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J. C. Morrison, Y. Guo, J. A. Dyck, W. O. Cepurna, T. Doser, E. C. Johnson; Increasing the Sensitivity of Detecting Cell Responses to Elevated Intraocular Pressure: A Comparsion of Whole Retina Tt Retinal Ganglion Cell Layer. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3698. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Microarray analyses of gene expression in rat retinas exposed to elevated IOP have identified many significantly regulated genes. However, because retinal ganglion cell (RGC) RNA was diluted with RNA obtained from the entire retina, specificity and sensitivity to RGC responses may be limited. In this study, we use laser capture microdissection (LCM) and microarray analysis to determine gene expression changes in the RGC layer in our glaucoma model and compare these to previous analysis in whole retina from similarly damaged eyes.
IOP was elevated in rats by unilateral injection of hypertonic saline and IOP response monitored for 5 weeks. Ethanol-fixed, paraffin-embedded retinal sections were prepared and RGC layers collected by LCM. RGC layer RNA from 6 fellow eyes and 6 eyes with >40% nerve injury was extracted, linearly amplified and independently compared using cDNA arrays. Data were normalized and significant changes determined using Significance Analysis of Microarray (q<0.02, > 1.3 fold change, median FDR 2%). Affected gene categories were identified by the gene ontology web tool DAVID. RGC layer expression changes were compared to our previous microarray analysis of similarly damaged whole retina RNA.
Microarray analysis of RGC layer RNA yielded 3726 genes with altered expression (2003 up, 1723 down), 5-fold more than the 632 significant genes (335 up, 297 down) found in whole retina analysis. Numbers of genes changed by 2-fold were 972 (777 up, 195 down) for RGC layer and 125 (101 up, 24 down) for whole retina. Relative expression changes in specific genes varied. For 3 genes highly upregulated in both RGC layer and whole retina, fold changes were: (ATF3) 28 and 3; (lipocalin) 20 and 8; and (IGFBP3) 9 and 8, respectively. Affected gene categories also varied. In the RGC layer, protein synthesis dominated 14 distinct upregulated biological processes, but did not appear among 9 affected processes in whole retina, of which immune response was first. Eleven processes were downregulated in the RGC layer compared to 3 in whole retina. Downregulated processes unique to the RGC layer were axon extension and dendrite morphogenesis, and generation of precursor metabolism and energy.
Microarray analysis of just the RGC layer of the retina refines detection of gene responses to elevated IOP. This improves the potential of determining cellular mechanisms in RGC and their supporting cells that could be targets for methods to enhance survival of RGC and preserve vision, even in cases of advanced injury.
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