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C.-Y. Liu, H. Liu, J. Zhang, Y. Hayashi, W. W. Y. Kao; Role of Keratocan-Expressing Cells in Corneal Homeostasis. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4826. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Keratocytes, consisting of the major cell population of vertebrate corneal stroma, play a pivotal role in corneal morphogenesis during development and homeostasis in adult. We examined the hypothesis that keratocytes are essential for the maintenance of cornea avascularity.in adult mice by ablation of keratocytes in adult Kera-Cre/DTR (diphtheria toxin receptor) bitransgenic mice in which the keratocytes express DTR and become susceptible to DT (diphtheria toxin).
A novel CAG-floxed STOP-DTRdsRed (STOP-DTRdsRed) transgenic mouse line was prepared by microinjection of a transgene containing "floxed-STOP" preceding-DTR-coral-derived red fluorescent protein (Invitrogen, Inc) fusion minigene under the control of the pCAG promoter,CMV enhancer/chicken beta actin promoter. The DTRdsRed transgenic mice were crossedwith Kera-Cre driver mice to generate Kera-Cre/STOP-DTRdsRed double-transgenic mice which express DTRdsRed fusion protein within the keratocan-expressing cells. Administration of DT via intrastromal injection caused keratocyte cell death in corneas of Kera-Cre/STOP-DTRdsRed double-transgenic mice. Contralateral eyes were injected with PBS. Confocal microscopy, immunohistochemistry, and biochemical analyses were used to examine the phenotypic changes in corneas after DT injection.
Confocal micrographs of the flat mounted corneas showed that the punctate pattern of red fluorescnce appeared in the stroma but not in epithelium or endothelium, suggesting that the DTRdsRed fusion protein was expressed on the cell surface of keratocytes with Cre recombinase activity. Keratocyte nuclei become fragmented within 24 hr after DT administration (1 pg) via corneal intrastromal injection. DT-injected corneas exhibit many apoptotic cells in keratocytes without inflammation. As a result, the cornea exhibits ulceration and neovascularization within two weeks of DT administration.
Kera-Cre/STOP-DTRdsRed double-transgenic mice may be a valuable tool to study keratocyte regeneration after corneal stromal injury. Our results suggest that keratocytes are essential in the maintenance of corneal homeostasis and avascularity.
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