gms | German Medical Science

Background: Ischemia plays an important role in several ophthalmological diseases. Retinal ganglion cells (RGCs) are the most sensitive cells to ischemia. To investigate neuroprotective agents and therapies against these ophthalmological diseases we developed an easy-to-use chamber 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 can not be tested in these models.

Methods: For this pilot-study, we incubated retinas at 37°C for five different time periods (45–120 minutes) under ischemic conditions. 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. Thereafter the organotypic cultures were incubated for 24, 48 or 72 h in an incubator under standard conditions. To analyze the amount of RGCs and apoptotic RGCs, the retinas were frozen and processed for cutting. RGCs immunohistology was performed with a Brna3a-antibody. Apoptotic cells were visualized via TUNEL-staining and overall cell amount via DAPI-staining.

Results: A time-dependant decrease in the amount of RGCs after 24, 48 or 72 h was observable. Moreover, also a decrease in the amount of RGCs that was dependant on ischemia duration was observed. Furthermore the amount of TUNEL-positive cells was ischemia dependant.

Conclusion: We successfully established an organotypic culture model for retinal ischemia. Any therapy can now be tested under ischemic organotypic conditions. Furthermore, we improved our chamber to allow manipulations (injection of drugs, exchange of the media or temperature adjustment) during ischemia.