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Cynthia Xin-Zhao Wang, Bogale Aredo, Tao Li, Rafael Ufret-Vincenty; Increased expression of inflammatory markers in the RPE of CfhTg mice and in RD8+/+ mice.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3280. doi: https://doi.org/.
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Our aim is to determine how the expression of genes by the RPE is altered by two different factors: 1. the 402H variant of complement factor H, and 2. the RD8 mutation of the Crb1 gene.
Using the SRIRS (Simultaneous RPE cell Isolation and RNA Stabilization) technique, we isolated RPE cells from mouse eyecups of 2 year old CfhTg/mCfhKO and C57BL/6J mice, and isolated RNA from these RPE cells. We ran Illumina microarray chips on these samples, and performed a pathway analysis on the resulting genes. QPCR was used to test a subset of these genes and also an independent group of candidate genes for qPCR analysis. We also isolated RNA from the RPE (and also the retina separately) of 8m RD8+/+ vs. B6-wild type (WT) mice and performed qPCR for candidate genes.
We were able to isolate enough RNA from the RPE layer (and overlying subretinal microglia) from individual eyecups to perform Illumina and qPCR testing. QPCR confirmed Illumina findings: an increased expression of CD68 and TREM2, and a decreased expression of SNCA in CfhTg mice vs. B6 controls. Some candidate genes were also found to be increased in CfhTg/mCfhKO mice including: IP-10, NLRP3 and C4. In RD8 +/+ mice, qPCR of candidate genes revealed an increased expression of Ccl-2, CFB, C3, NF-kB and CD200R in the RPE compared to WT mice. The retina of RD8 +/+ mice showed an increased expression of oxidative stress marker HO-1, and complement components C1q and C4 compared to WT.
The 402H variant of Cfh can lead to an increased expression of inflammatory markers in the RPE. The RD8 mutation, which causes a retinal degeneration, also leads to a distinct increase in inflammatory gene expression in the RPE, and an increase in oxidative stress and complement activation markers in the retina.
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