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S. Chifflet, C. Justet, J. A. Hernandez, V. Correa; Stimuli for Wound Healing Responses in Bovine Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1826.
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We have reported that membrane potential depolarization (MPD) and sodium increase in the border cells, due to augmented ENaC expression, start to develop one hour after wounding and play a role in the healing process of bovine corneal endothelial (BCE) cells in culture. Several factors have been proposed to trigger the healing response in epithelia: disruption of cell contacts, factors released by dead cells, mechanical stimuli and the onset of a fast calcium wave (FCW). The purpose of this work is to investigate their possible participation in the MPD and sodium and ENaC increase of the healing BCE cells.
Discontinuities on BCE monolayers were obtained by wounding (scratch assay) or by masking part of the substrate surface with a thin strip of parafilm (mask assay). In this latter model the border cells do not contact the parafilm strip, remaining some 10 µm apart. Fluorescent probes were used to detect MPD (oxonol V), sodium (SodiumGreen) and calcium (Fluo4) changes. Antibodies and FITC-phalloidin were used for fluorescence visualization. ATP, cell lysates and mechanical stimuli (syringe jet of buffer) were investigated as possible factors determining the ENaC-dependent modifications.
In the scratch assay, an FCW is produced immediately after wounding. In the mask assay no wave is observed following the removal of the parafilm strip or treatment with cell lysates. However, in this latter model an FCW can be induced by mechanical stimulation or ATP. In addition, no MPD and increase in intracellular sodium or ENaC expression can be observed until at least 20 hours after the removal. Correspondingly, no elements of the healing response (e.g., lamellar protrusions, cell migration) can be appreciated until this time. Stimulation with either of the considered factors (ATP, cell lysates or mechanical stimuli) immediately after strip removal did not introduce any modification to this dynamics.
In BCE cells in culture, none of the potential stimuli of the healing response considered in this work (interruption of cell contacts, ATP, cell lysates and mechanical stimuli) is by itself capable of eliciting a response similar to the one produced by mechanical wounding.
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