Corneal stroma contains many highly organized collagen fibrils,
16,17 which increase in the fibrotic process, and collagen is a well-established marker of fibrosis.
18 Here, we stained the collagen in corneal stroma using picrosirius red. A complication can arise because muscle, in addition to collagen fibers, can be stained with picrosirius red, so we used a polarization method where only collagen is detected.
19 Using nonpolarized light (
Fig. 5A), an increase in red staining in the cornea after alkali burn in both WT (II) and Nox4 knockout (IV) groups was observed, but such staining was clearly reduced in the absence of Nox4 (IV) when compared with WT (II). Switching to a polarized filter (
Fig. 5B), an increase of collagen fibers was observed after NaOH treatment in WT and Nox4 KO corneas (II, IV, respectively) compared with untreated corneas in each group (I, III, respectively). Again, there were less collagen fibers after NaOH in the Nox4 KO group (IV) compared to the WT-treated group (II). Total collagen content was significantly reduced in Nox4 KO cornea after alkali burn injury compared to the WT (
Fig. 5C). These results indicate that lack of Nox4 reduces collagen synthesis after alkali burn injury.