Inhibition of corneal fibrosis by Smad7 in rats after photorefractive keratectomy

Background Haze or corneal subepithelial fibrosis is one of the common complications after refractive surgery procedures, such as photorefractive keratectomy （PRK）, laser epithelial keratomileusis, and epipolis laser in situ keratomileusis, which would result in refractive regression, decreased visual quality, and corneal opacification. Haze directly resulted from corneal fibrosis mediated by transforming growth factor β （TGFβ）. SmadT, an inhibitory Smad, can inhibit TGFβ signal transduction. Recently, the effects of Smad7 on the inhibition of fibrosis in several organs have been studied, while little is known about the effects on cornea after PRK. This study was aimed to determine the effects of lentiviral-mediated Smad7 gene expression on corneal fibrosis in rats after PRK. Methods Four different experimental groups were established using right eyes of Sprague-Dawley rats. Thirty-two eyes underwent de-epithelialization only and served as a sham operation group （group 1）. Ninety-six eyes underwent PRK operation and were further divided into group 2 （the PRK group） without lentivector administration, group 3 （the Lv-blank group） with control lentiviral vector without Smad7 administration, and group 4 （the Lv-Smad7 group） with Smad7 expressing lentiviral vector Smad7 administration. At 1 day, 1 week, 1 month, and 3 months after PRK, the transfection efficiency was determined by measuring the fluorescence signal as well as Smad7 protein and mRNA levels. Corneas were further processed for immunoblotting to assess the phosphorylation of Smad2 as a downstream event of TGFβ/Smad signaling. The expression of fibrotic markers, such as c（-smooth muscle actin （c（-SMA）, Type III collagen （collagen III）, and cell cycle-related marker Ki67, was measured by quantitative real-time reverse transcription polymerase chain reaction （RT-PCR）. Results Lentivirus-mediated exogenous Smad7 gene expression in rat corneal tissue resulted in reduced activation of TGFβ/Smad signaling caused by downregulation of phosphorylation of Smad2. Smad7 also downregulated the expression of TGFβ2. Markers of cell proliferation and fibrosis, including Ki67, c（-SMA, and collagen III, were inhibited by Smad7 up to 3 months after PRK operation. Conclusion Smad7 gene transfer inhibits fibrogenic responses of cornea in rats after PRK.

Development of a novel two color tracer perfusion technique for the hydrodynamic study of aqueous outflow in bovine eyes

Background Elevation of intraocular pressure is usually associated with primary open angle glaucoma and caused by increased outflow resistance. A two-color fluorescent tracer technique was developed to investigate the hydrodynamics of aqueous humor outflow with changing intraocular pressure within the same eye, to better understand the relationship between outflow facility and effective filtration area. Methods Eighteen enucleated bovine eyes were first perfused at 30 mmHg with Dulbecco＇s phosphate-buffered saline containing 5.5 mmol/L D-glucose. After a stable baseline facility, red fluorescent microspheres （0.5 um, 0.002% v/v） were exchanged and perfused. Eyes in the one-color control group （n=6） were immediately perfused with fixative. In the experimental group （n=6）, eyes were perfused with green tracer after intraocular pressure reduced to 7 mmHg, while in the two-color control group （n=6）, eyes were perfused with green tracer with intraocular pressure remaining at 30 mmHg. All 12 eyes were then per＇fusion-fixed. Outflow facility was continuously recorded in all eyes. Confocal images were taken along the inner wall of the aqueous plexus and the percent of the effective filtration length （PEFL; length of inner wall exhibiting tracer labeling/total length of inner wall） was measured. The relationships between outflow facility and PEFL were analyzed statistically. Results No significant differences were found in baseline facilities （ul.min-1.mmHg-1） among the three groups （the experimental group： 0.93±0.12; the two-color control group： 0.90±0.19; the one-color control group： 0.98±0.13）. In the experimental group, the outflow facility was significantly higher at 7 mmHg （4.29±1.01） than that at 30 mmHg （1.90±0.67, P 〈0.001）, which corresponded to a significant increase in the PEFL at 7 mmHg （54.70±8.42） from that at 30 mmHg （（11.76±4.56）%, P 〈0.001）. The PEFL labeled by red fluorescent microspheres in the experimental group （（11.76±4.56）%） showed no significant difference from that of the one-color control group （（13.39±2.19）%, P=-0.473） or the two-color control group （（11.49±4.95）%, P=-0.930）. The PEFL labeled by green fluorescent microspheres in the experimental group （（54.70±8.42）%） was significantly higher than that of the two color control group （（37.34±8.17）%, P=0.010）. A positive correlation was found between outflow facility and PEFL（r=0.897, R2=0.804） in the experimental group. Conclusions Changes in aqueous humor outflow patterns before and after a change in intraocular pressure can be successfully distinguished within the same eye using our newly developed two-color tracer perfusion technique. The PEFL showed positive correlation with the outflow facility.