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Elaine Johnson, Dongseok Choi, William O Cepurna, Tiffany Choe, Diana Lozano, Shandiz Tehrani, Hari Jayaram, John C Morrison; UTILIZING RNASEQ TO DETERMINE INITIAL OPTIC NERVE HEAD (ONH) GENE EXPRESSION RESPONSES TO ELEVATED INTRAOCULAR PRESSURE (IOP). Invest. Ophthalmol. Vis. Sci. 2016;57(12):2536.
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© ARVO (1962-2015); The Authors (2016-present)
The changes in ONH gene expression associated with a single, axon-damaging exposure to elevated IOP are unknown. We used RNAseq to begin to determine the responses of the rat ONH to a controlled elevation of intraocular pressure (CEI) that results in minimal axon loss.
Anesthetized rats were exposed unilaterally, via anterior chamber cannulation, to an 8 hour CEI at 60 mm Hg. Rat arterial oxygen saturation, body temperature, blood pressure, heart and respiratory rate were monitored throughout exposure. RNAseq was used to determine gene expression in 4 CEI ONH collected immediately following exposure and compared to that in 4 control ONH (ONH from 4 naïve rats). RNA was extracted and individual ONH libraries were prepared. Unique RNAseq reads were determined and mapped to rat genes included in the Ensemb Rat genome (Rnor_6.0) and normalized by the trimmed mean of M-values method. For read counts/gene greater than 10 reads/million in an individual library, reads were further transformed by the voom method. Differential gene expression was assessed by an empirical Bayes method. Fold change > 2.0 and false discovery adjusted p-value < 0.05 were considered statistically significant. Computations were made using Bioconductor packages and R language. Significant gene clusters (Enrichment score > 1.3) were discovered using DAVID Bioinformatics tools.
CEI optic nerves from 16 additional rats demonstrated small focal areas of axon degeneration at 10 days post-exposure. Immediately following the same CEI exposure, we found 632 significantly up and 512 down regulated genes in the exposed group, compared to controls. The upregulated gene clusters were dominated by membrane associated, axonal, insulin pathway, phosphorylation, actin cytoskeleton and cytokine signaling process messages. Downregulated clusters included processes associated with blood vessel development, motility, adhesion, extracellular matrix, apoptosis, signal transduction and cytokine regulation.
Coupling the CEI model with RNAseq techniques has great potential for determining the sequelae of quantitative ONH gene expression responses associated with IOP-induced injury. The time course of responses following a single CEI exposure, compared to that of naive and anesthesia/cannulation controls, remains to be determined.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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