CHARACTERIZATION OF PHOTODYNAMIC ACTIONS OF ROSE-BENGAL ON CULTURED-CELLS

Purpose. The authors have previously reported successful photodynamic occlusion of corneal blood vessels using intravenous rose bengal and a rgon green laser irradiation. To explore the action mechanism of this novel technique of photothrombosis, they examined the photodynamic eff ect of rose bengal on cultured fibroblasts, smooth muscle cells, and v ascular endothelium-the cellular components of blood vessels. Methods. Five types of cells were exposed to different concentrations of rose bengal and argon green laser irradiation. The irradiated cell areas we re analyzed by fluorescence microscopy and fluorometry. Various potent ial quenchers and proteins were tested for their modulation of the pho todynamic action. Results. Upon irradiation with 16 W/cm(2) of argon g reen laser light in conjunction with rose bengal concentrations extend ing above 1 X 10(-4) M, all cultured cell types showed a dose-dependen t photobiologic effect characterized by constriction and detachment of the laser-irradiated cell region from the rest of the cell monolayer. In addition, there was dye photobleaching and development of a blue s hift of the fluorescence excitation and emission maxima in the irradia ted cell areas. Binding of rose bengal to intracellular components was demonstrated by fluorescence microscopy and by fluorometry showing a red shift of the excitation maximum compared to the maximum in solutio n. This binding was a prerequisite for expression of the described pho tobiologic effect, because polymer-conjugated rose bengal (Sensitox II ) failed to reproduce it. The addition of native or heat-inactivated b ovine serum albumin or catalase decreased this photobiologic effect al so owing to dye binding, as indicated by G-75 Sephadex gel filtration chromatography. Conclusion. These results indicate that the specific p hotobiologic effect of monolayer contraction, which simulates the vaso constriction seen during photothrombosis under argon green irradiation , appears to be caused by the photochemical interaction of rose bengal bound with intracellular components.