Hypothermia protects retinal ganglion cells against ischemia

Abstract

Purpose: Hypothermia has been shown to be neuroprotective in the therapy of ischemic stroke. Furthermore the retina is easily accessible for inducing hypothermia in an acute onset of ischemia like during central retinal artery occlusion. By using a cooled irrigation solution during pars plana vitrectomy the retina could be easily cooled down for a certain timespan. The best temperature for cooling the retina is so far unknown. To investigate potential neuroprotective therapies like hypothermia we developed an easy-to use chamber for 6-well plates with inserts for organotypic cultures.

Methods: To determine the optimal neuroprotective temperature we incubated retinas at 20, 30 and 37°C for 75 minutes under ischemic conditions. Hypothermia was induced for 4 hours (h) and the ischemia chamber was adjusted to the desired temperature before the experiments. For inducing ischemia 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 of ischemia. Then the 6-well plate was removed from the chamber and left under a sterile bench with no lid for 2 minutes to adjust the air in the well plate to normal conditions. Afterwards the 6-well plate was incubated at the desired temperature for 4 hours. Next the 6-well plates were incubated for 24 and 48 h in an incubator under standard conditions. For comparison other organotypic cultures were treated with 1 mM glutamate instead of ischemia but underwent the same hypothermia protocol. To analyze the amount of RGCs and apoptotic RGCs, the retinas were frozen and processed for cutting. RGCs immunohistology was performed with a Brn3a-antibody. Apoptotic cells were visualized via TUNEL-staining and overall cell amount via DAPI -staining. Cells were counted manually.

Results: With 20°C and 30°C hypothermia we observed a survival-rate that was up to 1.76x higher than in 37°C incubated ischemic cultures. For the glutamate treated probe the survival rate with 20°C for 4 h was the same as with untreated control samples 48 h after onset of the experiments. Furthermore the amount of TUNEL-positive cells was extremely reduced under hypothermic conditions.

Conclusions: In conclusion, hypothermia is neuroprotective to retinal ganglion cells against ischemia. Therefore, by a cooling the retina during pars plana vitrectomy the tolerance time against ischemia or glutamate stress could be increased.

Keywords: 531 ganglion cells • 548 hypoxia • 615 neuroprotection