Synergistic effects of combined cytotoxic and apoptosis-inducing drugs on Tenon's capsule fibroblasts in vitro and in vivo.

BACKGROUND: Highly toxic antimetabolites have gained access to routine clinical use to modulate and reduce the amount of postoperative scarring following glaucomatous filtering procedures. It could be speculated that by combining two different antiproliferative substances with different mechanisms of action total amounts of the substances could be decreased and side effects reduced. METHODS: Twenty-two substances were tested that had antiproliferative effects by acting cytotoxically, inhibiting growth factors, or inducing apoptosis. With combinations of each two substances, cell culture experiments using 3T3 and human Tenon's capsule fibroblasts were performed evaluating cell toxicity, proliferation and migration, the extent of free radicals, and the amount of apoptosis (TUNEL, electron microscopy). The five most potent combinations were used in an animal experiment with rabbits performing filtering procedures. The extent of episcleral scarring was evaluated by histopathology. RESULTS: The results of the various assays revealed consistently strong effects in 5 of the 462 combinations. Of these five combinations, two were highly effective in the rabbit model. Substances with strong effects when applied in combination included staurosporine, mitomycin, and CD95L. CONCLUSIONS: We found synergistic effects in assays that evaluated different aspects of cell function. The amount of scarring in an animal experiment was inhibited to a level comparable with a high single dose of mitomycin. Combination therapy of two antiproliferative acting substances may be a promising concept.

Postoperative application of mitomycin c improves the complete success rate of primary trabeculectomy: a prospective, randomized trial.

BACKGROUND: The intraoperative application of mitomycin c for primary trabeculectomy is associated with potentially sight-threatening side-effects. This study was performed to evaluate the pressure-lowering effect of postoperative application of mitomycin c for primary trabeculectomy and to evaluate the complications of this new technique. MATERIALS AND METHODS: A randomized, prospective clinical trial with 52 consecutive patients scheduled for glaucoma surgery in one large surgical center was performed. Patients underwent routine trabeculectomy. In group 1, mitomycin c (0.05 mg/ml) was applied topically to the filtering bleb for 5 min on the 3 days after surgery (postoperative application). In group 2, no mitomycin c was applied (controls). The IOP values, visual acuity, number of antiglaucomatous medications and complications were evaluated. RESULTS: Follow-up was evaluated up to 24 months for all patients. The mean intraocular pressure decreased from 31.1 to 15.4 mmHg in group 1 and from 24.8 to 15.6 mmHg in group 2 (P=0.79; t-test). The average number of medications decreased from 2.5 and 2.4 to 0.4 and 0.6 (P=0.53; t-test) in groups 1 and 2, respectively. No cases of hypotony maculopathy occurred. An individual decrease of more than 25% of the IOP was present in 84.6% in group 1 and in 53.8% in group 2 (P<0.017). Survival analysis for eyes with a complete surgical success revealed a better outcome of eyes in group 1 as compared to the eyes in group 2 (P<0.013; log-rank test). CONCLUSIONS: To our knowledge, this is the first prospective, randomized clinical trial to evaluate the efficacy of postoperative mitomycin c application in primary trabeculectomy. The application of mitomycin significantly reduced the IOP while not increasing the rate of complications.

Bullous keratopathy as a complication of trabeculectomy with mitomycin C.

BACKGROUND: The routine use of mitomycin C to enhance glaucomatous filtering surgery has found wide acceptance. Complications of the application of mitomycin C have been repeatedly noticed. We now report a previously undescribed complication showing a toxic effect of mitomycin C to the corneal endothelium. METHODS: Patients underwent routine trabeculectomy with mitomycin C. Following surgery, the eyes were examined by biomicroscopy and specular microscopy of the corneal endothelium. RESULTS: In two cases, we observed a partial decompensation of the corneal endothelium resulting in a well-demarcated clear zone of the cornea and a second zone with thickening of the cornea and a bullous keratopathy adjacent to the filtering bleb. The specular microscopy showed marked irregularities of the endothelial cells and areas of necrosis in those parts of the cornea close to the filtering bleb. CONCLUSIONS: The two cases described here demonstrate that if the corneal endothelium is already compromised before surgery, the application of mitomycin C may have an additional toxic effect on the endothelium and may result in a partial bullous keratopathy. Surgeons should be aware of this complication.

Ciliary body adenoma after application of mitomycin.

PURPOSE: To describe a ciliary body tumor that was detected in a rabbit eye. METHODS: For a specific experiment, the rabbit underwent trabeculectomy with mitomycin. The eye was evaluated by electron microscopy 5 weeks following surgery. RESULTS: A ciliary body tumor was discovered in close relation to pigmented epithelial cells. The tumor cells were large with a small amount of pigment and contained prominent vesicles. CONCLUSIONS: The development of a ciliary body adenoma may be caused by the application of mitomycin. Whether the demonstrated tumor was benign or malignant could not be determined. While it could not be proven that the tumor developed related to the use of mitomycin, this possibility may be important for surgeons using this substance.

Latanoprost stimulates secretion of matrix metalloproteinases in tenon fibroblasts both in vitro and in vivo.

PURPOSE: To investigate the presence and the possible role of different matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in Tenon capsule fibroblasts. These enzymes are essential for the control of tissue remodeling in the context of wound repair. This aspect is important to further the understanding of and possibly to influence the scarring process of filtering blebs after glaucoma surgery. METHODS: Untreated and latanoprost-treated human Tenon fibroblasts were examined for the presence of MMPs and TIMPs on the mRNA and protein levels. Assays performed included RT-PCR, real-time RT-PCR, immunocytochemistry, Western blot analysis, flow cytometry, and zymography. To investigate the changes in vivo, conjunctival specimens of rabbits treated with latanoprost eye drops were examined by immunohistochemistry. RESULTS: In all assays, both MMP-3 and TIMP-2 were detected. With the real-time RT-PCR technique, MMP-1, -2, -3, -7, -9, and -14 and TIMP-1 and -2 were detected. An upregulation of MMP-3 and TIMP-2 after latanoprost treatment of the fibroblasts was shown and found to occur on the mRNA and the protein levels. The upregulation of MMP-3 and TIMP-2 was confirmed in vivo. CONCLUSIONS: Tenon fibroblasts contain the ability on the mRNA level to synthesize all enzymes of the MMP and TIMP family that are related to remodeling of the extracellular matrix. The levels of MMP-3 and TIMP-2 increase after treatment with latanoprost. Tenon fibroblasts may be the target cells for attempts to influence the tissue levels of MMPs and TIMPs in the context of conjunctival wound healing after glaucoma surgery.

Intraoperative episcleral versus postoperative topical application of mitomycin-C for trabeculectomies.

OBJECTIVE: To assess the efficacy of a new application technique of mitomycin-C to enhance the outcome of filtering surgery in cases of complicated glaucoma. DESIGN: Randomized clinical trial. PARTICIPANTS: Fifty consecutive patients scheduled for glaucoma surgery in one large surgical center. INTERVENTION: Patients underwent routine trabeculectomy. In group 1, mitomycin-C (0.05 mg/ml) was applied topically to the filtering bleb on days 1, 2, and 3 after surgery (postoperative application). In group 2, mitomycin-C (0.2 mg/ml) was applied by means of to a sponge during surgery (intraoperative application). MAIN OUTCOME MEASURES: Preoperative and postoperative intraocular pressure (IOP) values, visual acuity, the need for antiglaucoma medication, previous surgical procedures, and the need for further surgical interventions were monitored. RESULTS: Sufficient follow-up data were available from 24 of 25 patients in each of the two groups. The follow-up ranged from 12 to 28 months and was evaluated at 12 months for all patients. The mean IOP decreased from 27.3 to 15.5 mmHg in group 1 and from 29.0 to 17.5 mmHg in group 2. The average number of medications decreased from 2.3 and 2.4 to 0.9 and 0.8 (P = 0.68; t test) in groups 1 and 2, respectively, at the 12-month visit. Hypotony was more frequent in group 2, in which the only case of hypotony maculopathy occurred. There was a tendency of more eyes with lower IOP values in this group. The rate of loss of visual acuity of more than 2 lines was higher in group 2. Failures were more frequent in group 2 (7 of 24) compared with group 1 (1 of 24) (P = 0.04, chi-square test). CONCLUSIONS: To our knowledge, this is the first prospective, randomized clinical study to evaluate the efficacy of this different technique of mitomycin-C application. The postoperative application of mitomycin-C was effective, having few failures. This application of mitomycin-C may be associated with a lower rate of complications when used in eyes with complicated forms of glaucoma.

Characterization of CD95 ligand (CD95L)-induced apoptosis in human tenon fibroblasts.

Toxic side effects of cytotoxic agents such as 5-fluorouracil or mitomycin-C in glaucomatous filtering procedures call for alternative approaches to control fibroblast proliferation. CD95L is a death ligand that triggers apoptosis in susceptible target cells. Apoptosis allows for the safe disposal of cells without damaging the surrounding tissue. The goal of this study was to characterize and to evaluate the CD95L induced cell death in cultured Tenon fibroblasts. Human Tenon fibroblasts were treated with different concentrations of CD95L. For comparison, murine NIH 3T3 fibroblasts were used. Immunohistochemistry and Western blot were used to investigate the CD95 and CD95L expression. Cytotoxicity was measured by crystal violet assay. Apoptosis was investigated using in situ DNA end labelling (TUNEL). DEVD-AMC caspase 3 like activity was measured and caspase 3 processing was studied by immunoblot and the use of the caspase inhibitor DEVD-CHO in cell culture assays. Tenon and NIH 3T3 fibroblasts express CD95 and CD95L. The authors found concentration dependent inhibition of proliferation after CD95L treatment. Tenon fibroblasts, but not NIH 3T3 fibroblasts, show synergy when combined with actinomycin D or cyclohexamide. CD95L treatment did not alter total protein or RNA synthesis. Cell death induced by CD95L was apoptotic and activated caspase 3, as TUNEL positive cells and the active fragment of caspase 3 were found. CD95L induced cell death could be inhibited by the caspase-inhibitor.Here, it is demonstrated that the CD95L induced cell death in cultured human Tenon fibroblasts is apoptotic and possibly mediated by the caspase 3 pathway. These results suggest that it may be possible to use CD95L in glaucomatous filtering procedures. In vivo studies are necessary for further evaluation.

Trabeculectomies in fellow eyes have an increased risk of tenon's capsule cysts.

OBJECTIVE: To assess outcomes and complications of primary trabeculectomies in fellow eyes in a large group of patients. The assumption was that first and fellow eyes undergoing fistulizing surgical procedures behave similarly in the postoperative period. DESIGN: Retrospective nonrandomized comparative trial (paired eye study). PARTICIPANTS: Over a 4-year period, 566 consecutive patients underwent primary trabeculectomy, all without antifibrotic agents. One hundred thirty-eight of these patients underwent bilateral surgery. INTERVENTION: A primary trabeculectomy was performed. MAIN OUTCOME MEASURES: Preoperative data, postoperative intraocular pressure, and visual acuity were monitored. In addition, complications and the need for consecutive surgical procedures were noted. RESULTS: The mean follow-up period of these trabeculectomies was 27.4 months (range, 7-62 months). Of the 124 bilateral adult cases, no statistical difference was found in intraocular pressures, number of antiglaucomatous medications, and success or failure rates between the two eyes. The need for enhancement procedures, such as needling or surgical excision of Tenon's capsule cysts, was significantly higher in fellow eyes than in first eyes (16 vs. 6 cases; P = 0.03; Mann-Whitney U test). Hypotony as a complication occurred more frequently in fellow eyes. CONCLUSIONS: Primary trabeculectomies performed in both eyes of patients have a remarkably similar clinical course. Failures of first eyes may be a reason to use antimetabolites in primary trabeculectomy of the fellow eye. The present data suggest that fellow eyes have a greater risk of Tenon's capsule cyst formation. This may be important for patient counseling before surgery. These results may additionally be important for clinical studies. Given that first and fellow eyes do not behave in an absolutely similar manner, study designs making intraindividual comparisons may not be feasible.

Melanocytoma of the choroid: angiographic and histopathologic findings.

A juxtapapillary choroidal melanocytoma that was clinically confused with a choroidal melanoma is described. The clinical examination of a 40-year-old female patient included ultrasonography, fluorescein, and indocyanine green angiography. Histopathologic and immunohistochemical study of the enucleated eye resulted in the identification of a markedly pigmented, 3.8 mm elevated choroidal melanocytic tumor observed nasally to the disk. A-scan ultrasonography showed a high initial spike with a low to medium internal reflectivity with decreasing amplitude. On fluorescein angiography, the tumor was surrounded by a rim of retinal pigment epithelium depigmentation with a well-defined comet-tail. The lesion was hypofluorescent in the early phases of the indocyanine green angiogram, but from the late venous phase appeared ill-defined hyperfluorescence. Histopathologic study revealed a juxtapapillary choroidal melanocytoma The pigmented tumor cells stained positive for HMB45 and vimentin but negative for S100, GFAP, NSE, and MNF116. No tumor cells stained positive for the proliferation marker Ki67. These findings conclude that choroidal melanocytoma may mimick choroidal melanoma. Abnormalities on fluorescein angiography may be a clinical hint to suspect a pigmented tumor different from a choroidal melanoma.