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Behrad Y. Milani, Abed Namavari, Maryam A. Shafiq, Mercede Majdi, Asadolah Movahedan, Hossein M. Sagha, Ali R. Djalilian; Effect of Sirolimus on Proliferation and Differentiation of Human Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1081.
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Corneal scarring is largely due to proliferation and differentiation of corneal fibroblasts into myofibroblasts. The purpose of this study was to first examine the effects of sirolimus (an inhibitor of the mTOR pathway) on human corneal fibroblast proliferation and differentiation. Next, we wished to investigate the effect of sirolimus in preventing stromal haze formation after Photorefractive Keratectomy in a rabbit model.
Human corneal fibroblast cultures were initiated from donated human corneal tissue. After passages 3, the fibroblasts were treated with Sirolimus (1 μg/ml) + TGF-β (1 ng/ml) or TGF-β alone as control. Cell proliferation and expression of the myofibroblast marker alpha-smooth muscle actin (αSMA) were examined by phase-contrast microscopy and immunostaining. For in vivo studies, 140 micron PTK was performed on the right eye of 12 New Zealand White rabbits. Six eyes received 1mg subconjunctival sirolimus immediately and 2 weeks following the surgery. Six eye received vehicle in the same fashion. Corneas were followed by slit-lamp microscopy and were graded for haze development weekly for 4 weeks. At one month post-surgery, the animals were euthanized and corneas were subjected to immunostaining for myofibroblast marker alpha-smooth muscle actin (αSMA), and Tunel assay for apoptosis.
Cell proliferation was significantly reduced in the Sirolimus group compared to control (6.2±1.6 vs. 33.3±5.4 cells/20x field). The percentage of SMA positive cells was significantly lower in the Sirolimus group compared to control (16.2% vs. 78.6%). Corneal haze was significantly less in the sirolimus group compared with the control group (0.75±0.4 vs. 2.17±0.7). The density of SMA+ cell/400x field in the central stroma was significantly lower in the sirolimus group compared with the control group (5.8±1.4 cells vs. 18.2±3.2 cells). Tunel assays and Clinical eye examination revealed sirolimus is nontoxic for the cornea.
Sirolimus appears to inhibit proliferation and differentiation of corneal fibroblasts into myofibroblasts in vitro. Furthermore, subconjunctival sirolimus is effective for preventing corneal stromal haze formation after PTK in a rabbit model.
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