P 19Preparing an organotypic culture of porcine retina: Variations of cytoskeletal components with increasing time of cultivation
S. Hagelstein, V. Frank, H. Eisner, H. Laqua, J. Winkler
Introduction: The aim of this study was to obtain an organotypic culture system of mature porcine retina explants and to describe individual structural components during in vitro cultivation.
Material and methods: The explants were transfered onto special carriers an cultivated at 5% of CO2 within an optimized tissue culture media. After fixation and embedding in paraffin we used immunohistological techniques in conjunction with epifluorescence microscopy to investigate the distribution of various cytoskeletal components.
Results: The structural integrity of the retina explants was examined over a defined period of time. After 1-7, 10 and 14 days of incubation, different cytoskeletal markers were applied, to describe structural variations. Conventional H&E-staining revealed an increasing desintegration of retinal layers with time. The inner nuclear layer was especially affected by these changes. Moreover, an invasion of cells into the nerve-fibre-Iayer (NFL) was observed. Tubulin-specific antibodies marked the axons of the NFL, the middle limiting membrane and the Muller cells. With increasing time of cultivation the tubulin-staining became more diffuse. However, the Muller cells and the NFL were still the dominating tubulin-expressing elements. Antibodies against vimentin and GFAP marked the radially orientated Muller cells. Between the days 6-14 the intermediate filaments became the dominating cytoskeletal components. In addition, we used antibodies against. ß-catenin to specifically stain the cell-cell-contacts between the Muller cells and the outer segments of the photoreceptors. This specific staining remained stable until day 14, visualized by a discontinuous reaction.
Conclusions: The characterization of retina-explants using immunocytochemical techniques clearly revealed the changes of cytoskeletal components with time. Intermediate filaments like vimentin became the dominating supporting elements, whereas the functional nerve fibres continuously degenerate.
Laboratory of Experimental Ophthalmology, Eye Clinic of the Medical, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck