Immunhistochemische Charakterisierung der organotypischen Retinakulturen von B6J.Sv129 Rpgrtm1Stie-Mäusen:

Beschreibung

Gene therapy through the use of targeted DNA double strand breaks and the cell’s own DNA repair mechanisms represents a possible therapeutic approach for X-linked retinitis pigmentosa that normally leads to blindness. Such gene therapeutic approaches could be tested during the organotypic culture of retinal explants from the Rpgr knock in mouse model. Pre-emptively, the need for characterization of the Rpgr-KI mouse during retinal culture arises. Retinae from 3 and 9 month old Rpgr-KI mice were cultured for up to 10 days. Fixation, embedment into freezing medium and preparation of frozen sections followed. Assessment of apoptosis took place via TUNEL-assay. Further staining was made via immunofluorescence and targeted neuronal structures (PKCα, CtBP2, β Dystroglycan), gliosis (GFAP, GS), micro glia (Iba1), the interphotoreceptor matrix (CD44) and multiple proteins for DNA damage detection and repair (γH2AX, pATM, 53bp1, BRCA1, Ku80, DNA-PKcs, CtIP). Nuclear rows in the ONL and INL were counted over the course of culture. These counts were subjected to a regression analysis. There was a clear correlation between time in culture and the ONL nuclear rows, which were reduced in both age groups the longer the time in culture was. Retinae retained their typical morphology during cultivation and via the structural proteins well known processes of retinal degeneration like sprouting and gliosis could be detected. The DNA damage detection and repair proteins inside the retina could be evidenced as well. Like in other retinal explant cultures degeneration of photoreceptors occurred, although the loss of photoreceptors in older Rpgr-KI retinae was faster and more severe than in younger retinae. This might be due to preceding and accumulative damage in vivo. Typical retinal degenerative processes took place earlier than in wild type mice. Most of the DNA associated proteins could be found inside the retina, albeit with only limited staining inside the ONL nuclei with their inverted chromatin structure. Ku80 was exempt from this and during the time course of cultivation showed a translocation from the OPL towards the ONL. The results show that the organotypic cultures of retinal explants from Rpgr-KI mice are vital with continuing remodeling processes as well as existent expression of the investigated DNA associated proteins and thus are viable for future gene therapeutic testing.

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Wilhelm Rheinhard

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Augen Retina Retinitis pigmentosa Gen-Therapie

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