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J. A. Dyck, T. Doser, W. O. Cepurna, Y. Guo, J. C. Morrison, E. C. Johnson; Interleukin-6, Leukemia Inhibitory Factor and Cell Cycle Gene Expression Upregulation Characterizes Early Optic Nerve Head Injury Produced by Intraocular Pressure Elevation in Rats. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3694.
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While the scleral portion of the optic nerve head (ONH) is thought to be the primary site of injury due to intraocular pressure (IOP) in glaucoma, the mechanism of injury is not well understood. In our most recent rat glaucoma model microarray studies, we identified sequential patterns of gene expression change in initial ONH segments injured by elevated IOP exposure. Among these, the cell cycle was the most upregulated biological process in early injury. Here we use quantitative reverse transcriptase-PCR (qPCR) to determine expression levels of genes representative of cell cycle progression and place these changes in context by measuring the levels of other genes implicated in glaucomatous injury.
Unilateral IOP elevation was produced in Brown Norway rats (n=44) by episcleral vein injection of hypertonic saline. RNA was extracted from the initial, 0.4 mm ONH segment and retrobulbar optic nerves post-fixed, cross-sectioned and graded for the extent of axonal degeneration on a scale of 1 (no injury) to 5 (total nerve involvement). The ONH RNA was linearly amplified and reverse transcribed for qPCR. ONH gene expression was compared between three groups: controls (n=12), early injury (grade < 2, n=20), and advanced injury (grade > 2, n=24). In early injury, less than 15% of optic nerve axons were morphologically altered. Statistical analysis included ANOVA, linear and non-linear regression.
In the early ONH injury group, the cell cycle genes, DNA topoisomerase 2a (6.1 fold), protein regulator of cytokinesis (8.4 fold) and separase (4.4 fold) were confirmed to be significantly upregulated. Early injury also upregulated interleukin-6 (IL6, 15.5 fold) and leukemia inhibitory factor (LIF, 5.4 fold). These changes were accompanied by a 50% downregulation of glial connexin 43 mRNA and occurred prior to significant alterations in neurofilament L and growth associated protein 43 axonal mRNA. In the advanced injury group, extracellular matrix components were significantly upregulated, as indicated by increased levels of periostin, fibulin 2 and matrix gla protein (18.1, 9.8 and 5.3 fold, respectively).
Both microarray and qPCR analysis has allowed the identification of cellular processes and specific gene expression changes accompanying early ONH injury induced by elevated IOP. This study confirms cell cycle progression as an early event in pressure-induced optic nerve injury and identifies the cytokines IL6 and LIF as potentially important mediators in the early injury process.
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