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. 2015 Oct 7;5:15005. doi: 10.1038/srep15005

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Copyright © 2015, Macmillan Publishers Limited

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(a) The corneal endothelial dysfunction model was created by mechanically removing the rabbit corneal endothelium. A total of 5.0 × 10⁵ high-CD or low-CD RCECs was injected, together with ROCK inhibitor, into the anterior chamber, followed by maintenance in a face down position for 3 hours (n = 6). The corneal endothelial dysfunction model in which RCECs were not injected was used as a control (n = 3). Representative slit lamp photograph images are shown. Corneal transparency was restored in endothelial dysfunction models by intracameral injection of high CD RCECs with ROCK inhibitor, while the controls exhibited hazy corneas due to corneal endothelial dysfunction. Interestingly, senescent RCECs with low CD were also able to restore corneal transparency. (b, c) The mean central corneal thickness and corneal volume evaluated by Pentacam^® at 7 and 14 days after cell injection are shown as a graph. The corneal thickness and corneal volume were significantly reduced in the eyes injected with high CD RCECs when compared to eyes injected with low-CD CECs. *P < 0.01, **P < 0.05. (d) Regenerated corneal endothelium following injection of high-CD and low-CD RCECs was evaluated by contact specular microscopy at 14 days. (e) The mean cell density of regenerated corneal endothelium was analyzed. The CD of the regenerated corneal endothelium was significantly higher in the eyes injected with high CD-CECs than with low-CD senescent CECs. **P < 0.05. (f) Function-related markers of CECs (Na⁺/K⁺-ATPase, ZO-1, and N cadherin) were immunostained in the regenerated corneal endothelium. Phalloidin staining was also performed to evaluate the actin cytoskeleton. Na⁺/K⁺-ATPase, ZO-1, and N-cadherin were expressed in all regenerated CECs in eyes injected with high-CD CECs, while expression of these markers was partially disrupted in the CECs in eyes injected with low-CD CECs. Actin was distributed in the cell cortex in the eyes injected with high-CD CECs, while cortical actin distribution showed irregularity and the presence of stress fibers in the eyes injected with low-CD CECs. Nuclei were stained with DAPI. Scale bar: 100 μm.