Cellular and Cytoskeletal Dynamics Within Organ Cultures of Porcine Neuroretina

Abstract

The aim of this study was to establish a retinal organ culture and to follow the cellular and cytoskeletal changes. For this purpose the authors detached porcine neuroretinas from the underlying pigment epithelium, and incubated them for 2 weeks under standardized conditions. After 3, 6, 10, and 13 days in culture the retinal tissues were fixed, embedded in LR-White resin or paraffin wax and processed for electron-, light-, immunofluorescence- and confocal laser scanning microscopy. Antibodies directed against α-tubulin, actin, glial fibrillary acidic protein (GFAP), vimentin, neurofilament200 and β-catenin were used to investigate the cytoskeletal changes over a certain period of time. After experimental detachment Müller cells quickly started to hypertrophy showing increased levels of intermediate filaments (i.e. vimentin and GFAP). The actin labelling of photoreceptor cells decreased concomitantly with a rapid degeneration of the outer segments. After 1 week of detachment the Müller cell cytoplasm revealed increasing amounts of actin and tubulin staining. Actin filaments appeared frequently organized in thick bundles across the full width of the retina, whereas increasing levels of tubulin shifted into the outer nuclear region especially concentrated near the outer limiting membrane. A prolonged time of explant culturing resulted in a discontinuous staining of β-catenin along the adherent junctions of the outer limiting membrane, followed by an outgrowth of Müller cell extensions into the subretinal space. Double staining of tubulin and cones showed that this outgrowth predominantly occurred between cone inner segments. The outer limiting membrane was penetrated by stalk-like structures, highly enriched with tubulin and associated with swollen tips, reaching into the subretina. Electron microscopy demonstrated in detail the focal disruption of the outer limiting membrane by Müller cell extensions and subsequent subretinal gliosis. The cytoskeletal reactions described here were compared with degenerative changes observed after induced retinal detachments.