Abstract
Purpose :
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.
Methods :
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.
Results :
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.
Conclusions :
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.