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Jose Hurst, Patricia Klemm, Matthias Blak, Thoralf Herrmann, Max Schultheiss, Karl-Ulrich Bartz-Schmidt, Guenther Zeck, Martin Stephan Spitzer, Sven Schnichels; Hypothermia protects retinal ganglion cells from cell death in an organotypic ischemia model. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4414. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion cell (RGC) death induced by ischemia causes irreversible loss of vision. Several ophthalmic diseases are affected by ischemia, including central retinal artery occlusion. We previously presented an ex-vivo organotypic ischemia model. In this study, we successfully tested hypothermia as a therapy for ischemia.
Retinal ischemia was mimicked in vitro using a custom-made ischemic chamber. In this chamber, retinal explants were kept under oxygen free conditions at 37°C. The chamber was streamed with N2 for 5 min, then immediately sealed and the retinas were incubated for 75 min. Hypothermic treatment over 4h starting directly after the preparation at 20°C or 30°C, by placing the retinal explants into a preheated incubator. After incubation the 6-well plate was adjusted to normal air conditions and incubated under standard conditions. To analyze the effect of hypothermia qPCR for ganglion cell marker and Western-Blot analyses were conducted. Moreover, retinal thickness via OCT measurements was determined. In addition, measurements with multi electrode assays (MEA) were performed to test for functionality of the RGCs.
Our ex-vivo retinal ischemia model showed a drastic reduction in retinal thickness after 24h and 48h. 30°C hypothermia counteracted the retinal thickness, hypothermia of 20°C rescued the loss in retinal thickness already after 24h and the neuroprotective effect was persistent after 48h.mRNA expression levels of RGC markers Nefh, Thy1 and Tubb3 were also highly reduced after ischemia and rescued by hypothermia of both 30°C and 20°C. A trend of microglial and astrocytic activation after ischemia, indicated by Gfap and Cd11b mRNA levels was observed, which was also counteract by hypothermia.After 48h of culturing the control and the ischemic retinas showed no activity of the RGC above 3 Hz indicating a substantial loss in functionality. The neuroprotective effect of hypothermia was detected as a significantly increased proportion of spontaneous RGC activity above 3 Hz.
Hypothermia for 4h was highly neuroprotective regarding retinal thickness, expression of neuronal mRNA and protein levels and spontaneous RGC spiking after retinal ischemia. Of the two tested hypothermic temperatures both were neuroprotective, however 20°C hypothermia showed a stronger neuroprotection that not only counteracted but protected from ischemia-induced retinal damage.
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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