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Natalie Wagner, Maurice Gammel, Sabrina Reinehr, Jose Hurst, Sven Schnichels, Burkhard Dick, Stephanie C Joachim; Establishment of an in vitro photoreceptor model suitable for AMD research. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1339.
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© ARVO (1962-2015); The Authors (2016-present)
The age-related macular degeneration (AMD) is a multifactorial disease; therefore, suitable models exist only to a limited extent. An established porcine degeneration model has been modified to enable improved photoreceptor cultivation and to make it applicable for AMD research.
Two methods, namely “filter” and “tweezers”, were tested to gain porcine neuroretina explants, with the ganglion cell layer facing down. Retinas were cultivated for 4 days and compared to explants gained via an established method, photoreceptors facing down. To characterize the explants optical coherence tomography (OCT; n=6/group), H&E staining, immunohistochemistry, and qRT-PCR (n=4/group) were performed. More specifically, morphology of cones (opsin), rods (rhodopsin), amacrine (calretinin), bipolar (Pkcα) and retinal ganglion cells (RBPMS, β-III-tubulin) was evaluated.
OCT analyses revealed a decrease of retinal thickness to a lower extent in “tweezers” compared to “filter” (p≤0.001) and “established” method (p=0.04). Moreover, measurements of retinal thickness via H&E staining showed for both new methods (filter: 28.6±1.1 µm; tweezers: 28.2±0.8 µm) a significantly improved photoreceptor structure compared to the established method (22.6±1.1 µm; filter: p=0.002; tweezers: p=0.003). Additionally, the rhodopsin+ area was significantly increased in the “filter” (6.9±0.2 area[%]/image; p=0.0005) and “tweezers” group (6.3±0.1 area[%]/image; p=0.048) in contrast to the established one (5.4±0.1 area[%]/image). In contrast, the number of cones was only higher in “tweezers” method (162.0±1.3 cells/mm) compared to the established one (141.1±2.5 cells/mm; p=0.035). The amount of amacrine, bipolar and retinal ganglion cells was unaltered. On mRNA level, we revealed an upregulation of rhodopsin (filter: p=0.048) and opsin (tweezers: p=0.045) in both new methods compared to the established one.
This project aimed to develop a more suitable in vitro photoreceptor degeneration model. The cultivation by the “tweezer” method led to a significantly improved morphology, which made it more comparable to the in vivo situation. Subsequently, to establish the AMD model a co-cultivation of neuroretina and RPE-cells will follow. Consequently, this system may serve as a new model for AMD drug screening.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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