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Konstantin Astafurov, Lampros Panagis, Lizhen Ren, Sandeep Kumar, Esther Yoon, John Danias; Effect of Short Term Elevation of Hydrostatic Pressure on Complement Gene Expression in Murine and Primate Retinal Organotypic Cultures. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3869.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate whether complement expression is regulated by short term elevation of hydrostatic pressure in mouse and monkey retinal explanted tissue cultures.
Whole mounted C57BL/6 mouse and Macaca radiata retina organotypic cultures were maintained under hydrostatic pressures of: 0, 15, 30 or 45mmHg for either 24 or 72 hours. All experiments were repeated 4 times. At the end of the experiment, RNA and protein was extracted from the explants and quantitative PCR (qPCR) was performed for C1q, C2, C3, C4, CFH, Thy1,Sncg and GFAP. Protein expression of C1q and C3 was examined using immunoblotting and normalized against beta-actin expression.
Quantitative PCR experiments for mouse and monkey retinal explant tissue cultures showed low levels of expression of the complement genes studied while no significant difference (ANOVA, p>0.05) was observed in the levels of C1q, C2, C3 and C4 under any of the conditions of hydrostatic pressure they were subjected to, for either 24 or 72 hours. Similarly, levels of C1q and C3 protein remained unchanged (p>0.05) in both murine and monkey tissue compared to controls that were not subjected to elevated pressure. Levels of both Thy1 and Synuclein-gamma gene expression were significantly decreased (2 way ANOVA, p<0.05) in mouse retinas subjected to 72 hours of culture compared to retinas cultured for only 24 hours. In monkey retina, levels of CFH were significantly downregulated in explants subjected to 45mmHg compared to those under ambient pressure (Fisher LSD, p<0.05).
The expression of most complement components in murine and primate retinal explants in culture is not influenced by short term rise in hydrostatic pressure.
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