RESUMEN
PURPOSE: Despite advances in intraocular lens design and material, posterior capsule opacification remains one of the major problems in modern cataract surgery. Therefore, the use of antiproliferative agents has been advocated. CD95 ligand (CD95L, Fas, Apo-1) 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 evaluate CD95L-induced cell death in cultured lens epithelial cells (LEC). METHODS: Expression of CD95 in untreated porcine LEC was investigated by flow cytometry. Cell death after CD95L or CD95 agonistic antibody treatment was assessed by crystal violet assay and DNA fragmentation was measured by comet assay. RESULTS: The presence of CD95 was observed in LEC. CD95L treatment resulted in a time--and concentration-dependent killing of LEC, which was synergistically enhanced by the addition of cyclohexamide. CD95L treatment induced DNA fragmentation. CONCLUSIONS: The present study confirms the use of apoptosis-inducing CD95L in the inhibition of LEC proliferation. Further studies are needed before clinical application of CD95L to inhibit posterior capsule opacification will be feasible.
Asunto(s)
Apoptosis/efectos de los fármacos , Células Epiteliales/patología , Cristalino/citología , Glicoproteínas de Membrana/farmacología , Receptor fas/biosíntesis , Animales , Células Cultivadas , Ensayo Cometa/métodos , ADN/análisis , Fragmentación del ADN , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Proteína Ligando Fas , Citometría de Flujo , Técnicas In Vitro , Ligandos , PorcinosRESUMEN
PURPOSE: The goal of this study is the characterization of the strong yellow fluorescence of oxidized melanin in the retinal pigment epithelium (RPE) and the choroid. METHODS: Naturally occurring melanin in the human retina and choroid was oxidized by exposing fixed and plastic-embedded sections of a human eye to light and hydrogen peroxide. Synthetic melanin was also oxidized in vitro by exposure to light and hydrogen peroxide. The fluorescence of oxidized melanin was examined by absorption spectroscopy, fluorescence spectroscopy, and fluorescence microscopy. RESULTS: Naturally occurring melanin oxidized in situ exhibited a lipofuscin-like yellow fluorescence. Oxidation of melanin in vitro degraded the melanin polymer, resulting in a fluorescent solution. Fluorescence spectroscopy gave an excitation maximum at approximately 470 nm and an emission maximum at approximately 540 nm for both natural and synthetic melanin. Increasing the time of exposure to light or hydrogen peroxide increased melanin fluorescence. CONCLUSIONS: The results indicate that the strong yellow fluorescence of melanin in the RPE and choroid in situ is a property of oxidized melanin and is not due to contamination of the melanin by proteinaceous or lipid materials. The data presented allow a reinterpretation of the results obtained from fluorescence investigations of melanin-containing tissue and suggest a link between melanin degradation and lipofuscin formation.