title: Confocal microscopic characterization of wound repair after photorefractive keratectomy.
abstract: OBJECTIVE
  Development of postoperative corneal haze and regression of refractive effect are unfavorable clinical complications of excimer laser photorefractive keratectomy (PRK). Although exact mechanisms remain to be elucidated, these outcomes have been attributed to post-PRK corneal wound healing. The purpose of this study was to evaluate corneal wound repair quantitatively after PRK in a rabbit model using a newly developed in vivo technique, termed confocal microscopy through focusing (CMTF).
  
  
  METHODS
  Twelve rabbit corneas received a monocular, 6-mm diameter, 9.0-diopter PRK myopic correction. Animals were evaluated sequentially up to 6 months after surgery by in vivo CMTF, which uses an image-intensity depth profile to measure epithelial and stromal thickness and uses corneal light reflectivity as an objective estimate of corneal haze. At differing temporal intervals, in vivo morphology was correlated with ex vivo histology using fluorescence microscopy.
  
  
  RESULTS
  One week after PRK, an acellular layer of 86 +/- 24 microns was found anteriorly in the remaining stroma, which demonstrated surgically induced keratocyte death. Underlying keratocytes became activated and migrated toward the wound bed; repopulation was completed within 3 weeks. One week after PRK, there was a significant increase (P < 0.001) in light reflections detected from the photoablated stromal surface (1745 +/- 262 U) and from the underlying activated fibroblasts (713 +/- 607 U). Corneal reflectivity peaked at 3 weeks (4648 +/- 1263 U) and decreased linearly to 889 +/- 700 U by 6 months after the PRK; this corresponded to a reflectivity six times greater than the level seen in unoperated corneas. Two weeks after PRK, initial corneal edema had resolved, revealing an actual ablation depth (maximal stromal thinning) of 118 +/- 8 microns. Starting at 2 weeks after surgery, the stroma underwent gradual rethickening that reached 98% of the preoperative thickness at 6 months after PRK; at that time, only 6% of the initial photoablation depth persisted. By contrast, the central corneal epithelium showed no significant postoperative hyperplasia.
  
  
  CONCLUSIONS
  Rabbit corneas treated by PRK showed a remarkable stromal wound-healing response that ultimately led to the restoration of the original stromal thickness by 6 months after surgery, demonstrating complete regression of the initial photoablative effect. Additionally, corneal wound healing was associated with increased light reflections from both the photoablated stromal surface and the activated wound-healing keratocytes underlying this area. Based on these findings, the authors hypothesize that the development of clinically observed corneal haze in PRK patients may be related, in part, to activation of corneal keratocytes and to putative changes in the extracellular matrix.
authors.affiliation: Department of Ophthalmology, Aarhus University Hospital, Denmark.
authors.forename: T
authors.initials: T
authors.lastname: Møller-Pedersen
authors.forename: H F
authors.initials: HF
authors.lastname: Li
authors.forename: W M
authors.initials: WM
authors.lastname: Petroll
authors.forename: H D
authors.initials: HD
authors.lastname: Cavanagh
authors.forename: J V
authors.initials: JV
authors.lastname: Jester
mesh_terms: D000818:Animals; D002455:Cell Division; D002465:Cell Movement; D003315:Cornea / Q000166:cytology* / Q000502:physiology / Q000601:surgery; D003318:Corneal Opacity / Q000473:pathology / Q000503:physiopathology; D003319:Corneal Stroma / Q000166:cytology / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D007091:Image Processing, Computer-Assisted; D054018:Lasers, Excimer; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009216:Myopia / Q000601:surgery; D018903:Photorefractive Keratectomy; D011817:Rabbits; D014945:Wound Healing