Characterization of cell cycle modifications induced by electric pulses in human corneal endothelium

Abstract

AbstractPurpose We recently described the possibility of gene electrotransfer into endothelial cells (EC) of human organ cultured corneas (He. OphthalmicRes2010;43:43). Parallel to the transfection of plasmids, trains of electric pulses alone also triggered mitosis of EC, despite human endothelium is normally considered unable to proliferate. Our aim was to further characterize the effect of electric pulses on the cell cycle of ECMethods The short‐term (3 days) and long‐term (30 days) effects on EC of 8 square pulses lasting 100 ms, at 1 Hz frequency and 125 mA intensity were studied on 20 human organ cultured corneas. EC proliferation was evaluated by quantification of Ki67 expression, a proliferation marker of G1 to M phase, and by 5‐Ethynyl‐2’‐Deoxyuridine (Clik‐it EdU) incorporation during DNA replication (S phase marker) EC viability was assessed using the triple labeling with Hoechst33342/Ethidium/Calcein‐AM with calculation of the viable EC density by image analysis (Pipparelli.IOVS2011;52:6018) and using an apoptotic marker, pSIVA‐IANBDResults Three days after stimulation by electric pulses, a significant activation of EC proliferation was observed with S‐phase revealed by EdU staining and G1 to M phase, including mitosis, revealed by Ki67 staining. 90% of proliferating EC were located in area with ECD bellow 2000cells/mm2. For long‐term effect, ECD did not increase and nuclear fragmentation and cell death were detectedConclusion Electric pulses activate the cell cycle of EC on ex vivo corneas but cell division seems to abort and result in cell death. Mechanisms of cell cycle trigger and of mitosis abortion are currently being studied.