AQP4 expression in muller cells is not influenced by LPS application in vitro and in vivo
Xiao-Qiang Liu1 Hideyuki Kobayashi2 Nobuhisa Nao-i3
1 Eye hospital of wenzhou medical college, 270 xueyuanxilu, wenzhou, 325000
2 Department of Pharmacology, Faculty of Medicine, University of Miyazaki,Japan
3 Department of Ophthalmology, Faculty of Medicine, University of Miyazaki,Japan
PURPOSE: Retinal edema is a common complication of ischemic and inflammatory ocular diseases. It is known that the swelling of muller cells contributes to the pathogeneses of retinal edema. The water channel protein aquaporin-4 (AQP4) is expressed in the endfeet of muller cells and has been assumed to play essential roles in retinal edema. In order to investigate whether AQP4 is involved in the retinal edema accompanied with inflammation, we studied the effects of lipopolysaccharide (LPS) on AQP4 expression in the muller cells of the retina.
METHOD: Muller cells were prepared by dissociating the retinas of the new born rats, and were cultured. The purity of the cultures was evaluated by immunocytochemical staining with anti-vimentin antibody and anti-glial fibrillary acidic protein antibody. Primary cell cultures or cells of passages 1-3 were used for experiments. Cells were exposed to a various concentrations of LPS for 24 hours or to 1ug/ml lipopolisaccharide up to 24 hours. The AQP4 protein level in muller cells treated with LPS was analyzed with immunocytochemistry and western blotting. For in vivo experiment, a single dose of 10 μg of diluted LPS solution was injected intravitreally into one eye of each rat to induce endotoxin-induced uveitis (EIU). The expression of AQP4 in the retina during EIU was detected by immunohistochemical staining and Western blotting.
RESULTS: The cultures reached confluence within 3-7 days to become a monolayer of epithelioid-like cells. Immunocytochemical staining showed that the cells were positive for vimentin and glial fibrillary acidic protein, the retinal glial cell markers. Immunocytochemistry and Western blot results showed LPS did not altered AQP4 expression in muller cells in culture.In the animal model of EIU, after an intravitreal LPS injection, the immunostaining for AQP4 maintained the same pattern during the different stages of EIU and only a slight reduction in immunoreactivity was observed in the inner plexiform from 1-7 days after LPS injection. Western blot results showed the expression of AQP4 protein was only slightly reduced in the early stage after LPS injection and then increased gradually and had nearly recovered to the normal level at 14 days after LPS injection.
CONCLUSION: These results suggest that the LPS application dose not alter the AQP4 expression in muller cells in vitro and in vivo. The changes of AQP4 expression in the muller cells may not be necessary for the the development and amelioration of retinal edema accompanied with inflammation.
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