Antigenic changes of rabbit retinal Müller cells in culture

Abstract

Purpose: To determine whether dissociated and cultured Müller cells from the avascular rabbit retina undergo the same phenotypic changes as Müller cells that are dissociated and cultured from a vascular retina.

Methods: Müller cells were dissociated from adult rabbit retinas by using an enzymatic digestion-mechanical trituration technique and a cell attachment method that provided Müller cell- enriched cell cultures. Indirect immunofluorescence localization of vimentin, glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), beta-amyloid precursor protein (beta-APP), and (alpha-smooth muscle actin (alpha-SMA) was carried out on Müller cells that were freshly dissociated, on those that had been in culture 2 and 6 days, and on confluent primary cultures and late-passage cultures. The specificity of the antibodies and changes in protein expression were examined by western blot analysis.

Results: The expression of vimentin, GFAP, GS, and beta-APP was present 2 days after dissociation and was retained through 6 days in culture, at which time alpha-SMA began to be expressed in a small number of cells. The confluent, primary cultures no longer expressed GS, but vimentin and beta-APP were still expressed, and the expression of alpha-SMA was increased. During the late-passage stage, the morphologic appearance of the Müller 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 beta-APP was maintained, whereas alpha-SMA was increased and appeared to be a major cytoskeletal protein.

Conclusions: Dissociated Müller cells that were maintained in culture underwent phenotypic changes that included a large, amorphous appearance; the loss of detectable vimentin, GFAP, and GS expression; the persistent presence of beta-APP; and the de novo appearance of alpha-SMA. The phenotypic and antigenic changes that occurred in cultured Müller cells from an avascular retina were similar but not identical to the changes observed in cultured Müller cells from a vascular retina.