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JE Gatlin, MW Melkus, A Padgett, HD Cavanagh, WM Petroll, JV Garcia, JV Jester; Multiphoton Confocal Imaging of Corneal Wound Healing Fibroblasts Using Gene Transfer of Green Fluorescent Protein (gfp) . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1712.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Numerous studies indicate that corneal fibroblasts play an important role in wound healing, however the dynamic cellular events underlying wound tissue organization and contraction remain unclear. This study focused on the development and characterization of a wound healing model utilizing retroviral gene transfer to mark corneal fibroblasts in vivo to permit three-dimensional imaging and localization of live wound healing fibroblasts. Methods: Cultured rabbit corneal fibroblasts were transduced with an RD114 pseudotyped MLV vector expressing EGFP and gene transfer efficiency was determined by flow cytometry. Subsequently, following lamellar keratectomy, rabbit corneas were exposed to vector supernatants to evaluate fibroblast labeling in vivo. Corneas were excised and fresh tissue was analyzed using epifluorescence microscopy and multiphoton confocal microscopy up to four months following transduction. Results: Cultured primary rabbit corneal fibroblasts were transduced in vitro up to 49%. In vivo, corneal fibroblast transduction was achieved by both topical application and immersion in vector supernatants. At 7 to 14 days post-transduction, EGFP expressing cells were detected at the wound margins, appearing in clusters suggestive of individual expanding fibroblast clones. Fibroblasts appeared broad, with a flattened, dendritic morphology quite different from spindle shaped fibroblasts observed in culture. Three-dimensional imaging using multiphoton confocal microscopy demonstrated fibroblasts to be extensively interconnected and multi-layered within the corneal wound. Interestingly, marked fibroblasts were not detected at 4 months post-transduction. Conclusion: These findings demonstrate the feasibility of using gene transfer to introduce and express fluorescent reporter proteins into wound healing fibroblasts in vivo. Additionally, our findings indicate that in vivo corneal fibroblasts appear morphologically distinct from cultured fibroblasts and that they may have a defined life-span within the wound. Using this approach, expression of reporter fusion-proteins important in fibroblast function may facilitate the molecular study of fibroblast activation and function during wound healing.
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