97th DOG Annual Meeting 1999
CHARACTERIZATION OF THE RETINAL PIGMENT EPITHELIUM (RPE) IN A PERMANENT PERFUSED CULTIVATING SYSTEM BY USING LASER EFFECTS
C. Framme, K. Kobuch, W. Herrmann, E. Eckert, W. W. Minuth, J. Roider
Background: A new cultivating system for retinal pigment epithelium is used to examine the effects of laser application to the RPE. To preserve the specific architecture and function of the tissue even outside the organ, the RPE was cultivated under permanent perfusion with medium.
Material and methods: RPE with underlying choroid was prepared from enucleated porcine eyes and fixed in a double ring system, the Minusheet. The tissue in this special holder system was placed in a specific two-compartment tissue container with a permanent perfusion line of medium at both sides, the upper RPE- and the lower choroid-side. Cultures were kept for 1, 3, 7 and 14 days and were examined histologically afterwards. Laser treatment was performed on each tissue-ring-system by application of 3 x 3 laser burns (t=0,1; Ø=200µm, distance about 600µm) using different energy levels (400-800mW). As a control non treated RPE was examined histologically after perfusion.
Results: The permanent perfused RPE showed regular appearance in histological sections. Up to 14 days of cultivation light microscopy revealed a normal matrix with a well differentiated RPE-monolayer. During laser treatment a marked lightning of the RPE with centrifugal spreading could be observed. By using higher levels of energy a contraction of the RPE towards the focal point was noticed. Seven days after perfusion histologic sections showed a detachment of the RPE from Bruch membrane and choroid and a cubic thickening of the RPE-cells. Tissue with a RPE-contraction during laser treatment revealed a complete destruction of the RPE-monolayer and of the proximal choroid.
Conclusion: Using this new cultivating system it was possible to preserve an intact RPE up to 14 days. The examination of laser effects on the fixed tissue was practicable. Histology revealed reactive proliferation of the RPE after laser application. These results show, that this organotypical RPE cultivation system with a permanent perfusion line is suitable to investigate laser tissue interactions. Further more this system seems to be useful for investigations of the RPE in AMD, RPE-transplantation and RPE toxicity.
University Eye Hospital, Franz-Josef-Strauss-Allee 11, D-93042 Regensburg