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N. Koizumi, Y. Nakano, Y. Sakamoto, N. Okumura, P. Dai, H. Tsuchiya, R. Torii, S. Kinoshita, T. Takamatsu; Corneal Endothelial Cell Proliferation is Enhanced by a Single Injection of Connexin 43 siRNA in a Primate Endothelial Injury Model. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3955. doi: https://doi.org/.
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In a rat corneal endothelial injury model, knockdown of connexin43 (Cx43) induces endothelial cell proliferation (Nakano et al, IOVS, in press). The current study was designed to investigate the cell proliferative effect of a single injection of Cx43 siRNA into the anterior chamber of primates with corneal endothelial injury.
Under general anesthesia, the central corneal endothelium of six eyes of three cynomolgous monkeys were damaged by transcorneal freezing with a 6mm diameter probe tip (four eyes) or a 3mm diameter tip (two eyes). Probes were applied for 15 seconds. Next, 100µm of aqueous humor was aspirated from the right eyes and replaced by 40µM of hCx43 siRNA (siRNA eyes, n=3). Control siRNA was injected into the left eyes (control eyes; n=3). On the third day after injection, animals were euthanized and corneal tissue was excised. Tissue obtained from each cornea was divided into four pieces to evaluate endothelial wound healing by immunohistochemistry for Ki67 and propidium iodide. The numbers of total cells and Ki67 positive cells were counted from the center through the limbal area.
In all eyes, Ki67 positive cells were detected in the leading edge of the endothelial wound. In two animals injured by 6mm tips, the Ki67 labeling index (Ki67 positive cells/total cells, %) was significantly increased in Cx43 siRNA eyes compared to control eyes (42.8±7.5, 23.8±14.0, respectively. p<0.05, Student’s t). Total cell numbers were not significantly different in these animals. In one animal injured by the 3mm tip, the endothelial wound was already closed on day 3 in the Cx43 siRNA eye with less Ki67 positive cells. The denuded wound area was still visible, however, in the control eye.
A single injection of Cx43 siRNA into the anterior chamber increases corneal endothelial cell proliferation in vivo, and accelerates wound closure in a primate endothelial injury model. Though long term observations are necessary, our results provide useful information for the possible future clinical application of Cx43 siRNA in tackling corneal endothelial dysfunction.
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