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E. R. Ritchey, A. J. Fischer; Immunohistochemical Markers After Corneal Insult in the White Leghorn Chicken. Invest. Ophthalmol. Vis. Sci. 2007;48(13):794.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the changes in the expression of multiple immunohistochemical markers in the corneas of White Leghorn Chicken (Gallus gallus domesticus) in response to acute corneal damage.
Postnatal day 7 white leghorn chickens were anesthetized with inhaled 2.0% isofluorane + 1.5 L/min2 O2 and 1 drop 0.5% topical proparacaine ophthalmic solution. A 4mm barraquer lid speculum was positioned under the superior and inferior eyelid. Central corneal incisions were made into the corneal stroma of one eye with a #23 scalpel blade. To identify proliferating cells, 2 micrograms of BrdU, diluted in 20 microliters of saline, were injected into the vitreous chamber at different times after the corneal incisions. Treated and control corneas were harvested at 1 day, 4 days, 7 days and 14 days after corneal incision was performed. Corneas were fixed in 4% PFA, cryoprotected in 30% sucrose in phosphate buffered saline and soaked in embedding medium. Twelve micron thick transverse corneal sections were cut perpendicular to the corneal incision. Immunohistochemical labeling was performed for various markers including BrdU, vinculin, caspase 3, vimentin, perlecan, and fragmented DNA.
Up-regulation of immunohistochemical markers is observed after corneal. The time course of up-regulation differs for each marker. TUNEL-positive cells are abundant the day after corneal insult, but are absent at 4, 7 and 14 days after insult. By comparison, levels of vinculin and vimentin are up-regulated by day 4 after the insult. Up-regulation of perlecan is seen at day 7. In addition, we find BrdU-labeled cells not only in the epithelium, but also in the stroma near the site of the injury.
In control corneas, BrdU positive cells are seen primarily in the corneal epithelium, perlecan expression is seen in the corneal endothelium, vimentin is seen in the corneal stroma and vimentin is expressed in the endothelium and stromal keratocytes. After incision, we see an accumulation of BrdU positive cells in the region. We find that incisions to the chicken cornea results in the accumulation of TUNEL (but not cleaved caspase3) positive cells, suggesting necrosis rather than apoptosis. In response to acute damage, stromal keratocytes rapidly up-regulate vimentin. Subsequently, the keratocytes proliferate, accumulate in the area near the incision, and then produce elevated levels of vinculin and perlecan. We propose that the chicken cornea is a good model to study acute damage and scarring.
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