Purpose: The tree shrew model of refractive development is particularly useful because, like humans, tree shrews have a fibrous sclera. Selective changes in some candidate extracellular matrix proteins and mRNAs have been found in the sclera during the development of and recovery from induced myopia. We undertook a more neutral proteomic analysis using two-dimensional gel electrophoresis and mass spectrometry to identify scleral proteins that are differentially expressed during the development of and recovery from lens-induced myopia.
Methods: Five tree shrews (Tupaia glis belangeri) wore a monocular -5 D lens for four days, starting 24 days after natural eye opening. At the end of this time, all treated eyes had partially compensated for the lens and were -3.5+/-0.7 D (mean+/-SEM) myopic relative to the untreated fellow control eyes. An additional five animals wore a -5 D lens for 11-13 days followed by four days of recovery without the -5 D lens. The amount of recovery was 1.6+/-0.4 D. Scleral proteins from both groups were then isolated and resolved by two-dimensional gel electrophoresis and spots that were differentially expressed were identified by mass spectrometry.
Results: The scleral protein profile typically displayed about 700 distinct protein spots within the pH 5-8 range. Comparison of the treated-eye and control-eye scleras of the lens-compensation animals revealed five spots that were significantly and differentially expressed in all five pairs of eyes; all were downregulated 1.2 to 1.7 fold in the treated eye. These proteins were identified as: pigment epithelium-derived factor (PEDF), procollagen Ialpha, procollagen Ialpha2, and thrombospondin I (two spots). In the recovering eyes, the two thrombospondin I spots remained lower in abundance while PEDF and the procollagens were no longer downregulated. In addition, 78 kDa glucose-regulated protein (GRP 78), a member of the heat shock protein 70 family, was slightly upregulated 1.3 fold.
Conclusions: We found consistent results across animals that were of a magnitude consistent with the physiologically small changes to the focal plane of these eyes. Changes in collagen confirm previous findings, but downregulation of thrombospondin I adds detail to our understanding of the chain of signals that regulates scleral creep rate. The differential changes in PEDF and GRP 78 were not expected based on previous studies and demonstrate the utility of the proteomic approach in tree shrew sclera.