ANTIGENIC CHANGES OF RABBIT RETINA MULLER CELLS IN CULTURE

PURPOSE. To determine whether dissociated and cultured Muller cells fr om the avascular rabbit retina undergo the same phenotypic changes as Muller cells that are dissociated and cultured from a vascular retina. METHODS. Muller cells were dissociated from adult rabbit retinas by u sing an enzymatic digestion-mechanical trituration technique and a cel l attachment method that provided Muller cell-enriched cell cultures. Indirect immunofluorescence localization of vimentin, glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), beta-amyloid precur sor protein (beta-APP), and (alpha-smooth muscle actin (alpha-SMA) was carried out on Muller cells that were freshly dissociated, on those t hat had been in culture 2 and 6 days, and on confluent primary culture s and late-passage cultures. The specificity of the antibodies and cha nges in protein expression were examined by western blot analysis. RES ULTS. The expression of vimentin, GFAP, GS, and beta-APP was present 2 days after dissociation and was retained through 6 days in culture, a t which time alpha-SMA began to be expressed in a small number of cell s. The confluent, primary cultures no longer expressed GS, but vimenti n and beta-APP were still expressed, and the expression of alpha-SMA w as increased. During the late-passage stage, the morphologic appearanc e of the Muller cell cultures was large and amorphous, with additional changes in antigenicity. Although there was loss of expression of the intermediate filament proteins GFAP and vimentin, the expression of b eta-APP was maintained, whereas alpha-SMA was increased and appeared t o be a major cytoskeletal protein. CONCLUSIONS. Dissociated Muller cel ls that were maintained in culture underwent phenotypic changes that i ncluded a large, amorphous appearance; the loss of detectable vimentin , GFAP, and GS expression; the persistent presence of beta-APP; and th e de novo appearance of alpha-SMA. The phenotypic and antigenic change s that occured in cultured Muller cells from an avascular retina were similar but not identical to the changes observed in cultured Muller c ells from a vascular retina.