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Sven Schnichels, Matthias Blak, Tanja Dorfi, Johanna Hofmann, Karl-Ulrich Bartz-Schmidt, Focke Ziemssen, Maximilian Schultheiss, Martin Spitzer; Establishment of a retinal ischemia organ culture model. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6333. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Ischemia plays an important role in several ophthalmologic diseases. To investigate neuroprotective agents and therapies against these diseases we developed an easy-to use chamber for 6-well plates with inserts for organotypic cultures. We decided to use organotypic cultures, because in-vivo models or primary cultures are very time-consuming, expensive and several therapies or agents cannot be tested in these models.
We incubated retinas at 37°C for different durations (45, 60, 75, 90 and 120 minutes) under ischemic conditions. Briefly, the chamber was streamed with N2 for 5 minutes, then the chamber was immediately sealed and the retinas were incubated for the rest of the designated time. After the incubation the 6-well plate was adjusted to normal air conditions and incubated for 24, 48 or 72h in an incubator under standard conditions. To analyze the amount of RGCs immunohistology was performed with a Brna3a-antibody. Apoptotic cells were visualized via TUNEL-staining and overall cell amount via DAPI-staining. Furthermore, Western-Blot analyses with GFAP- and Thy-1-antibodies were performed. Moreover, OCT measurements of the organ cultures were performed for up to one week. Additionally, comparisons with retinas treated with 0.5mM and 1mM glutamate were performed.
A time- and ischemia duration-dependant decrease in the amount of RGCs after 24, 48 or 72 h was observed. Moreover, the amount of TUNEL-positive RGCs was also ischemia duration- and time-dependant. The damage to the RGCs through 75 minutes of ischemia was comparable to the amount of damage by 1mM glutamate incubation for 24h (20.27 vs. 19.69) and 48h (13.41 vs. 14.41). In contrast, in glutamate treated retinas, only few apoptotic RGCs were found. The thickness of the retina significantly decreased ischemia duration- and time-dependant as observed with OCT-measurement.
We successfully established a cheap, reliable, reproducible, ease-to-use organotypic culture model for retinal ischemia. Any therapy can now be tested under ischemic organotypic conditions. We selected 75 minutes of ischemia for further studies, because approximately 50% of the RGC died compared to the control after 48 h. Moreover, the RGC-loss after 75 minutes of ischemia is comparable to the loss with 1mM glutamate. Results of a neuroprotective treatment with our chamber are shown on another poster from our group (Schultheiss et al.).
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