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Sven Schnichels, Sandra Kuehn, Adelina Jashari, Jose Hurst, Stephanie C. Joachim; A novel ex-vivo retina model for oxidative stress – chances for the replacement of animal experiments. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6415.
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© ARVO (1962-2015); The Authors (2016-present)
Oxidative stress is a major player in several ophthalmic diseases, including glaucoma, ischemia, AMD or diabetic retinopathy. To investigate potential therapies we aimed to establish an ex-vivo model with porcine eyes from the abattoir. On one hand, hundreds of pigs are daily sacrificed for the food industry and these eyes are simply discarded. On the other side, researchers kill many animals to test for potential medical therapies. Not only saving the lives of animals is a mayor advantage of using eyes form the abattoir, additionally, the pig eye is much more similar to the human eye than any rodent eye.
Organotypic cultures of porcine retina were cultivated and treated with different doses of H2O2 (100, 300 & 500 µM) for 3h on day 1. At day 3 and 8, retinas were cryo-conserved for histological (n=6-8/group), Western blot (n=6/group) and qRT-PCR (n=6/group) analysis. The apoptotic state of retinal ganglion cells (RGCs; Brn3a+cleaved Caspase 3) and the activation of macroglia (GFAP; vimentin) and microglia (Iba1; Fcγ) were analyzed. Further, the expression of PGP9.5, GFAP, CD11b, HSP70, VEGF, INOS, TNFα, IL1β & p21 were quantified via qRT-PCR.
The amount of Brn3a+ RGCs was reduced in all H2O2 treated groups (p<0.05). In accordance, the amount of apoptotic RGCs was higher compared to the control. A dose-dependent increase of microglia was observed. In contrast neither histological nor protein expression changes were found in regard to macroglia. Quantitative RT-PCR confirmed these findings. Treatment with H2O2 had a negative effect on the expression of PGP9.5 after 3 (1.4 - 2-fold reduction) and 8 days (1.4-fold reduction) of cultivation. At day 8, a higher activation of CD11b (1.5-1.7-fold) was observed. After 3 days, a loss of IL1β mRNA (1.5-2.6-fold) and a rise of iNOS mRNA (2-3-fold) was detected. However, this effect diminished at day 8. HSP70 expression also increased by 2.3 fold after 3 days for all concentrations.
A profound RGC death was observed after adding H2O2. Also inflammatory markers showed an expected response. An ex-vivo model for oxidative stress was therefore successfully established. The use of this model offers high chances to reduce the amount of animals used in retinal research. Moreover, the pig eye is anatomically and molecular closer to the human eye, thus offering higher chances of a successful prescreening of potential therapies.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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