Detection of corneal fibrosis by imaging second harmonic-generated signals in rabbit corneas treated with mitomycin C after excimer laser surface ablation.
OBJECTIVE
Recent studies have shown that confocal imaging of second harmonic-generated (SHG) signals can detect corneal collagen organization. The purpose of this study was to assess whether SHG signals can detect differences in corneal fibrosis after excimer laser surface ablation (photorefractive keratectomy [PRK]).


METHODS
Rabbits received 9-D PRK in one eye followed by treatment with either mitomycin C (MMC) or vehicle. Corneal haze was measured by in vivo confocal microscopy before and 2, 4, 8, and 12 weeks after surgery. Animals were then killed and corneas were evaluated by visible and nonlinear confocal microscopy.


RESULTS
PRK induced significant haze in vehicle-treated corneas that peaked at 2 weeks and remained elevated at 12 weeks after surgery. MMC treatment significantly (P < 0.05) reduced corneal haze at 2 weeks and was essentially normal by 12 weeks. Imaging of SHG signals in 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 normal orthogonally arranged collagen lamellae. MMC treatment showed normal collagen organization at the surface. Fibrotic tissue was associated with a high cell density and alignment of intracellular actin filaments with collagen fiber bundles. In MMC-treated eyes, an anterior acellular zone overlaid a sparsely populated stroma containing isolated and enlarged keratocytes.


CONCLUSIONS
Imaging of SHG signals provides a sensitive means for detection of corneal fibrosis after surface ablation and can be used to assess the effects of antifibrotic therapy on corneal healing after refractive surgery.
Farid
Marjan
M
Gavin S Herbert Eye Institute, University of California, Irvine Medical Center, Orange, California 92868, USA.
Morishige
Naoyuki
N
Lam
Larry
L
Wahlert
Andrew
A
Steinert
Roger F
RF
Jester
James V
JV
D000477:Alkylating Agents / Q000627:therapeutic use*; D000818:Animals; D003131:Combined Modality Therapy; D003315:Cornea / Q000378:metabolism / Q000473:pathology* / Q000601:surgery; D003318:Corneal Opacity / Q000175:diagnosis* / Q000378:metabolism / Q000628:therapy; D024022:Fibrillar Collagens / Q000378:metabolism; D005355:Fibrosis / Q000175:diagnosis / Q000378:metabolism; D054018:Lasers, Excimer; D018613:Microscopy, Confocal; D016685:Mitomycin / Q000627:therapeutic use*; D010590:Phalloidine / Q000031:analogs & derivatives; D018903:Photorefractive Keratectomy; D011817:Rabbits
D000477:Alkylating Agents; D024022:Fibrillar Collagens; C037191:fluorescein isothiocyanate-phalloidin; D010590:Phalloidine; D016685:Mitomycin