sirolimus and Corneal-Opacity

Topics: Administration, Ophthalmic; Animals; Burns, Chemical; Cell Proliferation; Cells, Cultured; Cornea; Corneal Injuries; Corneal Neovascularization; Corneal Opacity; Disease Models, Animal; Drug Carriers; Drug Compounding; Eye Burns; Fibroblasts; Fibrosis; Humans; Lipids; Male; Mice, Inbred BALB C; Nanomedicine; Nanoparticles; Sirolimus; Sodium Hydroxide; Wound Healing

To investigate the protective effect of rapamycin against alkali burn-induced corneal damage in mice.. BALB/c mice were treated with 0.1 N NaOH to the cornea for 30 seconds. Corneal neovascularization and opacity were clinically evaluated at 1, 2, and 4 weeks after chemical burn injury. Rapamycin was delivered topically to right eyes (1 mg/mL) and injected intraperitoneally (0.2 mg/kg) once a day. Concentrations of interleukin-6 (IL-6) and transforming growth factor-beta1 (TGF-β1) in the cornea were measured by enzyme-linked immunosorbent assay (ELISA). In vitro-cultured human corneal stromal cells were treated with 0 to 500 nM rapamycin for 3 days and then assessed by immunofluorescence staining of vimentin and alpha-smooth muscle actin (α-SMA). Western blotting assays for α-SMA, phosphorylated extracellular signal-regulated kinase (ρ-ERK 1/2), and total ERK 1/2 were also performed.. Corneal neovascularization and corneal opacity scores measured 4 weeks after the chemical burn corneal injury were lower in the rapamycin group than in the control group. Two weeks after the chemical burn injury, a significant elevation in the corneal IL-6 levels of the positive control group was observed, compared to the levels in the negative control group or the rapamycin group (P < 0.05). Corneal TGF-β1 levels were lower in the rapamycin-treated group than in the control group at 4 weeks after chemical burn injury (P < 0.05). Moreover, rapamycin inhibited TGF-β1-induced α-SMA expression and augmented ERK 1/2 phosphorylation.. Rapamycin treatment reduced corneal opacity and corneal neovascularization in BALB/c mice. Rapamycin protected the cornea from chemical damage via reduction of IL-6 and TGF-β1 expression. Rapamycin reduced α-SMA expression through the ERK 1/2 pathway.

Topics: Animals; Blotting, Western; Burns, Chemical; Cells, Cultured; Corneal Neovascularization; Corneal Opacity; Corneal Stroma; Enzyme-Linked Immunosorbent Assay; Eye Burns; Immunosuppressive Agents; Injections, Intraperitoneal; Interleukin-6; Male; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Signal Transduction; Sirolimus

Corneal stromal scarring partly involves the production of corneal myofibroblasts. The purpose of this study was to examine the effects of rapamycin (an inhibitor of the mammalian target of rapamycin [mTOR] pathway) on myofibroblast formation in vitro and in-vivo.. Human corneal fibroblasts were grown in culture and transformed into myofibroblasts using TGF-β (2 ng/mL). The phosphorylation (activation) of the mTOR pathway was examined by immunoblotting. Cell proliferation with and without rapamycin was examined by thiazolyl blue tetrazolium bromide (MTT) assay and Ki67 staining. The expression of the myofibroblast differentiation marker smooth muscle actin (SMA) was examined by immunostaining and immunoblotting. The functional effects of rapamycin were measured using a gel contraction assay. For in vivo studies, 140 μm laser ablation was performed on rabbit corneas followed by subconjunctival rapamycin or vehicle. Corneal haze development was graded at 4 weeks, while the expression of myofibroblast markers was examined by immunostaining and immunoblotting.. The TGF-β activated the mTOR pathway with peak phosphorylation at 2 to 4 hours. Treatment of corneal fibroblasts with rapamycin reduced their proliferation by 46% compared to control. Rapamycin significantly inhibited TGF-β-induced expression of myofibroblast markers (17.2% SMA positive cells with rapamycin compared to 69.0% in control). Rapamycin also significantly inhibited TGF-β-induced collagen gel contraction. In the rabbit eyes treated with rapamycin, corneal haze development was significantly less compared to controls (0.75 ± 0.4 vs. 2.17 ± 0.7).. Rapamycin appears to inhibit proliferation and differentiation of corneal myofibroblasts and, thus, may provide an effective therapeutic measure for preventing corneal scarring.

Topics: Animals; Blotting, Western; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cicatrix; Corneal Opacity; Corneal Stroma; Disease Models, Animal; Female; Humans; Immunosuppressive Agents; Myofibroblasts; Photorefractive Keratectomy; Rabbits; Sirolimus; Transforming Growth Factor beta

We investigated the effects of bevacizumab and rapamycin on central corneal opacity and apoptotic keratocyte number after photorefractive keratectomy (PRK) followed by ultraviolet B (UV-B) irradiation.. A total of 60 right eyes of Sprague-Dawley rats in four groups (n = 15 each) underwent PRK ablation to 80 μm with a 3-mm zone. Sponges soaked with 0.02% mitomycin C (MMC), 2.5% bevacizumab, 0.01% rapamycin, and balanced saline solution were applied for 2 minutes to these eyes in the MMC, bevacizumab, rapamycin, and control groups, respectively. At 3 weeks after PRK, all right eyes were exposed to 100 mJ/cm(2) UV-B irradiation. Biomicroscopy was used to determine the amount of haze, and TUNEL staining for apoptosis and histology were performed at 3, 6, and 12 weeks.. Contrary to the results at 3 weeks, central corneal haze, and apoptotic keratocyte and keratocyte number decreased significantly in the MMC, bevacizumab, and rapamycin groups compared to the control group, and the keratocyte number was lower in the MMC group than the bevacizumab and rapamycin groups at 6 weeks (all P < 0.05). At 12 weeks, the apoptotic keratocyte number was lower in the MMC, bevacizumab, and rapamycin groups than the control group, and the keratocyte number was significantly lower in the MMC than the rapamycin and control groups (all P < 0.05).. Intraoperative bevacizumab and rapamycin administration decreases central corneal haze and apoptotic keratocyte number after PRK. Bevacizumab and rapamycin may be safe alternatives to MMC during refractive surgery to prevent postoperative corneal opacity less affecting the keratocyte number.

Topics: Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal, Humanized; Apoptosis; Bevacizumab; Cell Count; Corneal Keratocytes; Corneal Opacity; Disease Models, Animal; Immunosuppressive Agents; In Situ Nick-End Labeling; Microscopy, Acoustic; Photorefractive Keratectomy; Rats; Rats, Sprague-Dawley; Sirolimus; Treatment Outcome; Vascular Endothelial Growth Factor A