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Y. Zhang, A. De Erkenez, K. Anderson, S. Hanks, B. Jaffee; Full Thickness Retinal Organ Culture: Model Development and Characterization. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5915. doi: https://doi.org/.
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The aim of this study was to establish an ex vivo organ culture model to study retinal diseases with a special focus on oxidative stress and complement activation.
Adult C57BL/6 mice and 6-8 weeks old Long Evan rats were sacrificed. The eyes were removed and incubated in Ham’s F12 medium supplemented with 1.2% proteinase K at 37oC for 15-20 min. The retina with attached RPE dissected in Ham’s F12 medium containing 10% bovine serum. Explants were cultured in serum free DMEM/F12 medium, supplemented with glutamine, B-27 and N-2 for up to 72 hours. To induce oxidative RPE degeneration, mouse explants were treated with 100 µM hydrogen peroxide (H2O2) for 24 hours, and cultured in serum free medium for 36 hours. To investigate the effect of complement activation rat explants were treated for 1, 3, 6, 18, 24, and 72 hours in the medium containing 10% rat serum and 2mg/ml zymosan. Explants without any treatment and supplemented with heat inactivated rat serum, served as controls. Pathological changes and complement activation on the explants was determined using a variety of RPE specific and inflammatory markers.
In the control groups, Zo-1 and occludin immunostained whole mounts appeared to have normal morphology at RPE cells and RPE junctions. Mouse explants treated with H2O2 showed high degree of swelling, disorganization and degeneration of a large number of RPE cells. Zo-1 and occludin immunolabeling displayed the disruption and the loss of the RPE junctions.The supernatants of the rat explants treated with Zymosan showed significantly increased levels of TNFα, IL-6 and MIP1α protein expression between 3 and 72 hours in culture. The highest increase of TNFα and IL-6 expression occurred at 18 hours. Western blot revealed a similar level of RPE65 expression in treated and untreated explants indicating that Zymosan does not compromise the integrity of the RPE cells.
The full thickness retinal explants developed from adult mouse and rat tissue maintained intact retinal structure and normal biological characteristics. Different manipulations of the explants induced pathological and biological changes that are identical to either oxidative RPE degeneration or complement-dependent inflammation of retinal tissues. The changes seen in this model have rapid onset and are highly reproducible. This study suggests that the full thickness retinal culture may be a significant tool for the screening and evaluation of compounds that target retinal disease.
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