Analyse verschiedener Netzhautkulturmodelle an der Netzhaut vom adulten Hausschwein

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.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2007

Author dc:creator

• Müller-Kämpf, Stefanie

Contributors dc:contributor

• Walter, Peter

• info:eu-repo/classification/ddc/570
• Netzhaut
• Stäbchenzelle
• Zapfenzelle
• Gewebekultur
• Zellkultur
• Immunohistologie
• Schwein
• Vimentin
• Glutaminsynthetase
• Glia
• Astrozyt
• Kollagen
• Fibrose
• Gliose
• Auge
• Pigmentepithel
• Aderhaut
• Biowissenschaften, Biologie
• RPE
• S100
• Choroidea
• Minuth
• Perfusionskultur
• choroid
• perfusion culture

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112664%22

OAI record oai:identifier

https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A50107