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Y. Guo, E. C. Johnson, J. A. Dyck, W. O. Cepurna, T. Doser, J. C. Morrison; Retinal Ganglion Cell Layer Gene Responses to Elevated Pressure and Progressive Axon Loss in Experimental Glaucoma. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3697.
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© ARVO (1962-2015); The Authors (2016-present)
Cell responses and interactions within the retinal ganglion cell (RGC) layer are a crucial component of glaucoma pathogenesis but poorly understood. Among these, the initial cellular events and underlying molecular mechanisms are particularly intriguing as they offer potential targets for neuroprotective intervention. In this microarray study, we examine gene responses to elevated IOP in RGC layer isolated from retina by laser capture microdissection (LCM) and compare early changes to those with more extensive axon loss.
IOP elevation was produced in Brown Norway rats by unilateral injection of hypertonic saline. RGC layers were obtained by LCM from 6 fellow, 9 early injury (<30% optic nerve axon degeneration) and 15 extensive injury eyes (>30% axon degeneration). RNA was extracted and amplified for cDNA microarrays of independent samples (N=30). Normalized array data were analyzed with SAM and ANOVA with post-tests (>1.4 fold change, p<0.05) and significant gene categories were determined using DAVID.
Early injury yielded 532 genes significantly altered in expression (225 up, 307 down). The major gene category upregulated by focal injury was transcription regulation, while downregulated categories were mitochondria, ribosome, proteasome, energy pathways, protein synthesis, protein degradation and synaptic transmission. With extensive injury, 1781 genes were significantly regulated (989 up, 792 down). Gene categories included upregulated immune response, cell adhesion and downregulated dendrite morphogenesis and axon extension. Interestingly, previously downregulated protein synthesis became the most significantly upregulated process here. A comparison of the specific genes between the two groups showed that 50% of the early changed genes were not present in the extensive injury group. Responses of these uniquely affected genes were dominated by downregulation of ribosomal genes and altered expression of insulin-signaling pathway genes. Insulin-like growth factor 2 was decreased by 48% while downstream genes phosphatidylinositol 3-kinase, 3-phosphoinositide dependent protein kinase and the regulatory subunit of protein phosphatase 1 were upregulated to at least 150%.
For the first time, we have successfully combined microarray analysis with LCM to study the response of RGC layer cells in a glaucoma model. The early gene expression changes appear more representative of RGC responses and further investigation may help identify critical cellular mechanisms as potential targets for neuroprotective therapy.
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