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:To evaluate the ocular toxicity of trypan blue (TB) injected into the vitreous cavity of rabbit eyes. TB is a dye that could be useful for staining epiretinal membranes during vitrectomy surgery.METHODS:Ten New Zealand White (NZW) rabbits underwent gas-compression vitrectomy. Rabbits were divided into three groups to receive injections of 0.1 ml basic salt solution, 0.1 ml of a 0.06% TB solution or 0.1 ml of a 0.2% TB solution. Ocular toxicity was assessed by slit-lamp biomicroscopy, ophthalmoscopy, electroretinography and histology.RESULTS:Transient posterior capsule opacification was noted in all animals. No significant reductions in a-wave or b-wave amplitudes were found in any of the animals. Light and electron microscopic examination of the inferior retina in the 0.2% TB-treated eyes showed damaged photoreceptors and marked disorganization. Immunohistochemical staining for rhodopsin was strongly reduced in those sections and staining for proliferation with Ki-67 was positive. No histological abnormalities were found in the upper retina of the 0.2% TB-treated eyes or in any part of the retina of the 0.06% TB-treated or control eyes. No histological abnormalities were found in any of the anterior chamber angle specimens.CONCLUSIONS:Although no signs of toxicity were found after the prolonged presence of TB at a concentration of 0.06% in the vitreous cavity of rabbit eyes, marked damage occurred in the lower retina of 0.2% TB-treated eyes. The short-term presence of TB at a concentration of 0.06% in the vitreous cavity is harmless to the rabbit eye but a higher concentration of TB could be unsafe.

We have established a new test concept for in vitro pharmacological trials. Our model employs tissue explants to test compounds for toxicity which arises with the metabolic interactions among different cell types. Microsurgical preparation of tissue explants avoids the destruction of the organ-specific tissue architecture. Explants were mounted in tissue carriers to improve nutrition and handling of the sample. To allow for the omission of serum supplementation of the culture medium, explants were cultured under continuous medium flow. Test substances are applied considering the polar architecture of most tissues in vivo, for example, covering the apical aspect of epithelia. In principal, all tissues obtained from any species, including man, can be used in this system. A trial application was performed with vitreous body substitutes, substances used in ophthalmology. One compound had passed cell culture tests, but caused massive blood vessel deterioration in vivo. Using our test system based on the developing renal vessel system, we could confirm, within 24 hours, severe vessel damage which resembles the injury suffered by the rabbit retina. We demonstrate that an improved tissue culture assay is a suitable tool for the detection of toxicity that remained unidentified in cell culture tests.

Kidney function depends on a well-developed vascular system. Any impairment of the blood supply disturbs the integrity and function of the organ. The differentiation of renal vessels has been investigation for many years, but little is known about the relationship between nephrogenesis and vessel development. In the present work the spatial organization of the differentiating vessels was analyzed in precisely oriented tissue sections and in optical sections acquired by laser scan microscopy. Developing vessels as well as small capillaries were visualized with two endothelium-detecting antibodies. Small vessels running in parallel towards the organ capsule were detected in numerous cortico-medullary-oriented tissue sections. Cross-sections of the nephrogenic zone showed a regularly arranged network, which was composed of cells detected by both monoclonal antibodies. Parts of this network were localized in regions of the nephrogenic zone which have been assumed to be free of vessels or vessel-like structures for a long time. These results were confirmed by the laser-scan-microscopic analysis of complete cortex explants. The extraordinarily regular arrangement of the endothelial network in the nephrogenic zone allowed us to reconstruct the developing vascular system. The results presented here underline the close relationship between nephrogenesis and vessel development.

A technique for the preparation of plasmodesmata within a cell wall fraction of Solanum nigrum tissue homogenates has been developed, featuring good ultrastructural preservation of plasmodesmatal structure. SDS-PAGE of protein extracts of this plasmodesmata-containing cell wall fraction revealed the distinct enrichment of two bands, with molecular weights of 28 and 43 kD.