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S.-W. M. Koh; A New Corneal Inflammation Model Using Human Donor Corneoscleral Explants and the Anti-inflammatory Effect of VIP Modulation of the Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1955.
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To demonstrate a new corneal inflammation model using human donor corneoscleral explants in organ cultures and the anti-inflammatory effect of vasoacive intestinal peptide (VIP) modulation of the corneal endothelium.
Paired human donor corneoscleral explants (either fresh or in preservation) were treated with either DPBS or H2O2 (1.4 mM)/DPBS (30 min; 37oC) and then placed in viewing chambers and incubated in 20 mM HEPES- buffered minimal essential medium (MEM) (24 h; 37oC) to induce sterile inflammation in corneas. To test the effect of VIP pretreatment of the corneal endothelium on corneal inflammation, prior to the H2O2 treatment either MEM/HEPES alone or that contained10 nM VIP was placed in the corneal cups of paired explants placed in viewing chambers and incubated for 20 min (37oC). The corneal cup-conditioned medium (300 µl) was collected and analyzed by ELISA for monocyte chemoattractant protein (MCP)-1 and transforming growth factor (TGF) β1.Cryosections were cut, blocked, and stained with either FITC- conjugated monoclonal anti-CD45 antibody or monoclonal anti-CD68 primary antibody, biotinylated secondary antibody, and FITC-conjugated extravidin, and examined under a fluorescence microscope. For immune cell number counting, photos (10) were taken at X200 magnification of each region of the stroma (anterior, middle, and posterior) of the central cornea. More, whole crneoscleral explants were quartered, stripped off the epithelium and the endothelium, immunostained with FITC-anti-CD45 monoclonal antibody, flat-mounted under a fluorescence microscope.
Human donor corneoscleral explants demonstrated an inflammatory response to H2O2 -induced cell injury. CD45/CD68-positive cells in the stroma of the inflamed corneas were more numerous than in the control paired corneas and appeared in the mid-stroma, which was normally devoid of immune cells. VIP pretreatment of the corneal endothelium in the corneal cups greatly reduced the numbers of immune cells (100% vs. 60%; p=0.003) in all three regions of the stroma, a result confirmed by flat-mounted corneoscleral explantst. ELISA also demonstrated the anti-inflammatory effect of VIP pretreatment of the corneal endothelium. While MCP-1 was down-regulated (100% vs 59%, p=0.03, N=5 pairs), the anti-inflammatory cytokine TGFß1 was up-regulated (100% vs 214%, p=0.076, N=5 pairs) by VIP-pretreatment.
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