triggering Of Corneal Endothelial Cell Mitosis By Electric Pulses

Abstract

Abstract: : Purpose: To report preliminary results of a new technique that, against all expectations, was able to trigger adult human corneal endothelial cell division. During gene electrotransfer experiments for another project, we constantly observed endothelial cells with a modified morphology of their cytoplasm and nucleus, which were proven to be dividing cells Methods: We retrospectively analysed all corneas electroporated with the same current parameters (8 pulses of 100ms, 1 Hz, 135 mA). On permanent flat mounted corneas, counterstained with hematoxylin, we counted the mitosis, along with the collection of donor age and endothelial cell density prior to electroporation, and also time between exposition to the electric field and observation. Results: All electroporated corneas (n=60) carried dividing endothelial cells. The first mitosis were observed 24 hours after electroporation and picked at 72 hours. Exceptionally, few mitosis were observed 28 days after electroporation. Their number was extremely variable between corneas (from 5 to 1000 mitosis per cornea), but markedly similar when paired corneas were observed in the same conditions. It did not correlate with donor age. Noticeably, dividing cells were mostly located at the periphery of the endothelium. Most of the dividing cells were in metaphase, or telophase. The triggering of mitosis was not associated with the presence of DNA for gene transfer since most of the dividing cells were not transfected, and mitosis were observed even without DNA Conclusions: This unexpected side effect of electroporation on human endothelial cells is particularly innovating but nevertheless remains questioning. A direct action on microtubule polymerisation by electric current had already been described in other cell models. Partial rupture of endothelial integrity could also trigger mitosis of neighbouring cells, like previously described after endothelial treatment with EDTA, but microscopic observation of the endothelial layer did not reveal endothelial disorganization. The peripheral location of dividing cells is in favour of the stimulation of hypothetical corneal endothelial stem–like cells, with a high inter–individual variability. Further experiments, using BrdU and Ki67 immunostaining are ongoing to clarify these points

Keywords: cornea: basic science • cornea: endothelium • cornea: storage