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Y Zhang, AR Caffé, S Azadi, T van Veen, B Ehinger, MT Perez; Neuronal Integration In Abutting-Retinas Culture System . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3440. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Limited integration is consistently observed between subretinal transplants and host retinas. We have therefore established an in vitro model system for the study of connectivity between two abutting retinas. Methods: Neuroretinas were dissected from transgenic mice expressing green fluorescent protein (GFP) and from wild type (WT) mice (5 days old and adult) and cut into 4 pieces each. Pieces from two different retinas (GFP/WT or WT/WT) were placed photoreceptor side down, side-by-side (contacting each other at the margins) or overlapping each other in organ culture for 7 days. Retinal cells and fibers were visualized by immunocytochemistry using antibodies against neurofilament (160kDa, NF+) and neuronal nitric oxide synthase (nNOS+), and by GFP fluorescence (GFP+). The presence of interconnecting fibers was examined in wholemount preparations and in cross-sections by fluorescence and confocal microscopy. Results: In side-by-side, GFP/WT pairs, GFP+ fibers were seen to have extended into the inner plexiform/ganglion cell layer of the WT-derived tissue. In both, GFP/WT and WT/WT pairs, numerous horizontal cell fibers (NF+) and amacrine cell fibers (nNOS+) could be seen to have crossed the interface between the two pieces, forming contiguous plexiform layers. In cultures of adult tissue, also NF+ ganglion cell fibers were seen to have grown across the margins. In overlapping pairs, NF+ and nNOS+ fibers displayed parallel plexiform layers, and no crossover of fibers was observed even if the inner retinal layers of the two pieces faced each other. Some integration was seen only in small areas where the structure of both retinal pieces appeared disrupted. Conclusion: The results demonstrate the ability of neurites to extend between abutting retinas and to make appropriate target choices when they are placed side-by-side. However, this ability is limited when they overlap each other, similar to what is observed in subretinal transplantation. Further investigation of the factors influencing neuronal connectivity by using this abutting-retinas culture system may help us to design effective protocols for retinal transplantation.
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