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Brigitte Muller, Birgit Lorenz, Knut Stieger; Time course of morphological changes in murine retinal explant cultures. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1342.
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The purpose of this study was to characterize the organ culture of murine neuroretina to establish survival and early degeneration patterns of neuronal cells to prove retinal explants suitable for therapeutic applications.
Neuroretina explants were prepared from wildtype C57Bl6 mice and evaluated after 2 to13 days in culture. Some eyes were used as fresh retina controls. Explants were cultured in Millicell® culture plate inserts with the photoreceptor layer facing the supporting membrane. Culture medium (DMEM) was maintained in contact with the membrane beneath the explant. Cryostat sections were prepared for TUNEL assay and immunohistochemistry. Neuroretinal modifications were evaluated after HE staining, TUNEL assay and after immunostaining for neurodegeneration markers and neuronal cell markers.
During the first week in culture, only minor morphological changes were detectable in retinal explant culture apart from disruption and truncation of photoreceptor outer segments. TUNEL-staining revealed isolated apoptotic nuclei in the outer and inner nuclear layer (ONL, INL). Immunolabeling with anti-AIF (apoptose inducing factor) showed no dislocation of AIF into the nuclear layers. Rod bipolar cells stained by PKCα appeared densely packed in the INL. Sprouting of individual dendrites into the ONL was visible occasionally. No remarkable reduction in the number of nuclei at both nuclear layers was obvious and the cells appeared densely packed. After two weeks in culture all outer segments were lost and inner segments were reduced to little spheres but were still immunopositive for rhodopsin and M-cone opsin. TUNEL-staining revealed numerous apoptotic nuclei in the ONL and INL, which was in line with translocation of AIF into the nuclear layers.
Our results indicate that retinal explants are in a “close to healthy” condition during the first week of in vitro culture. Degeneration processes during the second week of retinal explant culture might reproduce some cellular modifications occurring in vivo after retinal damage. Therefore murine retinal explant culture might be a suitable model system for therapeutic applications like retinal gene therapy. In addition, this approach can help reduce the numbers of animals used for gene therapeutic experiments.
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