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M. R. Frost, T. T. Norton; DIGE Analysis of Tree Shrew Sclera: Differential Protein Expression During the Development of, and Recovery From, Lens-Induced Myopia. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1739.
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© ARVO (1962-2015); The Authors (2016-present)
During experimentally induced emmetropization there is a regulated remodeling of the scleral extracellular matrix. A previous proteomic study of ours found changes in the expression level of some ECM proteins (Frost & Norton, Mol. Vis. 2007). We extended our earlier traditional 2D gel electrophoresis (2DGE) study to see if a more refined method, Difference Gel Electrophoresis (DIGE), could identify additional proteins that are involved in the emmetropization process.
Two groups of 5 tree shrews (Tupaia glis belangeri) wore a monocular -5 D lens, starting 24 days after natural eye opening. The induced myopia group wore the lens for 4 days and became -2.8 ± 0.5 D myopic (mean ± SEM) relative to the untreated fellow control eyes. The recovery group wore the lens for 11 days and compensated fully. After 4 days without the lens, the eyes had recovered by 2.7 ± 0.4 D. Scleral proteins were then isolated and resolved by DIGE. The resulting protein profiles were compared using DeCyder 6.5 analysis software to identify protein spots that were differentially represented between fellow induced myopia vs. control eyes and recovery vs. control eyes.
During myopia development, 2 protein spots were upregulated and 35 spots were downregulated in the treated eye relative to the control eye, including the 5 protein spots that we identified previously using the 2DGE approach (collagen I, thrombospondin I, and PEDF). This is consistent with previous results indicating that during myopia development there is an overall loss of extracellular matrix. During recovery, 10 spots were upregulated and 5 spots were downregulated. Three of the spots that differed during myopia development also differed in recovery; 2 remain downregulated during recovery (thrombospondin I; also reproduced from the previous study) whereas the third spot was downregulated during myopia development but upregulated during recovery.
The magnitude of the expression level changes was predominantly less than 2-fold, consistent with the relatively mild myopia and hyperopia used as stimuli in this study. The discovery of a further 44 differentially represented protein spots supplementary to our previous study, including several very low abundance spots, emphasizes the improved ability of DIGE to detect small biological changes compared to a traditional 2DGE methodology.
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