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This study utilizes high resolution multi-dimensional imaging to identify temporal and spatial changes in cell/extracellular matrix (ECM) patterning mediating cell migration, fibrosis, remodeling and regeneration during wound healing. Photorefractive keratectomy (PRK) was performed on rabbits. In some cases, 5([4,6-dichlorotriazin-2yl]-amino)fluorescein (DTAF) was applied immediately after surgery to differentiate native vs. cell-secreted collagen. Corneas were assessed 3-180 days postoperatively using in vivo confocal microscopy, and cell/ECM patterning was evaluated in situ using multiphoton and second harmonic generation (SHG) imaging. 7 days post-PRK, migrating fibroblasts below the ablation site were co-aligned with the stromal lamellae. At day 21, randomly patterned myofibroblasts developed on top of the ablation site; whereas cells underneath were elongated, co-aligned with collagen, and lacked stress fibers. Over time, fibrotic tissue was remodeled into more transparent stromal lamellae. By day 180, stromal thickness was almost completely restored. Stromal regrowth occurred primarily below the ablation interface, and was characterized by co-localization of gaps in DTAF labeling with elongated cells and SHG collagen signaling. Punctate F-actin labeling was detected along cells co-aligned with DTAF and non-DTAF labeled collagen, suggesting cell-ECM interactions. Overall, collagen lamellae appear to provide a template for fibroblast patterning during wound healing that mediates stromal repopulation, regeneration and remodeling.

Purpose: To evaluate the efficacy of the agent RGTA for epithelial, stromal and nerve regeneration after laser-induced corneal wounding in rabbits., Methods: After excimer laser wounding of the anterior cornea in 25 New Zealand white rabbits, topical RGTA or placebo was applied in a randomized, masked manner. Fluorescein epithelial staining was performed on the first 5 postoperative days. In vivo confocal microscopy of corneal subbasal nerves and stroma was performed preoperatively and on week 2, 4, 8 and 16. At 16 weeks, corneas were stained for beta-III tubulin expression., Results: Postoperatively, all epithelia had closed by at least 90% after the third postoperative day. No significant difference in epithelial wound size was found between RGTA and placebo-treated groups, and RGTA did not hinder fluorescein binding. After epithelial wound closure, corneas remained transparent to 16 weeks. By confocal microscopy, subclinical stromal haze was significantly deeper in placebo-treated eyes (mean depth 60 μm) relative to RGTA group (52 μm), p = 0.02. Regenerating beta-III tubulin-positive subbasal nerves were observed in all corneas, but partial masking by haze rendered quantitative analysis unreliable., Conclusions: RGTA restored stromal microarchitecture and reduced subclinical haze relative to placebo. The mild epithelial wound quickly healed regardless of treatment suggesting an optimal natural healing process in freshly wounded healthy corneas, and indicating that RGTA may be more suitable for healing of chronic or more aggressive wounds. Limitations of the rabbit model for nerve quantification in the presence of haze should also be recognized., (© 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Purpose: To study the histopathology of the remodelling process in the stroma after excimer-laser-induced corneal irregular injuries., Methods: Seven New Zealand white rabbits received in one eye a transepithelial plano photoablation (60 microm) and an additional plano ablation (25 microm). On the denuded stroma, an electron microscopy specimen grid was placed and another 25 microm ablation was applied to produce surface irregularities. Dichlorotriazinyl aminofluorescein (DTAF) was then applied for 45 seconds. Another seven right eyes of seven rabbits were ablated the same way but without using the grid, resulting in a plano ablation. All the rabbits were killed at weekly intervals after treatment. The harvested corneas from both eyes were further processed for haematoxylin-eosin staining and were also stained with monoclonal antibodies directed against Ki-67 antigen and alpha-smooth muscle actin (alpha-SMA). All specimens were examined under light and fluorescence microscope., Results: The corneal wounds were covered by epithelium during the first week. The 25 microm x 25 microm x 25 microm stromal irregularities were clearly discernible up to 3 weeks after treatment, during which time they melted and disappeared. A homogeneous zone was formed in which stroma cells laid down an initially disorganized stroma. This was sharply visible under a fluorescence microscope as a dark area between the dichlorotriazinyl aminofluorescein (DTAF) fluorescent stroma and autofluorescent epithelium. Very little response was seen in the plano-ablated wound microscopically and in terms of positive stained cells., Conclusion: As the irregularities are flattened and the homogenous zone becomes repopulated with keratocytes forming extracellular matrix material (ECM), the cornea regains its previous architecture in both groups. The irregular wound surface promotes wound-healing reactivity, a process that allows the cornea to compensate for the irregularities and heal to a functional state.

Excimer laser keratectomy is widely used to correct refractive errors. Several complications of excimer laser keratectomy are reported including corneal infection, regression, corneal haze formation, glare and halo. Most of the complications are closely related to the corneal stromal wound healing process. In order to perform the excimer laser keratectomy with minimum complications, we should understand the mechanism of the corneal stroma wound healing process. In addition, such knowledge will help us to regulate the corneal stromal wound healing process in the future. In the present article, we discuss the molecular mechanism of the corneal stromal wound healing process after excimer laser keratectomy and its regulation by anti-inflammatory agents.

Aims: To investigate if the amniotic membrane (AM) promotes epithelial migration while inhibiting stromal remodelling associated with corneal haze after excimer laser keratectomy., Methods: A wound 150 microm in depth and 6.0 mm in diameter was produced in 40 rabbits using an excimer laser. One eye was randomly chosen to be covered by the AM while the other eye served as a control. Epithelial wound healing was evaluated, together with any morphological changes of the anterior stroma connected with corneal haze. These morphological changes were histopathologically analysed using dichlortriazinyl aminofluorescein (DTAF), Masson trichrome staining, and an image analyser., Results: The AM group had a short latent phase followed by fast epithelial healing (p<0.001) during the early wound healing period and a significant decrease in the inflammatory response, together with a smaller change in the number of keratocytes than the control group. The mean thickness of the regenerated stroma was significantly thinner in the AM group than in the control group at 8 weeks (p<0.0001). The AM group had a more regular architecture of regenerated stromal lamella at 8 weeks and significantly less haze after 4 weeks than the control group (p<0.05)., Conclusion: Use of the AM as a dressing on a corneal wound created by excimer laser surgery, in which severe haze is expected, may induce rapid epithelial healing with less inflammatory response. The AM may inhibit the irregular synthesis of stromal collagen that is associated with corneal haze.

Purpose: We investigated the keratocyte response and stromal remodeling after corneal incision and photorefractive keratectomy to understand the histophysiological and immunohistochemical differences between these two types of surgery., Methods: Corneal incision or photorefractive keratectomy was performed in rabbits or rats. Then we chronologically observed the histological changes and the changes in the localization of extracellular matrix proteins., Results: In both types of surgery, the keratocyte population at the damaged stroma became sparse, and the cells began undergoing apoptosis immediately after the surgical procedure. Subsequently, activated keratocytes migrated into the acellular zone, and the cells formed multiple layers at the resurfaced subepithelial regions. We observed deposition of amorphous substances between keratocytes that had migrated, and stromal remodeling appeared to start. Three months after surgery, the corneal structure had recovered to a near-normal condition at the corneal incision. After photorefractive keratectomy, however, extracellular matrix proteins were strongly immunoreactive at the subepithelial regions., Conclusions: These results suggest that the stromal wound-healing processes are similar after corneal incision and photorefractive keratectomy. A corneal incision may induce a transient keratocyte response during stromal remodeling, but photorefractive keratectomy may induce a sustained keratocyte response.

Purpose: To evaluate and compare the corneal wound-healing process after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK)., Setting: Kangnam St. Mary's Hospital, Seoul, Korea., Methods: Two surgical procedures, PRK with the VISX Star excimer laser and LASIK with a MicroTech microkeratome, were performed in 24 rabbit eyes. In the PRK group (n = 12 eyes), the rabbit cornea was treated with a 20 microns ablation. In the LASIK group (n = 12 eyes), a 20 microns laser ablation was performed after a 150 microns thick hinged corneal flap had been made. During both procedures, dichlorotriazinyl aminofluorescien (DTAF) dye was applied to the ablated stromal bed; in the LASIK group, the stromal side of the corneal flap was also stained with DTAF to differentiate regenerated collagen from normal stromal tissue. Corneal wound healing was evaluated postoperatively at 1, 4, 8, and 12 weeks using light, electron, and fluorescence microscopy. The amount of regenerated stromal tissue and the number of keratocytes were analyzed by an image-analysis system., Results: In the PRK group, epithelial migration and regeneration were observed in the ablated area without any stromal regeneration 1 week postoperatively. However, newly regenerated, irregularly arranged stromal collagen, with epithelial hyperplasia in the ablated area, was observed 4 to 12 weeks postoperatively by light and fluorescence microscopy. The number of keratocytes in the surgical area was also increased. In ultrastructural observation using an electron microscope, the shape of keratocytes in the ablated area was changed, and the number of rough and smooth endoplasmic reticuli, ribosomes, mitochondria, and electron-dense vesicles in the cytoplasm were increased, suggesting that the cells were activated. In the LASIK group, there was no observed regenerated collagen between the corneal flap and the ablated stromal bed except in the wound margin. Lamellated, parallel collagen fibers in the cornealstroma were not disturbed. However, in the wound margin, corneal epithelial ingrowth between the flap and the stromal bed was observed, as was some regenerated stromal tissue. The amount of regenerated stromal tissue and the number of keratocytes in the wound area were statistically smaller than those in the PRK group (P < .05). Observation by electron microscopy showed no activated keratocytes, unlike in the PRK group. The collagen fibers in the wound area were parallel., Conclusion: Stromal wound healing in the LASIK group was minimal compared with that in the PRK group, except in the wound margin. These results may support the clinical findings of less corneal haze in the human cornea after LASIK.

Background: Transforming growth factor beta (TGF-beta) regulates extracellular matrix deposition, cell proliferation, and migration, and is expressed in cornea. TGF-beta is thought to be involved in the corneal wound healing process., Methods: The central corneal area (3 mm in diameter) of Lewis rats was ablated using PTK mode excimer laser and the wound healing process was observed at 12 and 24 h and 2, 5, 10, and 30 days after treatment. The expression of TGF-beta 1, -beta 2 and -beta 3, TGF-beta type I and type II receptors, alpha 3, alpha 5, beta 4 integrin subunits, laminin and fibronectin was studied immunohistochemically. Antibody neutralizing TGF-beta 1, -beta 2 and -beta 3 was administered intraperitoneally, 50 micrograms daily, for 5 days after the laser treatment to investigate the effects of TGF-beta function blockade., Results: At the leading edge of the regenerating epithelium, no TGF-beta type I and type II receptors and beta 4 integrin subunits were expressed after 24 h. Regenerating epithelium covered the ablated area after 2 days. An abnormal fibrotic layer was formed in the subepithelial area. This layer contained round-shaped cells in the stroma in the early stage (2-5 days after laser ablation) and spindle-shaped fibroblast-like keratocytes after 10 days. Laminin and fibronectin expression increased in the fibrotic layer. The increased stromal cells expressed TGF-beta isoforms and TGF-beta receptors. Neutralizing TGF-beta inhibited the stromal cell increase in the laser ablated area after 5 days., Conclusion: TGF-beta may be involved in epithelial cell migration and stromal cell reaction during the corneal wound healing process after excimer laser ablation in rat models.

Purpose: 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.

Background: Variability of outcome following refractive surgical procedures is affected by corneal wound healing. Interactions between the corneal epithelium and stromal keratocytes affect both stromal remodeling and healing of the epithelium. These processes contribute to regression of initial effect, surface irregularity, and stromal scarring that occur following excimer laser photorefractive keratectomy (PRK). I review recent discoveries related to stromal-epithelial molecular interactions that provide insights into the cellular responses to refractive surgical procedures., Results: Injury to the corneal epithelium stimulates programmed cell death (apoptosis) of the underlying anterior stromal keratocytes. I hypothesize that apoptosis of the keratocytes occurring immediately after epithelial injury associated with refractive surgical procedures initiates the subsequent wound healing response. Activated keratocytes subsequently repopulate the anterior corneal stroma where they produce collagen and other components associated with stromal remodeling. In addition, secretion of hepatocyte growth factor and keratinocyte growth factor by keratocytes increases after corneal epithelial wounding and these growth factors stimulate proliferation and inhibit differentiation of epithelial cells, effects which could promote epithelial hyperplasia associated with regression after photorefractive keratectomy., Conclusion: Corneal stromal-epithelial interactions help explain the different results that occur following excimer laser photorefractive keratectomy and laser in situ keratomileusis.

Purpose: We studied the efficacy and safety of a recent technique of keratomileusis for myopia, excimer laser in situ keratomileusis., Methods: We studied retrospectively 88 eyes of 63 patients who received excimer laser in situ keratomileusis with the Chiron Automated Corneal Shaper and the Summit OmniMed laser under a hinged corneal flap without sutures., Results: Mean follow-up was 5.2 months. Mean spherical equivalent of the manifest refraction before surgery was -8.24 diopters (range, -2.00 to -20.00 diopters). Mean spherical equivalent refraction after surgery was +0.22 +/- 1.42 diopters. Of 40 eyes with a baseline refraction from -2.00 to -6.00 diopters, 25 eyes (63%) had refraction within +/- 0.50 diopter of emmetropia, and 37 eyes (93%) had refraction within +/- 1.00 diopter. In eyes with baseline refraction of -6.12 to -12.00 diopters, postoperative refraction was within +/- 1.00 diopter in 19 (65%) of 29 eyes. In eyes with baseline refraction of -12.10 to -20.00 diopters, postoperative refraction was +/- 1.00 diopter in eight (43%) of 19 eyes. Overall, 64 (72.8%) of 88 eyes had a refraction within +/- 1.00 diopter after surgery. Between three weeks and five months after surgery the change in the mean spherical equivalent refraction was -0.61 diopter in the myopic direction. Uncorrected visual acuity after surgery was 20/20 or better in 31 eyes (36%) and 20/40 or better in 61 eyes (71%). Three eyes (3.6%) lost two lines or more of spectacle-corrected visual acuity, two from progressive myopic maculopathy and one from irregular astigmatism. No eyes had vision-threatening complications., Conclusion: Excimer laser in situ keratomileusis under a corneal flap can be an effective method of reducing myopia between -2.00 and -20.00 diopters, with minimal complications. Current surgical algorithms need modification to improve predictability of outcome. Stability of refraction after surgery requires further study.

The development of corneal haze in some patients treated with excimer laser photorefractive keratectomy (PRK) and the variable predictability of the refractive cut in microkeratome keratomileusis in situ prompted us to evaluate excimer laser in situ keratomileusis (LASIK) for myopia. The Automated Corneal Shaper (Steinway-Chiron) was used to create a hinged corneal flap of 130 to 160 microns thick. PRK was performed on the lamellar bed with a Summit OmniMed laser. Minimum follow up was 3 months. Postoperative results and complications in the first 124 eyes are presented. LASIK is an effective method to correct myopia.

Excimer laser in situ keratomileusis was done in 97 eyes of 59 patients to correct moderate and high myopia. Mean follow-up was 150 days. Mean preoperative myopia was -10.75 diopters (D). Mean reduction in myopia was -11.281 D (106.98%) at 1 month and -10.37D (98.417%) at 3 months. At 1-month, 41 eyes (42.27%) had a refraction within -1.00 D and 70 teeyes (72.16%) had a refraction within -2.00 D. At 3 months, 41 eyes (46.59% of 88 eyes) had a refraction within -1.00 D and 66 eyes (75% of 88 eyes) had a refraction within -2.00 D. Mean preoperative keratometric power was 44.00 D; mean postoperative keratometric power was 36.39 D. The keratometric change in power was 7.61 D. Preoperatively, 73 eyes (75%) had a spectacle corrected visual acuity of 20/40 or better; postoperatively, 64 eyes (65.63%) at 1 month and 75 eyes (77.01%) at 3 months reached 20/40 or better. The uncorrected visual acuity was 20/40 or better in 33 eyes (33.72%) at 1 month and in 49 eyes (49.96%) at 3 months. Mean ablation zone diameter was 4.92 mm. Astigmatism was treated separately in 10 eyes. Complications included optical zone decentration (3 eyes with more than 1 mm, epithelial implantation (1 eye), incomplete resection of the disc (2 eyes), severe hypotension, and irregular astigmatism (1 eye).