Search

Purpose: Repeated subconjunctival injections with 5-fluorouracil (5-FU) after trabeculectomy are used in glaucoma patients for the inhibition of overproliferation in wound site. Thus, a certain amount of the drug may penetrate into epithelial layer, where it causes toxicity to corneal epithelial cells. The aim of this study was to evaluate the toxic effects of 5-FU and mechanisms of drug-induced apoptosis in cultured corneal epithelial cells.Methods: Cellular damage and the caspase pathway were estimated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptotic characteristics were detected by flow cytometry, a TUNEL test, and western blotting in cultured corneal epithelial cells.Results: The results indicated that 5-FU was toxic to corneal epithelial cells in a time- and dose-dependent manner. Pretreatment with a general caspase inhibitor (Z-VAD-FMK), a caspase-8 inhibitor (Z-IETD-FMK), and a caspase-9 inhibitor (Z-LEHD-FMK) reversed 5-FU-induced cellular damage. Following exposure to 5-FU, a flow cytometric assay with MitoLight dye demonstrated the significant loss of mitochondrial membrane potential. A positive TUNEL test revealed that cellular DNA apoptosis occurred following exposure to 0.5, 1, and 5 mg/mL 5-FU for 15 h. Positive annexin V-FITC and negative propidium iodide (PI) staining indicated that the cell membrane exhibited apoptosis upon exposure to 1 and 5 mg/mL 5-FU for 15 h. The western blot assay demonstrated upregulation of the p21 protein but downregulation of the Bcl-2 proteins induced by 5-FU.Conclusion: These data reveal that 5-FU-induced cellular apoptosis in corneal epithelial cells may be mediated through caspase-8, caspase-9, and mitochondria-regulated pathways, as well as by upregulation of p21 and downregulation of Bcl-2-dependent signal transduction pathways. [ABSTRACT FROM AUTHOR]

It has been reported that retinoic acid (RA) may inhibit the growth of RPE and be used in the treatment of proliferative vitreoretinopathy (PVR). However, previous reports in this field have been conflicting. The main reason for these contradictory findings is that different methods for evaluating the effects of RA on RPE from different species have been used. In human specimens, only RPE from the donor eye (stationary) but not RPE from the PVR membrane (already at active proliferation status) have been tested. This study tested the effects of RA on the growth of RPE using a novel in vitro model: RPE from the PVR membranes, which simulates the in vivo situation of PVR patients better than RPE from the donor eyes. This study also used various methods to solve the conflicting results reported previously. We found that both all transretinoic acid (all-RA) and 13-cis-retinoic acid (cis-RA) can promptly (though not completely) inhibit proliferation of RPE (inhibition rate of 89%-90%) over a very wide range of concentrations (10-9-10-5 M) and various lengths of periods (2-12 days) in a dosedependent and time-dependent manner and without evident cytotoxic effects. Previously reported disadvantages discovered from the study of RPE from donor eyes, e.g., the absence of inhibitory effects on the early passages of cultured cells and inhibition occurring only after long-term treatment, do not present in RPE cells from the PVR membrane. The proliferation of RPE recovered from the inhibition by RA rapidly after the discontinuation of treatment, indicating that a continuous supply of the drug over a long period, i.e., until the breakdown of the blood-retinal barrier has been repaired, is essential for the success of drug treatment of PVR. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the effects of indocyanine green (ICG), which is a fluorescent dye recently used for staining during manipulation of the internal limiting membrane (ILM) in surgery, on cultured human retinal pigment epithelial (HRPE) cells. Different concentrations, up to 0.25% (weight/volume), of ICG were added to the culture medium, which contained the cultured human RPE cells. Then the cell-growth curve and cell viability were assayed with a direct cell count and MTS cell proliferation kit, respectively. Two (2) models of experimentations, a dose-response group and a short-term application group, were designed. We obtained data for IC50 values for 24-, 48-, and 72hour ICG treatments, which were 0.062%, 0.041%, and 0.035%, respectively. This implies that the longer the culture duration, the lower the concentration of ICG, which can inhibit the HRPE cells. In the group of 3-day sequential incubations with ICG treatment, a 0.01% concentration significantly inhibited HRPE growth. In addition, HRPE cells were damaged immediately after ICG treatment, and the damage was ICG dose-related. In the sequential 3-day incubation, compared to the control group, the surviving HRPE cells of each treated group had the same growth rate. In conclusion, ICG has a relatively toxic effect on cultured HRPE cells, even in much lower concentrations than clinical nondilution status (0.25%). Since an ICG toxic effect on HRPE cells was found in our study (in vitro), we highly recommend staining the ILM with as low a concentration of ICG as possible, and washing out the residual intravitreal ICG as thoroughly as possible after finishing the peeling of the ILM. [ABSTRACT FROM AUTHOR]

It has been well documented that RPE cells play an important role in the formation of proliferative vitreoretinopathy (PVR). We studied the effects of four most widely used anti-proliferative drugs, i.e., 5-fluorouracil (5-FU), daunomycin (DM), mitomycin C (MMC) and dexamethasone (DEX) in an in vitro model system (cultured human RPE). The cells treated with indicated concentrations of drugs for 48 h were harvested for DNA content analysis. In addition, a time course study with constant dosage of drugs was performed. The results revealed that, after incubation for 48 h, an increased percentage (31.1%) of S phase cells was noted with exposure to MMC (10 ng/ml). It was confirmed by 5-bromodeoxyuridine (BrdU) incorporation test that this increase does not result from higher transition rate from G1 to S phase. The cells of G2/M phase markedly increased from 13.8% to 29.7% with 10 ng/ml DM. We also demonstrated that 5-FU and MMC treatment led to cell accumulation at S phase and DM treatment resulted in cell accumulation at G2/M phase, These findings were compatible with their pharmacological mechanisms. Development of an in vitro model using cultured human RPE to study the effects of various anti-proliferative drugs on cell cycle can provide a rapid, safe and inexpensive method for selection of drugs used for management of PVR. [ABSTRACT FROM AUTHOR]

The effects of various antiproliferative drugs have been tested in numerous cell types in vitro, but a comparison of effects of these drugs on cultured human retinal pigment epithelium (RPE) has not been reported. We studied the effects of four most widely used antiproliferative drugs (5-FU, daunomycin, mitomycin and dexamethasone) in a new in vitro model system (cultured human RPE). Various concentrations and exposure periods were tested in four human RPE cell lines. 5-FU showed a dose-dependant growth inhibition, with an ID[sub 50] of 5.35 ± 2.38 × 10[sup -7] M after 4 days culture. A decrease of cell number occurred relatively later, the slope of the dose-response curve was less steep than that of others. Mitomycin C showed an immediate and strong growth inhibition and cytotoxic effects, with an ID[sub 50] of 3.73 ± 0.71 × 10[sup -9] M. RPE cultured with daunomycin showed an abrupt decrease of cell number from 10[sup -9] M to 10[sup -8] M, the ID value was 1.07 ± 0.23 × 10[sup -8] M. Dexamethasone showed a biphasic effect; it stimulated cell growth at 10[sup -7] to 10[sup -6] M and inhibited cell growth at 10[sup -4] M or higher, with an ID[sub 50] of 6.05 ± 1.61 × 10[sup -3] M. The advantages and disadvantages of these drugs and the prospective clinical application of these drugs for management of proliferative vitreoretinopathy (PVR) were discussed. Development of an in vitro model using cultured human RPE to study the effects of various antiproliferative drugs can provide a rapid, safe and inexpensive method for selection of drugs used for management of proliferative vitreoretinopathy. [ABSTRACT FROM AUTHOR]

The incidence of recurrent vitreous hemorrhage of proliferative diabetic retinopathy following posterior vitrectomy ranges from 29% to 75% in reported series. Fluid-gas exchange and vitreous cavity lavage are the popular methods of treating this kind of recurrent hemorrhage. The fluid-gas exchange cannot offer clear vision immediately after the procedure. To improve the function of the classic vitreous cavity lavage, we designed a volume homeostatic fluid-fluid exchanger - Chen's I/A device. Tissue plasminogen activator (t-PA) is a protease that preferentially converts fibrin-bound plasminogen to the active proteolytic enzyme, plasmin. It has been clinically and experimentally proven effective in lysis of postvitrectomy blood clot and fibrin formation. When the blood clot is formed in the vitreous cavity, intravitreal injection of t-PA can convert plasminogen to plasmin and remove the clot. From July 1999 to January 2000, ten eyes of postvitrectomy diabetic vitreous hemorrhage (PDVH) were collected. In each case, 4 days after intravitreal injection (IVI) of t-PA (30 μg), vitreous cavity lavage was performed with Chen's I/A device. Of these cases, 8 eyes (80%) experienced an immediate clearing of the vitreous cavity. Early complications included anterior hyaloid fibrovascular proliferation (2 eyes) and postoperative intraocular pressure elevation (3 eyes). On the basis of the results of this study, our conclusion is that volume homeostatic vitreous cavity lavage, combined with intravitreal injection of t-PA, is an excellent method for treatment of postvitrectomy diabetic vitreous hemorrhage but, in cases of PDVH with iris rubeosis, the advantage of this procedure is uncertain. [ABSTRACT FROM AUTHOR]

Focuses on a study which investigated the release of vascular endothelial growth factor within the subretinal fluid in eyes with retinal detachment. Introduction to vascular endothelial growth factor; Materials and methods; Results and discussion.