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L. Bellner, K. A. Patil, A. Halilovic, K. Castellano, G. Cullaro, M. W. Dunn, M. L. Schwartzman; Targeted Suppression of Heme Oxygenase (HO)-2 Gene Expression Impairs the Innate Anti-Inflammatory and Repair Responses of the Cornea to Injury. Invest. Ophthalmol. Vis. Sci. 2010;51(13):386.
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HO-2 is highly expressed in the corneal epithelium and is a component of the HO system that represents an intrinsic cytoprotective and anti-inflammatory system. We have shown that in HO-2 null mice epithelial injury leads to unresolved corneal inflammation and chronic inflammatory complications including ulceration, perforation and neovascularization. We explored whether a localized corneal suppression of HO-2 is sufficient for impairing the innate anti-inflammatory and repair capability of the cornea.
Silencing hairpin RNA (shRNA) was administered subconjunctivally (100 ng) as well as topically (100 ng) starting one day before corneal epithelial removal and once daily thereafter. The corneal epithelium was removed using an Alger Brush on anesthetized mice. Re-epithelialization was assessed by fluorescein staining. Inflammatory response was quantified by histology and MPO activity. Levels of mRNA were measured by RT-PCR.
The ability of the HO-2-specific shRNA to knockdown HO-2 RNA in vivo was confirmed by real-time PCR. Treatment of eyes with HO-2-specific shRNA delayed re-epithelialization when compared with the control shRNA treated group by 20, 30% and 40% at days 2, 4 and 7, respectively (p<0.05). Leukocyte infiltration decreased (p<0.05) in the HO-2 shRNA-treated group as compared to the control shRNA-treated group (30%, 50% and 25% at days 1, 2 and 3 after injury, respectively).
This study indicates that the impaired corneal wound healing in response to HO-2-specific shRNA was associated with a significant decrease in neutrophil infiltration bringing forth the notion that, to some degree, local influx is critical for healing. It is possible that corneal epithelial HO-2 plays an important role in attracting and perhaps regulating this critical wave of neutrophils to the cornea. The local knockdown of HO-2 impairs this ability while global knockout not only negates it but also alters the characteristics of the infiltrated cells. Additional studies are needed to explore the role of corneal HO-2 in regulating neutrophil infiltration and resolution.
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