Blockade of CD40-CD154 costimulatory pathway promotes long-term survival of full-thickness porcine corneal grafts in nonhuman primates: clinically applicable xenocorneal transplantation.

The porcine cornea may be a good solution for the shortage of human donor corneas because its size and refractive properties are comparable to those of the human cornea. However, antigenic differences need to be overcome to apply xenocorneal transplantation in actual clinical practice. We aimed to investigate the feasibility of full-thickness porcine corneas as human corneal substitutes using a CD40-CD154 costimulatory pathway blocking strategy in a clinically applicable pig-to-nonhuman primate corneal transplantation model. As a result, the mean survival time of the xenocorneal grafts in recipients who received anti-CD154 antibody-based immunosuppressants (POD318 (n = 4); >933, >243, 318 and >192) was significantly longer than that in controls (POD28 (n = 3); 21, 28 and 29; p = 0.010, log-rank test). Administration of anti-CD154 antibodies markedly reduced inflammatory cellular infiltrations (predominantly CD8 T cells and macrophages) into the xenocorneal grafts and almost completely blocked xenoantigen-triggered increases in Th1-associated cytokines, chemokines and C3a in the aqueous humor. Moreover, systemic expansion of memory T cells was effectively controlled and responses of anti-Gal/donor pig-specific antibodies were considerably diminished by programmed injection of anti-CD154 antibodies. Consequently, porcine corneas might be promising human corneal substitutes when the transplantation is accompanied by potent immunosuppression such as a CD40-CD154 costimulatory pathway blockade.

Susceptibility of porcine keratocytes to immune-mediated damage in xeno-related rejection.

We admixed cultured porcine keratocytes or corneal endothelial cells in the presence of human sera or peripheral blood mononuclear cells (PBMCs) for 4 to 72 hours to investigate their immune-related susceptibilities to xeno-related rejection. We evaluated complement deposition at 48 hours by flow cytometry after staining with the C3 anti-goat cy3 antibody. The inhibition of proliferation of porcine corneal cells by human sera was examined using the 3-[4,5-dimethy/thiazol-2,5-dephenyl tetrazolium bromide (MTT) assay over 24 to 72 hours. The amount of 51chromium (Cr)-release was estimated after a reaction between the porcine cells and human PBMCs for 4 hours. There was greater C3 deposition in keratocytes (60.2%) than in endothelial cells (26.9%; P = .05, Mann-Whitney U test). Both keratocytes and endothelial cells showed significant levels of proliferative inhibition over a period of 72 hours. The number of 51Cr-release cells on interleukin-2 addition was significantly higher among keratocytes (88.0%) than endothelial cells (51.4%) at a 1:100 target:effector ratio (P = .04, Mann-Whitney U test). Our present data suggested that porcine keratocytes might be key target cells in xeno-related rejections when the porcine cornea is transplanted to primates.

In vitro effects of aminoglycosides and fluoroquinolones on keratocytes.

PURPOSE: Commonly used fluoroquinolones are reported to have less of an effect than aminoglycosides on corneal epithelial cells. The purpose of this study was to assess the effects of these antibiotics on stromal keratocytes in vitro. METHODS: Cultured rabbit keratocytes were incubated with various concentrations of gentamicin, tobramycin, ofloxacin, norfloxacin, and ciprofloxacin. Evaluations were performed by means of phase-contrast microscopy and [3H]thymidine uptake assay after 24 hours and 48 hours of incubation with the drug (concentrations from 3 to 0.003 mg/ml). RESULTS: At a concentration of 3 mg/ml, all three fluoroquinolones inhibited keratocyte proliferation significantly more than either aminoglycoside after 24 hours (P<0.001) and after 48 hours (P<0.001). In contrast to the aminoglycosides, all three fluoroquinolones induced a dose- dependent inhibition of proliferation after 24 hours. Even at the lowest concentration (0.003 mg/ml), ofloxacin and norfloxacin inhibited keratocyte proliferation significantly (P=0.001) compared to control after 24 hours. Concentrations of fluoroquinolones ranging from 0.09 to 0.24 mg/ml produced a 50% inhibition of proliferation, a level of inhibition not observed with any tested concentration of aminoglycosides. After 24 hours, all three fluoroquinolones, but neither of the aminoglycosides, showed moderate to severe signs of cytotoxicity at a concentration of 3 mg/ml. CONCLUSIONS: Relative effects of fluoroquinolones and aminoglycosides on epithelial cells and stromal keratocytes appear to be different. This might have an impact on choosing the optimal antibiotic drug to be applied prophylactically in clinical situations in which the epithelium is absent, such as after photorefractive keratectomy or chemical burn.

Prevention of keratocyte loss after corneal deepithelialization in rabbits.

PURPOSE: To determine if keratocyte loss, after removal of the corneal epithelium, can be prevented by a collagen shield used alone or in combination with a topically applied corneal preservation medium. METHODS: Twenty-four New Zealand white rabbits were divided into six groups of four rabbits each. The central 6 mm of corneal epithelium was removed from one eye of each animal by means of a blunt spatula. Postoperatively, these eyes were treated every 4 hours for 24 hours with the following: group 1, balanced salt solution (BSS) drops (Akorn Inc, Metairie, La); group 2, Optisol drops (Chiron IntraOptics, Irvine, Calif); group 3, a collagen shield soaked in sterile BSS plus BSS drops; group 4, the same collagen shield soaked in Optisol plus Optisol drops; group 5, a different collagen shield soaked in sterile BSS plus BSS drops; and group 6, the same collagen shield soaked in Optisol plus Optisol drops. The animals were killed at 24 hours after surgery, the corneas were fixed, and keratocytes within the anterior and posterior cornea, beneath the epithelial defect, were quantitated. Four untreated rabbits served as controls. RESULTS: Optisol drops alone, applied every 4 hours after deepithelialization, did not prevent keratocyte loss to a greater extent than did BSS drops alone (P = .96). Both collagen shields soaked in sterile BSS plus BSS drops every 4 hours were associated with less keratocyte loss than were BSS or Optisol drops alone. Both collagen shields soaked in Optisol plus Optisol drops every 4 hours were most successful at minimizing keratocyte loss (P = .0002 and P = .001). CONCLUSION: After corneal deepithelialization, use of a collagen shield in combination with topical application of a corneal storage medium may minimize keratocyte loss and may thus be beneficial after refractive surgery.

Effect of diclofenac, ketorolac, and fluorometholone on arachidonic acid metabolites following excimer laser corneal surgery.

OBJECTIVE: To compare the ability of several topical anti-inflammatory agents to modulate the production of prostaglandin E2 after excimer laser ablation in rabbit cornea. METHODS: Adult New Zealand white rabbits were subjected to phototherapeutic keratectomy with a commercially available excimer laser. Prostaglandin E2 and leukotriene B4 were detected by enzyme-linked immunoassay, and leukocyte infiltration was determined histologically. RESULTS: Prostaglandin E2 and leukocyte infiltration increased in the cornea after excimer ablation. Treatment with topical fluorometholone and diclofenac sodium significantly reduced prostaglandin E2 levels. Corneas treated with diclofenac had significantly higher levels of leukocyte infiltration than those treated with ketorolac tromethamine. No changes in leukotriene B4 levels were detected in this model. CONCLUSIONS: As a group, topical anti-inflammatory medications tend to lower prostaglandin E2 levels in rabbit corneas subjected to excimer ablation, but differ in their ability to reduce polymorphonuclear leukocyte infiltration. Further work is needed in this model to understand how these drugs alter leukocyte infiltration of the remaining stromal bed.

Development of a newly designed double-fixed Seoul-type keratoprosthesis.

OBJECTIVE: To develop a newly designed double-fixed keratoprosthesis (Seoul-type keratoprosthesis [S-KPro]) and to assess its mechanical stability and biocompatibility. METHODS: Twenty-five rabbits were divided into 4 groups by fixation technique, amniotic membrane (AM) implantation, and skirt material. The eyes were studied with the use of slitlamp, light, and electron microscopy. Stress testing was performed. In addition, 2 human subjects underwent S-KPro implantation. Best-corrected visual acuity was checked, and ophthalmic examination was performed. RESULTS: The average retention period of the group receiving double-fixated polyurethane-S-KPro with AM was longer (>24 weeks) than that of the others. Fibroblast invasions were found in polyurethane pores but not in polytetrafluoroethylene (Gore-Tex) pores on light microscopy. The minimal pressure that induced aqueous leakage was greater than 250 mm Hg in all of the tested eyes. Two human subjects have maintained a good postoperative condition for 18 and 8 months. CONCLUSIONS: The double-fixation technique of applied S-KPro and AM appears to be helpful in improving the stability of the keratoprosthesis. Polyurethane with relatively large pore size (40 microm) may be used successfully as a material for the keratoprosthesis skirt. CLINICAL RELEVANCE: Our results may be important for improving the clinical outcome of keratoprosthesis.

Keratocyte-populated collagen gel as an in vitro model of excimer laser keratectomy.

BACKGROUND: To develop an in vitro model to study the effects of excimer laser keratectomy on corneal stromal cells, we evaluated two types of collagen gel populated with keratocytes. METHODS: Keratocyte-populated collagen gels were prepared with type I collagen in 6-well plates or in culture plate inserts, the bottom of which consisted of a nitrocellulose membrane, contained within 6-well plates. The gels were ablated by the 193-nm excimer laser, set to ablate 50, 100, or 200 microns deep, and was observed under a phase-contrast microscope for 2 days. RESULTS: Keratocytes cultured in collagen gel developed cytoplasmic processes and formed networks of interconnected cells. Cells within the ablated area in the 6-well plates began to lose their cytoplasmic processes and became round approximately 3 hours after excimer laser ablation. These cellular changes were more prominent in the gels ablated to a depth of 200 microns. Cells outside of the ablation zones in the 6-well plates and the culture plate inserts remained intact. CONCLUSIONS: These results suggest the use of keratocyte-populated collagen gel as an in vitro model of cellular response to excimer laser keratectomy and also suggest that gel prepared in culture plate inserts is the preferred method.

Dichlorotriazinyl aminofluorescein inhibits human keratocyte proliferation in vitro.

PURPOSE: Dichlorotriazinyl aminofluorescein (DTAF) has been used to stain corneal stromal collagen as part of in vivo experimentation. Toxicity of this drug, if present, might alter the observed wound healing. To determine if this drug has any deleterious effect on keratocytes, we evaluated it in vitro. METHODS: Human keratocytes in 96 well plates were exposed to different concentrations of DTAF (10e-7, 10e-6, 10e-5, 10e-4, 10e-3, 10e-2, and 10e-1 mg/ml of media). Exposure times of 1 and 24 hours at each concentration of DTAF were evaluated. The cell number was measured 1 and 3 days after exposure to the drug using a coulter-counter and a hemocytometer. RESULTS: The proliferation of keratocytes after 24 hours of exposure to the drug was inhibited in a dose dependent manner by DTAF, but 1 hour exposure of keratocytes to the drug did not inhibit keratocyte proliferation. CONCLUSION: These results suggest that DTAF has inhibitory effects on human keratocyte proliferation after 24 hours of exposure, while exposure limited to 1 hour does not induce such a change.

Effects of gamma-interferon on keratocyte-induced collagen gel contraction and keratocyte proliferation.

PURPOSE: Gamma-interferon has been shown to be an effective immunoregulatory polypeptide that can modulate fibroblastic response. We investigated the effects of gamma-interferon on keratocyte proliferation and keratocyte-induced collagen gel contraction. METHODS: Gamma-interferon in concentrations of 0.01, 1, 100, and 1000 U/ml of media was added to keratocytes embedded in polymerized type I collagen and the gel area was measured after 5 days with an image analysis system. The rate of keratocyte proliferation within and outside the collagen gel under the influence of gamma-interferon was also investigated. RESULTS: Keratocyte-induced collagen gel contraction was significantly inhibited at all concentrations above 0.01 U/ml. The keratocyte proliferation was not affected by low and moderate concentrations and was significantly stimulated at concentration of 1000 U/ml. CONCLUSION: Keratocyte-induced collagen gel contraction is inhibited by gamma-interferon and the mechanism of this effect is not inhibition of keratocyte proliferation by gamma-interferon.

Holmium:YAG laser thermokeratoplasty for astigmatism in rabbits.

BACKGROUND: Holmium:YAG laser thermokeratoplasty has generated considerable interest as a technique for correcting hyperopia. In this study, the effect of holmium:YAG laser on inducing astigmatism according to application patterns was evaluated. METHODS: An experimental study based on the results of astigmatic holmium:YAG laser thermokeratoplasty using the Summit OmniMed laser system (Summit Technology Inc, Waltham, Mass) in 36 rabbit eyes is presented. We divided the rabbits into four groups: arcuate, reverse arcuate, linear, and control group according to application patterns. All rabbits were followed for 3 months and cycloplegic refractive measurements were carried out. RESULTS: The average surgically induced astigmatism was 1.86 diopters (D) for the arcuate group, 2.93 D for the reverse arcuate group, and 1.31 D for the linear group. No significant complications related to the operation were noted. CONCLUSION: The reverse arcuate pattern of holmium:YAG laser thermokeratoplasty is most effective in inducing astigmatism in rabbits.

Comparative experiments for in vivo fibroplasia and biological stability of four porous polymers intended for use in the Seoul-type keratoprosthesis.

AIMS: To evaluate in vivo fibroplasia and biological stability of porous polymers intended for use in the Seoul-type keratoprosthesis (S-KPro). METHODS: Four porous polymers (polypropylene, two kinds of polyethylene terephthalate (PE70 and PE50), and polyurethane) were investigated. Discs of polymers were inserted into the corneal stroma of rabbits for a 2 and 5 month period. Corneal oedema and neovascularisation were evaluated. The fibroplasia and collagen deposition were examined under light and transmission electron microscopy. S-KPros, whose skirt was made of four types of polymer, were implanted into the rabbits' eyes. The retention time and complications were evaluated. RESULTS: Neovascularisation and corneal oedema were found in all of the disc inserted eyes, but the corneal oedema subsided within 2 months in most of the eyes. The mean number of fibroblasts increased significantly in polypropylene and PE50 disc inserted eyes compared with polyurethane disc inserted eyes. Plentiful collagen deposition was also found in both polypropylene and PE50 disc inserted eyes. Mean retention time in the polypropylene SK-Pro implanted eyes was longer than that of the other eyes (20.7 weeks). The PE70 skirt induced corneal melting around the prosthesis. CONCLUSION: Polypropylene encourages fibroblast ingrowth and shows good biological stability when used as a skirt material in S-KPro.

Oxygen free radical damage in the cornea after excimer laser therapy.

AIMS/BACKGROUND: To evaluate the extent of oxygen radical damage in the cornea after excimer laser ablation. METHODS: The 193 nm argon fluoride excimer laser was programmed for an average fluence of 150 mJ/cm2, with a firing rate of 5 Hz and an ablation zone diameter of 6 mm. Phototherapeutic keratectomy was performed to remove 30 microns of epithelium and 50 microns of stroma from the corneas of New Zealand white rabbits. Oxidative tissue damage after laser was determined by measuring oxidised lipids (conjugated dienes and ketodienes) in corneal lipid extracts, and by fast blue B staining to localise the lipid peroxide in the tissue. RESULTS: Conjugated diene levels were 3.73 (SD 0.56) nmol per hemicornea in ablated corneas and 1.99 (0.33) nmol per hemicornea in normal corneas (p = 0.0044). Ketodiene levels were 2.72 (0.38) nmol per hemicornea in treated corneas and 0.91 (0.12) nmol per hemicornea in normal corneas (p < 0.001). Fast blue B staining disclosed that the tissue damage occurred primarily on the surface of the ablated cornea. CONCLUSION: The presence of lipid peroxidation in the superficial corneal stroma in excimer laser treated corneas was demonstrated. This lipid peroxidation could be from oxygen free radicals generated by the infiltrating polymorphonuclear cells at the site of tissue damage.

In vitro and in vivo study of lens refilling with poloxamer hydrogel.

AIM: To evaluate the compatibility of poloxamer hydrogel as a material for an injectable intraocular lens, in vivo and in vitro. METHODS: The appropriate concentration of poloxamer hydrogel was determined for injection by examining the transparency and gelling temperature of this material, assessing the lens capsule refilling technique, and studying the postoperative findings in a rabbit model. RESULTS: Poloxamer hydrogel showed excellent transparency and 25% was identified as an appropriate concentration for the lens refilling material. The authors developed a technique for injecting the material in vivo and obtained excellent short term results. CONCLUSIONS: Poloxamer hydrogel was identified as an appropriate material for direct lens refilling, and the developed injection technique produced excellent short term results.

Short-term effects of flurbiprofen and diclofenac on refractive outcome and corneal haze after photorefractive keratectomy.

PURPOSE: To evaluate the short-term effects of topical nonsteroidal anti-inflammatory drugs (NSAIDs) on refractive outcome and corneal haze after excimer laser photorefractive keratectomy (PRK) according to the degree of myopia and to compare the results with those of topical steroids. SETTING: Seoul National University Hospital, Seoul, Korea. METHODS: Patients were divided into two groups: low to moderate myopia (-6.00 diopters [D] or less) and high myopia (greater than 6.00 D). Then, each patient was randomly assigned to one of three drug subgroups for initial management (4 months post-PRK): corticosteroids (fluorometholone 0.1%); flurbiprofen sodium 0.03% (Ocufen); diclofenac sodium 0.1% (Decrol). Follow-up was 6 months. RESULTS: In eyes with low to moderate myopia, the steroid and diclofenac subgroups had significantly different refractions 2 and 4 months postoperatively but no difference at 6 months; subjective haze grading was consistently lower in the steroid subgroup than in the NSAID subgroups (flurbiprofen, diclofenac) after 2 months. In eyes with high myopia, the steroid subgroup had significantly less myopic regression after 3 weeks and lower subjective haze after 2 months than the NSAID subgroups. The steroid subgroup had severe myopic regression or corneal haze less frequently than the NSAID subgroups. CONCLUSION: Topical NSAIDs were less effective than topical steroids in reducing myopic regression and haze after PRK, especially in highly myopic eyes.

Bacterial keratitis after photoreactive keratectomy in a young, healthy man.

A 34-year-old man who had excimer laser photorefractive keratectomy (PRK) for myopia developed bacterial keratitis from Pseudomonas aeruginosa. He was treated with intensive topical and systemic antimicrobial agents. The eye recovered an uncorrected visual acuity of 20/30. Bacterial keratitis can occur in young, healthy patients after PRK, especially when a bandage soft contact lens is used without appropriate prophylactic measures.

Changes in corneal epithelial barrier function after excimer laser photorefractive keratectomy.

PURPOSE: To use fluorophotometry to measure corneal epithelial barrier function after excimer laser photorefractive keratectomy (PRK). SETTING: Seoul National University Hospital, Seoul, Korea. METHODS: Twenty-five eyes of 21 patients (13 women, 8 men) had PRK to correct myopia. Corneal epithelial healing time was measured and corneal epithelial permeability to sodium fluorescein evaluated by fluorophotometry 1, 2, and 3 weeks after surgery. RESULTS: Epithelial permeability showed a statistically significant increase 1 week after surgery and returned to its preoperative level 1 week later. Comparative studies according to epithelial healing day and corrected diopter showed results that were not statistically significant (P > .05). CONCLUSION: These results suggest that PRK delays complete reconstruction of corneal epithelial barrier function. In humans, the corneal epithelium regained its normal barrier function 2 weeks after PRK. Thus, at least during these weeks, care should be taken to minimize further epithelial trauma.

Effect of poly(ethylene glycol) graft polymerization of poly(methyl methacrylate) on cell adhesion. In vitro and in vivo study.

PURPOSE: To investigate the effect of surface modification of poly(methyl methacrylate) (PMMA) by poly(ethylene glycol) (PEG) grafting on cell adhesion. SETTING: Department of Ophthalmology, Seoul National University Hospital, Seoul, Korea. METHODS: The PMMA surface was oxidized with ozone, and PEG acrylate was then graft polymerized. To verify the PEG grafting on the surface, the oxygen content was measured by electron spectroscopy for chemical analysis. The contact angle was measured using the Wilhelmy plate method. The adhesion of keratocytes on modified PMMA was investigated in vitro. Cultured rabbit keratocytes (4 x10(5) cells/mL) were layered on each PMMA disk, cultured in a carbon dioxide incubator for 24 hours, harvested by trypsinization, and counted. A commercially available intraocular lens was modified as described and then inserted in the anterior chamber of a white rabbit. The cell adherence pattern on the modified IOL was examined by scanning electron microscopy. RESULTS: The PEG-grafted PMMA revealed a higher oxygen content and lower dynamic receding contact angles than the untreated PMMA. The mean number of adhered cells was 72.5 +/- 22 x 10(4)/mL for untreated PMMA. After PEG grafting of 1 hour and ozone oxidation of 2 hours, the adherent cell counts significantly decreased to 6.5 +/- 1.7 x 10(4)/mL and 7.6 +/- 1.6 x 10(4)/mL, respectively (P =.002). Scanning electron microscopy showed small round cells sparsely scattered on the modified PMMA in contrast to the untreated PMMA. CONCLUSION: Surface modification of PMMA using PEG grafting reduced cell adhesion. This may decrease the incidence of retroprosthetic membrane formation after keratoprosthesis surgery.

Effect of artificial tears on cultured keratocytes in vitro.

We investigated the effects of artificial tears on cultured rabbit and human keratocytes in vitro. The cells were exposed to seven nonpreserved commercially available artificial tear formulations and examined under phase-contrast microscopy for 60 min. After 5-min exposures to the solutions, rabbit keratocytes were fixed for transmission electron microscopy (TEM). In rabbit keratocytes, phase-contrast microscopy and TEM demonstrated that Aqua Site (CIBA Vision Ophthalmics, Atlanta, GA, U.S.A.), Hypo Tears PF (Johnson & Johnson, Claremont, CA, U.S.A.), and Tears Naturale Free (Alcon, Humacao, Puerto Rico, U.S.A.) immediately induced intracytoplasmic vacuoles and cell swelling, and subsequent cell degeneration. Rabbit cells exposed to the other artificial tears, which contained Ca2+ and did not contain EDTA, maintained their normal shape and appearance for 60 min. Phase-contrast microscopy of human keratocytes showed that Aqua Site and Hypo Tears PF induced mild and delayed cellular swelling, but the other artificial tears tested did not affect the cell shape for the entire 60-min observation period. Electrolyte balance and osmolarity of artificial tears appear to be critical for keratocyte survival. Maintenance of keratocyte integrity may be an important factor to consider when selecting an artificial tear preparation to be used when corneal epithelium is not intact.

Comparison of in vitro antiproliferative effects of steroids and nonsteroidal antiinflammatory drugs on human keratocytes.

Corticosteroids are currently administered after photorefractive keratectomy (PRK) to reduce corneal haze and myopic regression, However the beneficial effects of corticosteroids are controversial, and they are associated with many side effects. In this study we compared the in vitro antiproliferative effects of nonsteroidal antiinflammatory drugs (NSAIDs), diclofenac and flurbiprofen, with those of dexamethasone on human keratocytes. Human keratocytes were incubated with various concentrations of diclofenac, flurbiprofen, and dexamethasone. The control samples were incubated under the same conditions except for the absence of drugs. Proliferation of the keratocytes was measured by [3H]thymidine uptake into DNA on days 1, 2, and 4. Diclofenac was the most potent agent, inducing dose-dependent inhibition at concentrations > or = 10(-1) mM on days 1 and 2 and > or = 10(-5) mm on day 4. Flurbiprofen followed closely, inhibiting keratocytes at and above 1 mm on day 1, 10(-1) mm on day 2, and 10(-4) mm on day 4. Dexamethasone was the least effective, exhibiting inhibition at and above 25 mM on day 1, 5 mM on day 2, and 1 mM on day 4 (Wilcoxon rank sum test, P < or = 0.05). The ID50S reflect the same trend (day 4: diclofenac = 0.03 mM, flurbiprofen = 0.2 mM, dexamethasone = 3.2 mM). In addition, diclofenac and dexamethasone showed time-dependent antiproliferative effects. These results indicate that NSAIDs are more potent than corticosteroids in inhibiting proliferation of human keratocytes in vitro, and suggesting a potential use of NSAIDs in modulating corneal wound healing after PRK.

Effect of the application of human amniotic membrane on rabbit corneal wound healing after excimer laser photorefractive keratectomy.

PURPOSE: We investigated the influence of amniotic membrane application on corneal wound healing after excimer laser photorefractive keratectomy (PRK) in rabbits. METHODS: PRK of -9.9 D with an optical zone of 5.0 mm was performed on each right eye of 34 pigmented rabbits, which had been divided into two groups. In 17 eyes, preserved human amniotic membrane was applied in such a way that it covered the entire cornea for 48 h (amniotic group), and the other eyes formed the control group. The area of epithelial defect, inflammatory cell infiltration, the number of anterior stromal keratocytes, and corneal haze were evaluated. RESULTS: In all animals, the epithelium had healed completely within 3 days, and there was no difference between the two groups. At postoperative 12 and 24 h, the numbers of stromal inflammatory cells in the amniotic group were significantly lower than those in the control group (p = 0.009), and the numbers of anterior stromal keratocytes were significantly higher (p < 0.05). At postoperative weeks 2, 4, 6, 8, and 12, corneal haze scores in the amniotic group were lower than those in the control group (p < 0.05), and at postoperative week 12, the number of anterior stromal keratocytes in the amniotic group was significantly lower than that in the control group (p = 0.002). CONCLUSION: The application of amniotic membrane after PRK reduces keratocyte proliferation and corneal haze during corneal wound healing, possibly by reducing the infiltration of inflammatory cells and loss of keratocytes in the ablation area during the early postoperative period.