A Proteomic Approach to Investigate Mechanisms of Ocular Axial Length Control

Abstract

Abstract: : Purpose: Ocular axial length is an essential parameter of ocular optics and is tightly controlled throughout development and adulthood in many vertebrates so as to set the focal plane of the eye onto the retina (Emmetropia). Disturbance of this control will lead to defocus and vision impairment as the retina drifts behind the focal plane (myopia) or lingers in front of it (hyperopia) . Chicken are a very useful model in this field, as their ocular axial length can be manipulated by altering their visual environment. In an effort to analyze the molecular mechanisms of emmetropization, we undertook an extensive proteome analysis of chicken retina and sclera using high-resolution 2D-gel electrophoresis and mass spectrometry. Methods: 10-day old chicks were subjected to three types of experimental manipulations: Form deprivation myopia (FDM) with translucent goggles, lens-induced myopia (LIM) with -15D lenses and lens-induced hyperopia (LIH) with + 15D lenses. These treatments were applied only to one eye of each chick, the contralateral eye as well as eyes from animals without any visual manipulations being used as a control. The treatment was applied either for 1 day or 7 days, and the retina and sclera of both eyes were dissected. Solubilized tissue samples were applied to different narrow-range pH gradient strips for isoelectric focusing as a first dimension and subsequent separation according to mass by SDS-PAGE as the second dimension. The 2D gels thus obtained were stained in Sypro Ruby, digitized and analyzed using the Melanie 3 software to localize differentially expressed spots. Finally these spots were picked, subjected to trypsin digestion and identified by peptide mass fingerprint using MALDI mass spectrometry or by partial sequencing using nanospray MSMS whenever necessary. Results: Our first results prove the feasibility of the study: 1.On the total set of gradients >10'000 proteins can be visualized; 2.The tissue samples yield consistent and reproducible protein patterns; 3. A reasonable number of specific differences are observed. At this point we established a list of several differentially expressed proteins which are of potential use as markers or targets for myopia or hyperopia. Conclusion: The proteomic approach is a promissing tool that may yield new insights on the mechanisms that control ocular axial length and emmetropization.

Keywords: myopia • animal model • gene/expression