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S. Yeh, R.M. Corrales, C.S. de Paiva, W.J. Farley, D.Q. Li, M.E. Stern, S.C. Pflugfelder; Apoptosis of Ocular Surface Cells in Experimental Dry Eye: Comparison of C57B6 and Balb–c Mouse Strains . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4434.
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Purpose: To compare the level of ocular surface apoptosis in C57B6 and Balb–c mouse strains at 5 and 12 days in an experimental model for dry eye. Methods: Aqueous tear production and clearance were inhibited by subcutaneous scopolamine injection and exposure to an air draft for 5 and 12 days in 4–6 week old C57B6 and Balb–c mice. Eyes and ocular adnexa were excised, cryosectioned, and evaluated for apoptosis by terminal deoxynucleotidyl transferase–mediated dUTP–digoxigenin nick end labeling (TUNEL) assay and immunohistochemical assay for activated caspase–3, an effector in the apoptosis cascade. Apoptotic cells in different regions of the ocular tissue were quantitated with epifluorescence microscopy. Hoescht 33342 staining was used to examine nuclear morphology of apoptotic cells. Results: After 5 days of dry eye induction, apoptosis was observed in the central corneal epithelium of C57B6 mice (mean 19.7 cells, SD 7.3, n=3) and the Balb–c mice (mean 28 cells, SD 8.2, n=3). At day 12, apoptosis of central corneal epithelial cells in C57B6 mice (mean 20.3, SD 1.15, n=3) was not significantly different from the day 5 levels. In Balb–c mice, the level of apoptotic corneal epithelial cells at day 12 (mean 11.7, SD 7.4, n=3) was decreased from day 5 (p < 0.08). Following 5 days of dry eye induction, apoptosis was also observed in the bulbar conjunctival epithelium of C57B6 (mean 19.7 cells, SD 6.4, n=3) and Balb–c mice (mean 36.7 cells, SD 9.5, n=3). At day 12, apoptosis of bulbar conjunctival epithelial cells in C57B6 mice (mean 28, SD 9.8, n=3) was not significantly different from day 5. Apoptosis levels in Balb–c mice at day 12 (mean 22.7, SD 4.5, n=3) were decreased from day 5 (p < 0.06). Immunohistochemical assay for caspase–3 revealed increased immunoreactivity in regions of increased apoptosis. Characteristic nuclear morphologic changes were observed in these areas with Hoescht 33342 staining. No significant differences were observed between dry eye and control groups in the corneal stroma, bulbar conjunctival stroma, or lid margin. Conclusions: Initially, both C57B6 and Balb–c mice demonstrated ocular surface apoptosis in the central cornea and bulbar conjunctiva after 5 days of dry eye induction. The Balb–c mice appeared to recover from apoptosis in both the central corneal and bulbar conjunctival epithelium at 12 days, while ocular surface apoptosis persisted in C57B6 mice. Strain differences in ocular surface apoptosis may exist between mice. Apoptosis may play a key role in the pathogenesis of dry eye and may prove to be a therapeutic target.
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