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J. Zhao, T. Nagasaki; Characterization of a Cytotoxic Activity in the Mouse Tears . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2505.
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Purpose: We showed previously that the mouse tears were cytotoxic to the corneal stromal cells. This study was carried out to characterize this activity to help identify its molecular nature. Methods: Cytotoxicity of the mouse tears was determined ex vivo by using corneal stromal and endothelial cells as target cells. To test the stromal cell cytotoxicity, central corneal epithelium was gently removed in a freshly sacrificed mouse, and test samples were placed on the denuded stroma after enucleation, followed by an incubation at 37°C for 1-2 hours. To test the endothelial cell cytotoxicity, a corneal cup together with about 1 mm of sclera was dissected and placed with the epithelium side down in a round bottom container, and test samples were dropped on top of the endothelium, followed by a 30-min incubation at 37°C. Cell death was determined by either penetration of ethidium homodimer-1 into cells in a live tissue or DAPI nuclear staining in fixed cells. High power images of cell nuclei indicated different stages of apoptosis progression as determined by their morphology. Results: Under ex vivo assay conditions, the mouse tears were found to be cytotoxic to not only the corneal stromal cells but also to the corneal endothelial cells at similar concentrations. The mode of endothelial cell death appeared to be apoptosis as judged by gradual condensation and segmentation of the nuclei, followed by their disappearance. The corneal epithelial cells, on the other hand, were not affected by the tears. Stromal cell death was detected when the tears were applied to the bare stroma for as short as 3 minutes followed by a rinse and a 2-hour incubation. The tears failed to trigger apoptotic reactions when an incubation was carried out at 4°C instead of 37°C. Both the stromal and the endothelial cytotoxicity were heat sensitive and partially blocked by aprotinin or leupeptin, suggesting an involvement of serine protease in the expression of cytotoxicity. Some commercially available proteases, including bovine trypsin, porcine pancreatic kallikrein, human plasmin, human tissue plasminogen activator and human urokinase, triggered a varying degree of apoptotic and other damages to both stromal and endothelial cells, but their relationship to the tear cytotoxicity is not clear because none exactly duplicated the effect of the mouse tears. Lipopolysaccharides from Salmonella minnesota and egg white lysozyme were ineffective in cell killing at physiological concentrations. Conclusions: The cytotoxic activity in the mouse tears is effective toward corneal stromal cells and endothelial cells but not to corneal epithelial cells. A proteolytic action seems to be involved in the expression of the tear cytotoxicity.
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