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In Germany approximately 17.000 people go blind per year. Mainly because of degenarative retinopathias, while current available therapy strategies are limited and mostly failing to effect a cure. For some of this people, there is a new chance to gain their eyelight again within the Projekt RETINA-IMPLANT. In this project researchers are develloping a retina protheses. One part of this work are long time biocompatibility experiments. Therefore, the silicone coat of the protheses is implanted for different periods in one eye of a cat, pig, minipig or rabbit. Afterwards the animal is killed and both eyes are explanted and histologically examinated. The biological attachment of the silicone coat on the retina should be through a gliosis and not through a fibrosis. The histological examination is not always able to clearly distinguish between gliosis and fibrosis. So immunhistological methods are an optimal supplement to standard histological examinations. In routine procedures, material for such examinations is embedded in paraffin. But with this method, it is not possible to investigate the retina-implant-scaffolds. Paraffin is too smooth. We need a much harder embedding material like Technovit®8100 or 9100. Till now, there is no information about the immunhistological staining method with Technovit®8100 or 9100, except for the product information about Technovit®8100. So the goal of this study is to establish the immunhistological staining method with Technovit®8100 and 9100 on normal eyes without any implantations. Therefore, normal eyes from rabbit, swine and minipig were embedded in paraffin and normal eyes from rabbit and swine were embedded in Technovit®8100 and 9100. Afterwards serial sections of 2 µm were made from all embedded eyes succeded by immunhistological labelling. Additionally, sections of 2 µm thickness from a rabbit eye (RI014/02) embedded in paraffin which has a fibrosis were immunhistological labelled. Staining results were compared, depending on the embedding material and the possibility to clearly distinguish between gliosis and fibrosis. Staining results from the eyes embedded in paraffin served as negative control. The outcome of the experiments is, that with immunhistological labelled sections from eyes embedded in Technovit®8100, we could not clearly identify structural changes like we have within a gliosis or fibrosis. Another disadvantage with this embedding material was, that for positive staining results higher antibody concentrations are needed and it is not possible to get any positive results with the labelling method with DAP. Meanwhile staining results from immunhistological labelled sections from eyes embedded in Technovit®9100 show the same qualitity with similar antibody concentrations like labelling results from eyes embedded in paraffin. Therefore the immunhistochemistry with Technovit®9100 is suitable for the exact characterization between gliosis and fibrosis. The disadvantage of this method is the really costly and time-consumingembedding of the material in Technovit®9100, which takes more than four weeks. So at the time, there is no optimal embedding material for exploring tissue-implantat-scaffolds with immunhistological labelling.

Retina tissue culture represents an in vitro system to analyze neuronal as well as glial interactions and molecular processes. In the present work different retina tissue culture systems from the adult porcine eyes were examined by immunohistology and results compared to freshly isolated retina as positive control. The control retina displays histological characteristics of in vivo retina, except for an early degeneration of photoreceptor outer segments and a minor reduction of ganglion cell bodies. Astrocytes in control retina could be visualized reproducibly by both GFAP antibodies in immunohistology, whereas variable expression patterns of GFAP and Vim were found in Muller cells depending on variations in antibody specificity. Muller cells in control retina showed GFAP1 labelling however no clear anti-GFAP2 staining could be detected; anti-Vim1 antibody labelled apical Muller cell end feet whereas anti-Vim2 antibody displayed staining of the entire Muller cell. According to the literature S100b-antibodies detect Muller cells and astrocytes in freshly isolated retinal tissue whereas glutamin synthetase antibodies label Muller cells exclusively. Comparison of immunohistological results from freshly isolated, control retina to healthy retina (in vivo situation) verified their identical histological characteristics, except for photoreceptor degeneration. In the present work semiquantitative image analysis of fluorescence labelled, immunhistological sections were used for comparison of different retina cultures to freshly isolated retina. The comparison of this semiquantitative data to the literature proved that anti-GFAP2 and also anti-Vim1-labelled structures of the inner plexiform and the inner nuclear layer correspond to the actual volume of Muller cells. The semiquantitative results for anti-GFAP1, anti-Vim2, anti-S100b and anti-GS staining indicate higher Muller cell volumes than those published previously. The retinas were cultured without RPE over a period of one, 3 and 5 days. For these cultures a reduction in ganglion cell nuclei, a progressive degeneration of photoreceptor cells and an increase of Muller cell hypertrophy was observed over the cultivation period. Over the culture period of one and 3 days an increase of GFAP, Vim, S100b and glutamin synthetase could be observed whereas after 5 days cultures showed further increase of GFAP, S100b and GS as well as reduction of Vim in Muller cells and astrocytes. After 5 days of culture cellular mechanisms were identified supporting neuronal survival; however at this time an onset glial proliferation was observed. In this study, in contrast previous publications Muller cells did not display co-expression of GFAP and Vim; however this is explained by the fact that these intermediate filaments represented by a group of polypeptides. Various binding options to these polypeptides for different antibodies may result in variable immunohistological results. Furthermore is the relation of GFAP expression to Vim expression object to change in the time course of retina culture. In addition the co-culture of retina/RPE-choroid complexes was established. For this technique retina as well as the RPE-choroid complex were isolated separately and cultured for 3 days after juxtaposing both tissues in their original orientation. It has been shown that RPE co-culture supports survival of retinal cells. It could be observed improved ganglion cell and photoreceptor survival, limited nuclei loss in both nuclear cell layers, and decreased hypertrophy of Muller cells in retina-RPE cultures compared to retinal cultures alone. In addition, retina-RPE cultures were characterized by reduced up-regulation of glia specific proteins such as GFAP1, GFAP2, Vim1 and Vim2. Dynamic retina cultures in Minuth chambers displayed a reduced degeneration and proliferation of retinal cells, which can be explained by improved medium and oxygen supply as well as physiologic pressure conditions within the chamber. It is known from the literature that cultivated Muller cells induce gliotic membranes following in vivo injection and express collagen I abundantly. It has been shown in this study that the addition of Muller cells to the apical surface (ILM) of retina cultures as well as the addition of collagen I to the culture medium results in histological characteristics similar to epiretinal and intraretinal gliosis observed in vivo. Therefore this culture system can be used as in vitro model for retinal gliosis. Immunohistochemistry of Co-Cultures with Muller cells showed an increase in all glial specific proteins such as GFAP, Vim, S100b and GS in apical Muller cell processes up to the INL. The addition of collagen I, known to induce proliferation in pure Muller cell culture, resulted in increased GFAP- and S100b expression in apical Muller cell processes as well as a reduction of Vim, S100b- and GS expression in basal Muller cell processes.