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Manasee Gedam, Imran Ahmed Bhutto, Gerard A Lutty, Malia Michelle Edwards; Vitreal cytokines stimulate glial membrane formation. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5390.
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© ARVO (1962-2015); The Authors (2016-present)
We recently described membranes on the vitreoretinal surface of aged human retinas with increased severity associated with advanced AMD. This study tested the roles of vitreous and individual cytokines elevated in AMD vitreous in promoting glial membrane formation.
The vitreous and retina were isolated from normal aged control and AMD donor eyes. The vitreous was homogenized, spun to remove debris and frozen. Retinas were stained for vimentin (Müller cells). The MIOM-1 immortalized human Müller cell line was used to analyze Müller cell membrane formation in vitro. Vitreous (diluted 1:1 in media) or individual cytokines (VEGF or IL-6 at 1-50 ng/ml) were assessed in the Boyden chamber migration assay. After 24 hrs, migrated cells were fixed and stained for vimentin and DAPI. Wells were imaged at low magnification and migrated cells counted in Image J. The effects of cytokines [VEGF or IL-6 (0.1-100 ng/ml)] on proliferation were determined by counting cells 48 hrs after treatment. A minimum of nine wells per treatment were analyzed for each assay. Müller cells exposed to VEGF or IL-6 (100 ng/ml) were stained for GFAP, vimentin, glutamine synthetase and nestin to check for activation or morphology changes. Images were collected on a Zeiss 710 confocal microscope.
Retinas from donors with no ocular disease had a few glial cells on the vitreo-retinal surface. The corresponding vitreous stimulated glial migration but the migrated cells were rounded and clustered. By contrast, retinas with advanced AMD had large preretinal membranes. The vitreous from these eyes stimulated MIOM1 migration. Migrated cells had extensive processes which often overlapped. VEGF and IL-6 both caused a significant, dose-dependent increase in MIOM1 migration. Migrated cells had elongated processes typical of Müller cells but lacked the membrane-like appearance seen in wells treated with vitreous samples. Neither VEGF nor IL-6 stimulated MIOM1 proliferation or changes in protein expression or morphology.
The vitreous from eyes with advanced AMD contains factors which stimulate Müller cell membrane formation in vitro, which could explain the increased incidence of glial membranes on the vitreoretinal surface. VEGF and IL-6, which are elevated in AMD vitreous, may contribute to glial migration through the inner limiting membrane. Other factors, or a combination of factors, are required to stimulate proliferation and membrane formation.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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