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G.J. Quin, M.C. Gillies; Proteomic Comparison of Diabetic versus Non–Diabetic Rat Retinae . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3247.
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Purpose: Direct comparison of the proteins expressed in diabetic retinae versus non–diabetic retinal tissue is likely to provide insights into the pathogenesis of diabetic retinopathy. We used proteomic analysis to detect and identify retinal proteins that are structurally modified or altered in level of expression by diabetes. Methods: Diabetes was induced in a total of 3 Dark Agouti 8 week old male rats via a single intraperitoneal injection of streptozotocin. Aged matched controls were given sham injections of an equal volume of the vehicle buffer. The severity of the diabetic state was ameliorated by weekly injections of 2 units Protophane insulin. After 10 weeks of diabetes with regular confirmation of elevated blood sugar levels, the animals were sacrificed with immediate enucleation and excision of retinae. Three retinae from each group were pooled and probe sonicated to provide two samples for comparison. The samples were first loaded and run on IPG strips for isoelectric focussing, then separated on 17cm graded polyacrylamide gels by electrophoresis. The resultant 2–D gel protein profiles were then made visible by FX scanning after Sypro ruby fluorescent staining. Comparison of diabetic and non–diabetic gels was performed with the aid of BioRad PDQuest analysis software. Results: A prominent widespread down–regulation of protein expression was found in diabetic rat retinae. We detected up–regulation of 7 proteins in diabetic retinae, down regulation of 87, and disappearance of 49. The identification of these proteins is proceeding through MALDI–TOF and Q–TOF mass spectrometry. Conclusions: The systematic proteomic analysis of diabetic retinal tissue has provided a clear alteration in protein expression that warrants further examination in conjunction with genomic analysis of gene expression profiles.
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