cells), parvalbumin (bipolar and amacrine cells), recoverin (rod and cone photoreceptors), rhodopsin (rods), transducin (cones), vimentin (Müller cells) and synaptophysin (synapses).All proceedings and animal treatment were in accordance to Declaration of Helsinki.
The E17 control retina consisted of a neuroblastic cell layer (NBL). The E20 and P3 controls displayed a NBL and a multi-layered ganglion cell layer. The P 6, 10 and 13 control retinas displayed outer and inner nuclear layers with a row of single cells at the inner margin.Explants kept 7 DIV from the E17 retinas consisted of a NBL with double foldings, whereas the E20 explants had a nucleated layer. The E17 explants displayed a small number of pycno-tic cells at the innermost part, whereas the E20 explants showed a more profound degenera-tion in this area.Retinas kept 14 DIV had several double foldings and rosettes. The E17 explants displayed an outer nuclear layer and a layer of single cells at the inner retina, while the E20 specimens con-sisted of a nucleated layer.For immunohistochemic results see table below.
We found the age of the embryonic retina when placed in culture and the time in vitro to infuence the differentiation of the embryonic retina. This indicates a possibility to produce a customized graft for retinal transplantation.
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