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A.E. Hutcheon, K.C. Sippel, J.D. Zieske; Restoration of Barrier Function in the Cornea . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3824.
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Purpose: Previously we have studied the wound healing process by examining proliferation, growth factors and transcription factors in both the corneal epithelium and stroma; these studies have indicated that wound healing in the cornea is a complex process with multiple interactions. Our current study examines the possibility of a correlation between the restoration of barrier function and basement membrane reassembly in the cornea after wounding. Methods: Adult Sprague-Dawley rats were anesthetized and a 3mm superficial keratectomy was performed. The eyes were allowed to heal from 4 hours to 8 weeks. Four rats for each time point had the right eye wounded, and the contralateral eye served as a control. At the time of sacrifice, EZ-Link Sulfo-NHS-LC-Biotin (Pierce; Rockford, IL) was applied to all eyes, experimental and control, for 15 minutes. This compound does not penetrate through intact tight junctions. After 15 minutes, the eyes were rinsed with 1x PBS, blotted, enucleated and frozen in OCT on dry ice. Six µm sections were cut and indirect immunofluorescence was performed with anti-laminin (Dako; Carpenteria, CA), a marker of basement membrane, and anti-streptavidin (Jackson ImmunoResearch; West Grove, PA), a marker of biotin. The tissue was rehydrated, blocked and incubated for 1 hour at room temperature with anti-laminin. After an hour, the tissue was rinsed, blocked and incubated with anti-rabbit IgG and streptavidin-TRITC for another hour at room temperature. Tissues were then rinsed and coverslipped with Vectashield mounting media containing DAPI (Vector Labs; Burlingame, CA), a marker of nuclei. Results: Upon wounding, biotin appears to penetrate into the stroma subjacent and slightly peripheral to the wound area. This pattern is present from 4-32 hours post-wounding; however, as the cornea heals, the basement membrane reassembles, as shown by laminin localization. The area of biotin localization appears to decrease at these time points. By 48 hours, the basement membrane has fully reassembled throughout the entire wound area and no biotin is present in the stroma. Conclusions: Reformation of the basement membrane appears to correlate with the restoration of the barrier function in the cornea.
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