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Ekaterina Beglova, Sumon Roy, Kyle Trudeau, Argyrios Chronopoulos, Sayon Roy; Effect of Cx43 Downregulation on Development of Microvascular Lesions in the Rat Retina. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3556.
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Recent studies have shown that high glucose and diabetes contribute to reduced Cx43 protein levels in retinal vascular cells, induce apoptosis, and promote loss of retinal endothelial cells and pericytes, a hallmark of diabetic retinopathy (DR). While these findings suggest a close association between reduced Cx43 and retinal vascular cell loss, it is unclear if Cx43 downregulation alone is sufficient to promote characteristic vascular lesions of DR. In this study, we determined whether downregulation of Cx43 expression with siRNA strategy in rat retinas induces apoptosis and promotes the development of acellular capillaries (AC) and pericyte loss (PL) in rat retinas.
Sprague Dawley rats were divided into three groups comprising of six animals in each group: normal untreated rats as control, rats intravitreally injected with Cx43 siRNA, and rats intravitreally injected with scrambled siRNA as control for siRNA specificity, and studied at 1, 2, and 3 month time points; animals studied at second and third month time points received additional injection(s), administered at monthly intervals. Retinas isolated from these animals were subjected to trypsin digestion for isolation of intact retinal capillary networks. The retinal capillary networks were then stained with periodic acid Schiff’s and hematoxylin, digitally photographed, and images analyzed for AC and PL. In parallel, TUNEL assays were performed to identify cells undergoing apoptosis.
Analysis of retinal capillary networks showed significant increase in the number of AC and PL in animals injected with Cx43 siRNA at all three time points (1, 2, and 3 months) compared to those of normal or scrambled siRNA-treated animals. A significant increase in the number of apoptotic cells, AC and PL was observed by the end of 1 month of Cx43 siRNA injection that remained throughout the second and third month. The data from the second and third month time points, suggested increasing number of AC and PL with longer duration of Cx43 downregulation. No significant difference in the number of apoptotic cells, AC and PL was observed between normal and scrambled siRNA-treated capillary networks at any of the three time points.
These findings provide further support that Cx43 downregulation alone may be sufficient to produce AC and PL in the retina, and that the severity of these lesions is likely to increase in a time-dependent manner with continued retinal Cx43 downregulation.
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