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Asadolah Movahedan, Neda Afshar, Hossein Sagha, Robert Lavker, Ali Djalilian; Conditional Deletion of Notch1 in Mouse Corneal Epithelium Leads to the Loss of the Epithelial Barrier Function. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2564.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the mechanisms that lead to keratinization after conditional deletion of Notch1 in the corneal epithelium.
Notch1 was conditionally deleted in 3 month old Notch1 flox/flox, K14-Cre-ERT +/- mice using intra-peritoneal injection of 1 mg/day 4-hydroxy-tamoxifen(4-OHT) for 5 days. Eyes were serially monitored after 4-OHT treatment by slit lamp examination and fluorescein staining. Aqueous tear measurement was performed using phenol red thread test. Barrier function was also assessed by the penetration of sulfo-NHS-LC-Biotin. Immuno-fluorescence staining for ZO-1 was used to assess tight junctions in corneal epithelial cells cultures isolated from conditional Notch1-/- and wildtype mice.
Slit lamp examination after tamoxifen injection indicated progressive increase in corneal fluorescein staining before the onset of corneal opacification and keratinization. Sulfo-NHS-LC-Biotin penetrated into the corneas confirming the barrier impairment. The mean aqueous measurement for Notch1 KO group with evidence of corneal keratinization was significantly higher than wildtype mice (7.45 ± 2.3 mm vs 3.66 ± 1.4 mm) (p= 0.002) indicating increased lacrimal production. Histologically, progressive loss of meibomian glands was observed in the Notch1 KO mice (as described by Vauclair et al before), however this did not correlate with the epithelial barrier changes. There was no apparent difference in the goblet cells between conditional Notch1 KO and wildtype mice. In a calcium switch experiment in vitro Notch1 -/- cells demonstrated delayed membrane localization of ZO-1 compared to wildtype cells.
Conditional deletion of Notch1 in corneal epithelium leads to an impairment in the epithelial barrier in part due to impaired tight junction formation. These findings highlight the role of Notch1 in epithelial differentiation and suggests that intrinsic defects in the epithelial barrier may be a contributing factor to the development of squamous metaplasia and inflammatory keratinization in these mice.
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