PURPOSE:To study the effects of conventional laser application on the retinal pigment epithelium (RPE) in a perfusion tissue culture model of porcine retinal pigment epithelium without overlying neurosensory retina. METHODS:RPE with underlying choroid was prepared from enucleated porcine eyes and fixed in a holding ring (Minusheet). Specimens were then placed in two-compartment tissue culture containers (MinuCell & Minutissue, Bad Abbach, Germany) and were cultured during continuous perfusion with culture medium at both sides of the entire specimen, the upper RPE and the lower choroid (12 specimens out of 6 eyes). Cultures were kept for 1, 3, 7 and 14 days and were examined histologically. Laser treatment was performed on each tissue ring by application of 3 x 3 laser burns one day after culture began (argon ion laser, wavelength: 514 nm, pulse duration: 100 ms; spot size: 200 microm) using different energy levels (400-1,000 mW); (16 specimens out of 8 eyes). RESULTS:During laser treatment a marked lightening of the RPE with centrifugal spreading was observed. Using higher levels of energy, a contraction of the RPE towards the center of the laser spot was noticed. One day after laser photocoagulation histology revealed destruction of RPE; within 3-7 days of culture, migration and proliferation of neighboring cells was observed in several lesions. After 7 days the initial defect of the irradiated area was covered with dome shaped RPE cells and after 14 days multilayered RPE cells were showing ongoing proliferation. However, there were also cases without proliferation after laser treatment. The non-treated, continuously perfused RPE showed regular appearance in histological sections: during the first 7 days of culture, light microscopy revealed a normal matrix with a well-differentiated RPE monolayer. Subsequently proliferation even without treatment was observed and after 14 days the RPE became multilayered. CONCLUSION:It was possible to study the early healing response to the effect of laser treatment using the permanently perfused tissue culture system. A marked proliferation and repair of the laser defect could be observed in several but not all lesions. After 14 days even without laser treatment a proliferative multilayered RPE was present. Although this limits the use of the system for longer than 7 days, it seems to be useful for investigation of RPE-related disorders.

Background: The pathogenesis of central serous chorioretinopathy is poorly understood. It is believed to be due to dysfunction of the retinal pigment epithelium and/or choroid and has been associated with elevated levels of epinephrine and administration of corticosteroids. Epinephrine and corticosteroids have previously been shown to induce apoptosis (programmed cell death) in various types of cells. The objective of this study was to investigate whether these agents can induce apoptosis in cultured retinal pigment epithelium cells. This may help elucidate the pathogenesis of central serous chorioretinopathy.Methods: Third-passage porcine retinal pigment epithelium cells were grown to confluence and incubated for 1-7 days in culture medium containing epinephrine (10(2)-10(9) pg/ml) or a corticosteroid, dexamethasone (4-4x10(4) ng/ml). The cultures were evaluated for apoptosis by phase-contrast microscopy and in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Results: Epinephrine (7x10(7)-10(9) pg/ml) induced apoptosis in a dose- and time-dependent manner. Exposure to lower concentrations of epinephrine(10(2)-6x10(7) pg/ml) and all tested levels of dexamethasone did not result in apoptosis.Conclusion: Retinal pigment epithelium cells may undergo apoptosis following exposure to elevated levels of epinephrine. These findings suggest a possible pathophysiologic mechanism for the development of central serous chorioretinopathy.