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M Stepp, S Pal Ghosh, A Pajoohesh-Ganji; Dynamic Changes in 9 Integrin Localization during Reepithelialization in the Cornea Revealed using Whole Mounts . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4212.
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Purpose:The number of α9 positive epithelial cells at the limbal region around the periphery of the mouse eye varies with most of the positive cells being found in the nasal quadrant of tissue.Our goal here is to assess whether the up-regulation of the α9 integrin observed in response to wounding occurs uniformly throughout the entire corneal epitheliun or whether it is restricted to regions adjacent to those containing numerous α9 integrin positive cells Methods: Balb/c mice were used at 8 weeks of age for manual debridement wounds and animals sacrificed at different times after wounding. We assesed small (1.5mm) and large (2.5mm) wounds at 0, 6, 12, 18, 24, 48, and 72 hrs as well as at 1 and 2 wk after wounding. No fewer than 3 corneas were used for each time point studied. Corneal tissues were processed for whole mount immunofluorescence microscopy; tissues were stained with α9 integrin and nuclei were revealed using propidium iodide. After staining, tissues were cut at 4 peripheral locations so that they could be flat mounted onto black filters for viewing using confocal microscopy. Results:. In small wounds, α9 integrin begins to be upregulated as early as 12 hrs and is abundant within and between epithelial cells behind the leading edge of migration at 18 hr. However, expression is regionalized so that tissues adjacent to those having numerous α9 integrin positive cells in the nasal-most quadrant of the limbus express more α9 integrin during migration. Cells at the limbal region do not become upregulated for α9 integrin during reepithelialization of small wounds. At the leading edges, α9 integrin appears late during reepithelialization at sites where adjacent tissues merge immediately prior to closure. For the large wounds, we see abundant α9 integrin in migrating cells as early as 12 hr after wounding and expression is sustained. Conclusion:Contrary to our previous results obtained using frozen tissue sections, we observe increased α9 integrin localization during migration after both small and large corneal wounds. For molecules whose localization is not uniform around the corneal periphery, whole mount confocal microscopy provides a necessary tool for studies of differential protein localization. Research supported by RO1 EYO8512-12 (MAS).
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