Model Of Posterior Capsule Opacification Research Articles

Tissue culture of bovine lens as an in vitro model for posterior capsule opacification and the effects of pranoprofen on the cell confluence

To establish an in vitro model of posterior capsule opacification (PCO) by culturing the posterior capsule of bovine lens, to observe the proliferation and differentiation of lens epithelial cells and to study the influence of serum and pranoprofen eyedrops on cell confluence of this model. The bovine lens posterior capsule was spread on the surface of a 25 ml culture flask with cell layer upward. DMEM with 0%, 10% and 20% fetal calf serum was used as culture medium. The cell coverage and confluence time on the posterior capsule were observed by inverted microscope and the cell morphology was observed by Giemsa staining and scanning electron-microscope. Pranoprofen was added to the culture medium at a concentration similar to the aqueous humor concentration (0.23 mg/L), which was presented at 4 hours after the instillation of pranoprofen eyedrops. The difference of confluence time between the treated group and the control group was compared. The lens epithelial cells migrated and proliferated rapidly on the posterior capsule from the equatorial region to the center. The cell coverage was increased and the confluence time was shortened with the increase of serum concentration (P < 0.05). The PCO and wrinkles were presented. Pranoprofen at 0.23 mg/L could inhibit the confluence of lens epithelial cells (P < 0.01). The in vitro model for PCO was an useful method to study the mechanism of PCO formation. Pranoprofen can inhibit the proliferation of lens epithelial cells and is a safe and efficient drug for preventing the occurrence of PCO and can be used as a routine medication after the cataract operation.

Effect of TGF-β2 and Anti–TGF-β2 Antibody in a New In Vivo Rodent Model of Posterior Capsule Opacification

This study evaluated the effect of transforming growth factor (TGF)-beta2 and anti-TGF-beta2 antibody in a rodent model of posterior capsule opacification (PCO). An extracapsular lens extraction (ECLE) was performed in 72 Sprague-Dawley rats. At the end of the procedure, 10 microL TGF-beta2 (TGF-beta2-treated group), fetal calf serum (FCS)/phosphate-buffered saline (PBS; FCS/PBS-treated control group), a human monoclonal TGF-beta2 antibody (anti-TGF-beta2-treated group), or a null control IgG4 antibody (null antibody-treated control group) was injected into the capsule. Animals were killed 3 and 14 days postoperatively. Eyes were evaluated clinically prior to euthanatization, then enucleated and processed for light microscopy and immunohistochemistry afterward. PCO was evaluated clinically and histopathologically. Student's t-test and chi(2) were used to assess differences between groups. There were no statistically significant clinical or histopathological differences in degree of PCO between the TGF-beta2- and FCS/PBS-treated groups at 3 and 14 days after ECLE. Nor were there differences between the anti-TGF-beta2- and the null antibody-treated groups, with the exception of the histopathology score for capsule wrinkling 3 days after ECLE (P = 0.02). alpha-Smooth-muscle actin staining was observed in the lens capsular bag only in areas where there was close contact with the iris. No sustained effect of TGF-beta2 or anti-TGF-beta2 antibody on PCO was found in rodents at the dose and timing administered in this study. Iris cells may play a role in the process of epithelial mesenchymal transition linked to PCO.

Prevention of posterior capsule opacification by the induction of therapeutic apoptosis of residual lens cells

Posterior capsule opacification (PCO) is a common complication of cataract surgery. Using adenovirus(Ad)-mediated gene transfer, we overexpressed the proapoptotic molecules p53, procaspase 3, Bax, and TRAIL to induce therapeutic programmed cell death of residual lens cells to prevent PCO. Overexpressed TRAIL did not induce apoptosis in cultured rabbit lens cells or in human lens cells. Overexpressed p53 induced apoptosis of lens cells in vitro and ex vivo, but was unable to prevent PCO in vivo. Overexpressed procaspase 3 was associated with engagement of many components of the apoptotic pathway, including cleavage of intracellular caspase targets such as PARP and inter-nucleosome DNA fragmentation. Even when only slightly overexpressed, Bax caused apoptosis of transduced rabbit and human lens cells by engaging the mitochondrial pathway, including catalytic activation of the caspases. A single in vivo injection of Ad vectors expressing either Bax or procaspase 3 into the capsular bag at the end of phacoemulsification prevented PCO in rabbits. These experiments show that Ad-mediated Bax or procaspase 3 overexpression is capable of inducing therapeutic programmed cell death in vitro and in vivo in residual lens cells and preventing PCO in a rabbit model of PCO. Manipulation of proapoptotic molecule expression could be a novel gene therapy approach for prevention of PCO.

Posterior Capsule Opacification in Mice

To present a new model of posterior capsule opacification (PCO) in mice. An extracapsular lens extraction was performed in 28 consecutive mice. Animals were humanely killed 0 and 24 hours and 3 and 14 days after surgery. Eyes were enucleated and processed for light microscopy and immunohistochemistry. In 20 animals (71%), the eye appeared well healed before death. In 8 animals (29%), postoperative complications were noted. All animals developed PCO 2 weeks after surgery. Immediately after extracapsular lens extraction, lens epithelial cells were present in the inner surface of the anterior capsule and at the lens bow. At 24 hours, lens epithelial cells started to migrate toward the center of the posterior capsule. At 3 days, multilayered lens epithelial cells throughout the lens capsule and capsular wrinkling were apparent. Lens fibers and Soemmerring ring formation were observed 14 days after surgery. CD45(+) and CD11b (+) macrophages were found in greater numbers 24 hours and 3 days after surgery (CD45(+), P = .04 and P<.001, respectively; and CD11b(+), P = .01 and P = .004, respectively). The number of CD45(+) cells remained statistically significantly higher (P = .04) 14 days after surgery. In mice, PCO occurs following extracapsular lens extraction and is associated with low-grade but significant macrophage response. The use of genetically modified mice to evaluate the pathogenic mechanisms of PCO and search for new therapeutic modalities to prevent or treat PCO is now possible.

A new model of posterior capsule opacification in rodents.

To describe a new model of posterior capsule opacification (PCO) in rodents An extracapsular lens extraction (ECLE), by continuous curvilinear capsulorrhexis and hydrodissection, was performed in 42 consecutive Brown Norway rats. Animals were killed at 0, 6, and 24 hours and 3, 7, and 14 days after surgery. Eyes were enucleated and processed for light microscopy and immunohistochemistry. In 34 (81%) of the animals the operated eye appeared well healed before death, with a clear cornea and a well-formed anterior chamber. In eight (19%) there was no view of anterior segment structures because of hyphema, fibrin, or corneal opacification. PCO was clinically evident 3 days after ECLE and was present in all animals at 2 weeks. Immediately after ECLE, lens epithelial cells (LECs) were present in the inner surface of the anterior capsule and lens bow. Twenty-four hours after surgery, LECs started to migrate toward the center of the posterior capsule. At 3 days, multilayered LECs, some spindle shaped, were present throughout the lens capsule. Capsular wrinkling was apparent. Lens fibers and Soemmering's ring were observed in all animals 14 days after surgery, indicating some degree of cellular differentiation. Activated macrophages were present in greater numbers at 3 and 14 days after surgery (P < 0.05), when proliferation and migration of LECs appeared to be greatest, and lens fiber differentiation was evident, respectively. In rodents PCO occurs after ECLE and is associated with low-grade inflammation, mostly of mononuclear macrophages. Although no intraocular lens implantation was performed, this model appears to be valuable for studying the sequence of events that leads to PCO after cataract surgery and the extracellular matrix cues that promote lens fiber differentiation.

Effect of surgical technique on in vitro posterior capsule opacification

Purpose To compare the effect of different cataract extraction surgical techniques on residual lens epithelial cell (LEC) density and cell regrowth rates using an in vitro model of posterior capsule opacification (PCO). Setting Comparative Ophthalmology Research Laboratories, North Carolina State University, Raleigh, North Carolina, USA. Methods Lens capsule explants were prepared from freshly enucleated canine globes after extracapsular cataract extraction (ECCE), phacoemulsification, or phacoemulsification followed by capsule vacuuming. Initial cell density on the capsule and cell proliferation were determined by phase contrast microscopy. The effects of the surgical technique on time to confluent growth of the cells across the posterior lens capsule were determined. Results Residual cell density on the remaining anterior capsule immediately after lens removal was 31.6% ± 19.3%, 16.1% ± 8.9%, and 7.7% ± 5.7% in the ECCE, phacoemulsification, and phacoemulsification/capsule-vacuuming groups, respectively. Time to confluence (range 5.0 to 6.3 days) was not significantly different among the 3 groups when the lens capsules were cultured in serum-supplemented media. The confluence rate was significantly longer (by approximately 5 to 7 days) in the phacoemulsification/capsule-vacuuming group than in the other 2 groups when the capsules were cultured in serum-free media. Conclusions Phacoemulsification with and without anterior and equatorial capsular vacuuming led to less initial LEC density in the capsular bag than ECCE. However, because cell proliferation rates among the 3 groups were only marginally affected, near 100% removal of LEC at the time of cataract extraction may be necessary to prevent PCO.

Adenovirus-mediated suicide gene transduction: feasibility in lens epithelium and in prevention of posterior capsule opacification in rabbits.

The most common complication of cataract surgery is the development of posterior capsule opacification (PCO). Hyperplasia of the lens epithelium is one of the main cellular events following phacoemulsification, and has been found to be an important feature contributing to opacification of the posterior capsule. Adenoviral vector-mediated transfer is a suitable method for transducing the herpes simplex virus thymidine kinase gene (HSV-tk) into proliferating cells, allowing for the selective killing of these cells by ganciclovir (GCV) treatment. To determine the potential of gene transduction for lens epithelial cells, we studied the transduction of rabbit lens epithelial cells with adenoviral vectors containing either the Escherichia coli beta-galactosidase (lacZ) gene or the HSV-tk gene in vitro and in vivo in an experimental model of PCO. The efficiency of lacZ gene transfer in rabbit lens epithelial cells was at least 95% both in vitro and in vivo. In vivo transduction with HSV-tk adenoviral vector followed by GCV treatment significantly inhibited the development of PCO (p<0.001). These results suggest that adenoviral vector-mediated transfer of HSV-tk into the proliferating lens epithelial cells is feasible and may provide a novel therapeutic strategy for PCO.

Retrovirus-mediated transfer of a suicide gene into lens epithelial cells in vitro and in an experimental model of posterior capsule opacification

PURPOSE. The most common complication of cataract surgery is the development of posterior capsule opacification (PCO). Hyperplasia of the lens epithelium is one of the main cellular events following phacoemulsification and was found to be an important feature contributing to opacification of the posterior capsule. We investigated the feasibility of killing the residual lens epithelial cells by retroviral-mediated transfer of the herpes simplex virus-thymidine kinase (HSV-tk) gene, a well-studied suicide gene, into rabbit lens epithelial cells followed by ganciclovir (GCV) treatment. METHODS. The capacity of retroviral vectors to transfer genes into rabbit lens epithelial cells was determined either in vitro (culture of rabbit lens epithelial cells) or in vivo (experimental model of PCO in rabbits) using cDNA encoding the ß-galactosidase (LacZ) reporter gene. To evaluate the efficiency of suicide gene therapy (infection with retroviral vectors encoding the HSV-tk gene followed by GCV treatment) we determined the sensitivity of HSV-tk infected lens epithelial cells to different concentrations of GCV in vitro. Then, in an experimental model of PCO, rabbits were treated with HSV-tk retroviral vectors at the end of the surgery and they received repeated intracameral and intravitreal injections of GCV at the concentration determined by the in vitro experiments. RESULTS. Infection efficiency using LacZ retroviral vectors was about 29% in vitro and 10% in vivo. After infection of the HSV-tk cDNA in vitro, the cell killing effect of GCV was evaluated. A significant enhancement (four- to five-fold) of the cell sensitivity to GCV was shown in FLY-DFGtk as compared with mock infected (P < 0.01) cells even without selection of the HSV-tk positive cells. The GCV concentration leading to 50% reduction in cell number (IC50) was 50 µg/ml. In vivo infection with a HSV-tk vector led to the tk gene transfer into lens epithelial cells. Despite this local HSV-tk gene expression, we could not prevent capsule opacification. CONCLUSIONS. Lens epithelial cells were successfully infected both in vitro and in vivo by ß-galactosidase and HSV-tk genes via retroviral vectors. In vitro infected lens epithelial cells displayed a strong sensitivity to GCV treatment. In vivo, we could not prevent capsule opacification in the rabbit model, very likely due to the limited level of the HSV-tk gene expression. However, our results suggest that virus-mediated suicide gene therapy might be a feasible treatment strategy to prevent capsule opacification with a more powerful vector.