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M. Farid, N. Morishige, A. Wahlert, R. F. Steinert, J. V. Jester; Detection of Corneal Fibrosis Following Excimer Laser Surface Ablation (PRK) Using Second Harmonic Generated (SHG) Signals. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4021.
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Recent studies have shown that confocal imaging of SHG signals can detect corneal collagen organization. The purpose of this study was to assess whether SHG can detect differences in corneal fibrosis induced by excimer surface ablation treated with and without mitomycin C (MMC).
Ten rabbits received 9 diopter PRK in one eye followed by treatment with either MMC (0.02% for 10 seconds) or vehicle (5 rabbits each). Corneal haze was measured by in vivo confocal microscopy pre-, 2, 4, 8 and 12 weeks post-operatively. Animals were then sacrificed, corneas fixed and evaluated by imaging SHG signals from collagen using a ti:sapphire femtosecond laser. Collagen organization was also correlated with cell density by en bloc staining for nuclei and cell cytoskeleton using Syto-59 and phalloidin.
PRK induced significant haze in vehicle treated corneas that peaked at 2 weeks (4,511 + UAUC) and remained elevated at 12 weeks post surgery (1,470 + 737 UAUC). MMC treatment significantly (p < 0.05) reduced corneal haze at 2 weeks (1,759 + 801 UAUC) and was essentially normal by 12 weeks (215 + 61 UAUC). In normal eyes imaging of SHG signals detected wide bundles of collagen fibers organized into orthogonally arranged lamellae. In contrast, vehicle treated eyes showed an anterior layer of collagen forming a honeycomb network blending into a dense mat of irregularly arranged collagen fibers that overlaid the deeper, stromal lamellae. MMC treatment showed normal stromal lamellae at the surface. Fibrosis in the vehicle treated eyes was associated with a higher cell density compared to normal and alignment of intracellular actin filaments with collagen fiber bundles. In MMC treated eyes, there was an anterior acellular zone that overlaid a sparsely populated stroma containing isolated and enlarged keratocytes.
Imaging of SHG signals provides a sensitive means for detection of corneal fibrosis following surface ablation and can be used to assess the effects of anti-fibrotic therapy on corneal healing post refractive surgery.
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