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Lars Bellner, Michael W. Dunn, Michal L. Schwartzman; Heme Oxygenase (HO)-2 Deficiency Leads To The Development Of Epithelial Defects In The Injured Cornea. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1984.
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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 epithelial injury in HO-2 null mice leads to chronic inflammatory complications including ulceration and neovascularization (Seta et al, 2006) and further that knockdown of HO-2 in human corneal epithelial cells resulted in a significant reduction in healing rate following injury due to inhibition of migration (Halilovic et al 2010). We evaluate the effect of HO-2 deletion and biliverdin application on the development of corneal defects.
HO-2 null mice were treated with biliverdin (100 µl of 100 µM ip once daily; and topical (10 µl of 100 µM), or its vehicle (HBSS, pH 7.4) one hour prior to injury and t.i.d. thereafter. The corneal epithelium was removed using an Alger Brush in anesthetized mice. Re-epithelialization was assessed at days 2, 4 and 7 by fluorescein staining. Infiltrating neutrophils were detected through GR1-staining of histological sections. TUNEL and DHE staining was used to show apoptotic changes and oxidative stress, respectively. QPCR was used to detect the mRNA levels of NADPH oxidases 2 and 4, and MMP- 2 and -9.
After epithelial removal, non-penetrating ulcerations developed in less than 25% of injured eyes of WT mice, compared to 90% of injured eyes of HO-2 null mice. In HO-2 null mice the average area of the defect was 31.7%±4.9% and 32.3%±3.2% (n=8-10), at days 4 and 7 after injury, respectively. biliverdin-treatment significantly accelerated wound closure when compared with vehicle-treated group reducing the size of the ulceration, 17.5%±2.6% and 13.8%±3.3% (n=8-10), at days 4 and 7, respectively. Biliverdin-treatment also caused a drop in the number of inflammatory cells, lessened oxidative stress as shown by a 4-fold reduction of DHE intensity of histological slides at day 4 after injury, and NADPH-oxidases -2 and -4 mRNA levels were reduced by 50% at day 7. TUNEL staining showed a significant reduction in the number of dead cells per histological section.
The demonstration that the inflammatory responses, including the infiltration of inflammatory cells and the oxidative stress are exaggerated in HO-2 knockout mice, together with the development of epithelial defects strongly supports the notion that the HO system is critical for controlling the inflammatory and repair response. Biliverdin/bilirubin, potent intrinsic antioxidants and anti-inflammatory molecules, may offer a novel strategy to treat corneal conditions.
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