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N. Morishige, N. Yamada, S.-I. Teranishi, T.-I. Chikama, T. Nishida, J. Takahara; Subepithelial Fibrosis Detected by Second Harmonic Generation Imaging Microscopy in Corneal Stromal Edema. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4527.
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Corneas with or without stromal edema were examined by second harmonic generation (SHG) imaging microscopy to characterize stromal collagen organization.
Tissue buttons from 31 corneas with stromal edema and eight normal corneas were fixed, and 3-mm-square blocks were cut and stained with phalloidin to visualize the cytoskeleton. The blocks were examined by SHG imaging with a laser confocal microscope and mode-locked titanium:sapphire femtosecond laser. Samples were scanned to a depth of 150 µm from the surface of Bowman’s layer, and SHG forward and backscatter signals were collected. Phalloidin staining was detected by conventional laser confocal microscopy. The three-dimensional (3D) structure of the anterior segment of the cornea was reconstructed from stacked SHG images.
The SHG forward scatter signal, which was generated by collagen lamellae of the stroma, appeared normal in corneas with stromal edema. The number of areas with a weak SHG backscatter signal, which was nonspecifically generated by collagen, was decreased in tissue with edema. 3D reconstruction showed adherence of interwoven collagen lamellae in the anterior stroma to the amorphous SHG structure of Bowman’s layer in both normal and edematous corneas. Irregular SHG signals at the level of Bowman’s layer were observed in corneas with stromal edema; 3D images revealed that these signals were actually localized above Bowman’s layer and were indicative of subepithelial fibrosis. Phalloidin staining showed transdifferentiation of stromal cells into fibroblastic cells in corneas with edema. The incidence of subepithelial fibrosis or of fibroblastic cells was increased beginning 12 months after the onset of clinical stromal edema.
SHG imaging of the anterior segment of corneas with stromal edema revealed a normal appearance of interwoven collagen lamellae in the anterior stroma as well as the development of subepithelial fibrosis at 12 months after the onset of edema, suggesting that stromal edema is a progressive disease.
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