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Sandra Kuehn, Jose Hurst, Adelina Jashari, Kathrin Ahrens, Marc-Ilan Wunderlich, Burkhard Dick, Sven Schnichels, Stephanie C Joachim; NMDA triggered microglia activation in a porcine retina organ culture. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4255.
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© ARVO (1962-2015); The Authors (2016-present)
The NMDA induced retina toxicity is applied for in vivo or in vitro models. The advantage of this porcine organ culture is a fast and easy analysis of unidentified therapy agents. Therefore, the reaction of retinal and glia cells to different doses of NMDA need to be identified for porcine retinas before testing therapy agents.
Organotypic cultures of porcine retina were cultivated and treated with different doses of NMDA (0 (Co), 50, 100 and 200 µM) on day 2 and 3. At day 7, retinas were cryo-conserved for histological (n=4/group), Western blot (n=3/group) and RT-PCR (n=6/group) analysis. The retinal ganglion cells (RGCs, NeuN) in apoptosis (cleaved Caspase 3+NeuN) were counted. Additionally, the signal area of the astrocytes (GFAP) was analyzed and the protein level was detected. The microglia (Iba1) in an active state (CD16/32) were counted. Groups were compared with student’s t-test.
Compared with the Co group a trend to less RGCs were seen in NMDA treated groups (50 µM: 84.3±3%, p=0.07; 100 µM: 84.4±6.9%, p=0.1; 200 µM: 85.9±4.8%, p=0.09). Yet, in the 100 and 200 µM group an increase of apoptotic RGCs could be measured (100 µM: 188.5±11.1%, p<0.001; 200 µM: 145.9±10.1%, p=0.001). The 50 µM group exhibited no apoptosis signs (p=0.6). Apoptosis was accompanied by an increase of Iba1+ microglia (100/200 µM: p<0.001). Some microglia expressed the CD16/32 receptors (100 µM: p<0.001; 200 µM: p=0.02). The 50 µM NMDA had no effect on the microglia cell count or activity (Iba1/CD16/32: p>0.05). While the microglia increased, the GFAP area decreased (50 µM: p=0.01; 100 µM: p=0.008; 200 µM: p<0.001). A similar development could be seen via GFAP Western blot analysis (50 µM: p=0.007; 100 µM: p=0.004; 200 µM: p=0.1).
The cultivation time seems to be too short for the total degeneration of the RGCs. However, a significantly higher apoptosis rate in the RGCs could be measured. The apoptosis mechanisms activated the microglia, whereas the macroglia, as a carrier of the NMDA receptor, are already affected by the toxic NMDA mechanisms. A similar glia reaction could be seen in the in vivo NMDA model. In conclusion, we evaluated the optimal NMDA concentration for a significant apoptosis induction in RGCs and macroglia, which does not cause a non-irreversible damage. This progression rather resembles cell death in chronic damage models.
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