Gs. Mcgillem et al., ANTIGENIC CHANGES OF RABBIT RETINA MULLER CELLS IN CULTURE, Investigative ophthalmology & visual science, 39(8), 1998, pp. 1453-1461
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.