Stromal wound healing explains refractive instability and haze development after photorefractive keratectomy: a 1-year confocal microscopic study

PURPOSE: To evaluate the mechanism(s) producing refractive instability and corneal haze development after photorefractive keratectomy (PRK). DESIGN: Prospective, nonrandomized, comparative case series, self-controlled. PARTICIPANTS: Seventeen eyes of 17 patients with low- to moderate-grade myopia (-2.88 to -9.13 diopters [D]) were included. METHODS: Surgical intervention was a standardized, 6-mm diameter PRK procedure using the Meditec MEL 60 excimer laser (Aesculap-Meditec, Heroldsberg, Germany). The photoablation center was evaluated before surgery and at 1, 3, 6, 9, and 12 months after PRK using rapid, continuous z-scans of confocal images, termed confocal microscopy through focusing (CMTF). MAIN OUTCOME MEASURES: Simultaneous epithelial and stromal thickness analysis and objective assessment of corneal light backscattering were obtained from digital image analysis of the CMTF scans. Corneal reinnervation and anterior stromal keratocyte density and wound healing morphologic features were evaluated on high resolution, in vivo confocal images. Manifest refraction was measured and corneal clarity was graded by slit-lamp biomicroscopy. RESULTS: Epithelial thickness averaged 45+/-10 microm at 1 month, 50+/-8 microm at 3 months, and 52+/-6 microm at 12 months after PRK, as compared with 51+/-4 microm before surgery, demonstrating complete restoration of the preoperative thickness without compensatory hyperplasia. Interestingly, epithelial rethickening had no significant correlation with refractive regression. By contrast, stromal regrowth (from 1-12 months) averaged 6+/-12 microm (range, 27 microm thinning-22 microm rethickening) and correlated closely (r = 0.84, P<0.001) with changes in refraction that averaged 0.84+/-1.23 D, ranging from -1.63 D (hyperopic shift) to +3.38 D (myopic regression). Stromal rethickening increased proportionally with the actual photoablation depth (r = 0.63, P<0.01); linear regression analysis suggested an average regrowth rate of 8% per year for the entire study group. Stromal rethickening was not associated with CMTF haze development over time, suggesting that haze and regression were caused by two independent wound healing mechanisms. In agreement with these findings, all "hazy" corneas showed increased numbers of anterior stromal wound healing keratocytes with increased reflectivity of both nuclei and cell bodies, suggesting that cellular-based reflections, as opposed to extracellular matrix deposition, are the major origin of increased corneal light scattering after PRK. CONCLUSIONS: Taken together, these data indicate that keratocyte-mediated regrowth of the photoablated stroma appears to be the main cause of myopic regression in humans treated with a 6-mm diameter PRK, whereas hyperopic shifts appear to be a direct consequence of stromal thinning. By contrast, the corneal epithelium appeared to restore its preoperative thickness without contributing significantly to the refractive changes after PRK. Finally, this study also provides strong evidence that the development of haze after PRK is directly associated with increased cellular reflectivity from high numbers of wound healing keratocytes.     

Laser epithelial keratomileusis for myopia

PURPOSE: To describe a new technique for excimer laser corneal surgery: laser epithelial keratomileusis (LASEK). METHODS: We report 76 eyes with spherical equivalent refractive myopia ranging from -8.00 to -22.00 D (mean -11.00 +/- 3.00 D). Using ethanol applied over an 8.5-mm-diameter area for 20 seconds, the epithelium was detached and photorefractive keratectomy (PRK) was performed. The epithelium was reapplied to the ocular surface and covered with a therapeutic soft contact lens. Treatments were carried out with the Nidek EC-5000 excimer laser. RESULTS: At a mean of 803 days after LASEK, mean spherical equivalent refraction was -1.80 +/- 2.40 D (range -9.27 to +2.00 D). Stability was reached in approximately 60 days. Fifty-nine percent of the epithelial flaps were easy to detach intact during surgery and 62.7% of patients reported no postoperative pain. Eighty-four percent of eyes had the therapeutic bandage lens removed by the fourth postoperative day. Twelve percent of eyes had incomplete epithelial flap removal. Epithelium can fall off the cornea during the healing phase. No significant subepithelial haze was seen in 95% of eyes. CONCLUSION: With LASEK, it may be possible to treat a larger diameter ablation zone in thinner corneas without the problems of a LASIK stromal flap and with less postoperative pain than PRK, with minimal subepithelial haze.     

Comparison of wound healing after photorefractive keratectomy and laser in situ keratomileusis in rabbits.

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.     

Mitomycin C alters corneal stromal wound healing and corneal haze in rabbits after argon-fluoride excimer laser photorefractive keratectomy.

PURPOSE: To investigate the effects of mitomycin C (MMC) on rabbit cornea wound healing after photorefractive keratectomy (PRK). MATERIALS AND METHODS: Rabbit corneas were stained with dichlorotriazinyl aminofluorescein immediately after PRK. MMC was applied to the right eye and phosphate-buffered salt solution (PBS) to the left. Corneal epithelial wound healing rate and corneal haze were examined. Ultrasound pachymetry was performed. Stromal collagen regeneration was evaluated by fluorescent microscopy. We used terminal deoxyribonucleotidyl transferase-mediated D-uridine 5'-triphosphated-digoxigenin nick-end labeling (TUNEL) assay and transmission electron microscopy (TEM) to evaluate keratocyte apoptosis. RESULTS: In eyes treated with MMC, there was no delay to the healing rate of corneal epithelial wound, and less haze 4 weeks after PRK. Ultrasound pachymetry showed thinner corneal thickness in MMC-treated eyes at week 4. Corneal stromal thickness regression was less in MMC-treated eyes observed by fluorescent microscope at week 4. Keratocyte apoptosis was noted in both MMC- and PBS-treated eyes by TUNEL assay and TEM observation. This study discovered the phenomenon that MMC prolongs keratocyte apoptosis. CONCLUSIONS: Applying MMC after PRK is an effective method to decrease haze formation and corneal stromal thickness regression in rabbit corneas. The effect may be related to MMC prolonging keratocyte apoptosis.     

Roughness of excimer laser ablated corneas with and without smoothing measured with atomic force microscopy

PURPOSE: To analyze the surface roughness of porcine corneas after excimer laser ablation with and without the smoothing procedure by means of atomic force microscopy. METHODS: Excimer laser photorefractive keratectomy (PRK) was performed on eight porcine corneas. Immediately following the procedure, smoothing was performed on four corneas using a viscous solution of 0.25% sodium hyaluronate. The corneas were examined in balanced salt solution after fixation in 2.5% glutaraldehyde solution using atomic force microscopy. Quantitative analysis of the ablated stromal surface topography was performed using the section analysis module of the atomic force microscopy software. Repeated measurements were made over small areas (< or =50 microm2) near the center of each ablation, with a vertical resolution of <1 nm. RESULTS: Images of the ablated stromal surface showed undulations and granule-like features on the ablated surface of the specimens. The specimens on which the smoothing procedure was performed (root-mean-square [RMS] rough: 0.152 +/- 0.014 microm) were more regular (P < .001) than those on which PRK alone was performed (RMS rough: 0.229 +/- 0.018 microm). CONCLUSIONS: Atomic force microscopy analysis requires a simpler preparation of the specimens with respect to that necessary for scanning electron microscopy; for this reason, atomic force microscopy techniques are more reliable for the study of biological surfaces and prove to be a feasible method to establish the differences when comparing different laser techniques. Our investigations highlight that although the laser cut of scanning-spot excimer laser systems is precise in removing even the smallest amounts of tissue, the smoothing technique may still be useful to reduce post-ablation roughness.     

Wound healing in rabbit corneas after photorefractive keratectomy and laser in situ keratomileusis

PURPOSE: To compare the wound-healing process in the rabbit cornea after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK) with the same refractive correction. SETTING: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. METHODS: Adult albino rabbits (N = 24) were used. One eye of each animal had PRK or LASIK with the same refractive correction. Each animal was killed after an interval of up to 6 months. The expression pattern of corneal stromal injury-related molecules with the 2 treatments were compared. Paraffin sections of the cornea were processed immunohistochemically for alpha-smooth muscle actin (alpha-SMA), collagen type IV [alpha1(IV)](2),alpha2(IV), and heat shock protein (HSP) 47 as well as other HSPs. Sections were also examined after hematoxylin and eosin or periodic acid-Schiff staining. RESULTS: Hematoxylin and eosin staining showed the central epithelium to be thick in PRK-treated corneas. The thick epithelium was restricted to the area around the corneal flap edge adhesion in LASIK-treated corneas at 3 months. Periodic acid-Schiff staining showed an absence of or interruption in the epithelial basement membrane in PRK-treated corneas for up to 6 months. Heat shock protein 47 was detected in keratocytes on day 3 but not after that in PRK-treated corneas. There was no difference in the expression of other HSPs. Alpha-smooth muscle actin was expressed in keratocytes repopulated in the central anterior cornea of PRK-treated corneas at 28 days. Keratocytes with immunoreactivity for these 2 proteins were not seen in LASIK-treated corneas. Collagen IV [alpha1(IV)](2),alpha2(IV) was not detected in either group of corneas. The central epithelium became transiently thicker in PRK-treated corneas. CONCLUSION: Keratocyte responses to laser stromal ablation were more marked in corneas treated with PRK than in those treated with LASIK.     

Apoptosis,necrosis, proliferation,and myofibroblast generation in the stroma following LASIK and PRK

The aim of this study was to semi-quantitatively analyze stromal cell apoptosis, stromal cell proliferation, and myofibroblast generation over time points from 4hr to 3 months in rabbit eyes having photorefractive keratectomy (PRK) or laser in situ keratomeliusis (LASIK). Stromal cell necrosis and inflammatory cell infiltration were also studied. PRK for low myopia (-4.5diopters [D]), PRK for high myopia (-9.0D), and LASIK for high myopia (-9.0D) were performed in rabbit eyes, and corneas were obtained for examination at 4, 24, and 72hr, 1 and 4 weeks, and 3 months after surgery. A total of 144 rabbits were included in the study. Stromal cell apoptosis, proliferation, and myofibroblast generation were evaluated semi-quantitatively by TUNEL assay, immunocytochemical analysis of Ki67, and immunocytochemical analysis of alpha-smooth muscle actin, respectively. Stromal cell necrosis and characteristics of other cell types in the stroma were evaluated by electron microscopy. Keratocyte apoptosis and the subsequent proliferation and generation of myofibroblasts were qualitatively and quantitatively different in PRK for high myopia compared to either PRK for low myopia or LASIK for high myopia. Stromal cell necrosis becomes a significant form of cell death by 24hr after injury and may involve corneal fibroblasts, myofibroblasts, and inflammatory cells. Large numbers of polymorphonuclear cells and monocytes invade the cornea by 24hr after surgery and persist for over 1 week. The qualitative and quantitative differences in the cellular wound healing response after PRK for high and low myopia and LASIK for high myopia are likely determinants of the clinical differences in refractive outcome and some of the complications, such as regression and haze, seen after these procedures.     

Immunohistology of corneal wound healing after photorefractive keratectomy and laser in situ keratomileusis. wachtlin@ukbf.fu-berlin.de.

PURPOSE: The aim of the study was to compare corneal wound healing after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK) using conventional, immuno- and enzymohistologic methods. METHODS: Sixteen white Russian rabbits in each group underwent PRK or LASIK. Keratocyte density was recorded from 1 week to 6 months post-operatively on conventional histological sections. Immunohistologic cellular fibronectin and tenascin were used as markers of early epithelial and stromal wound healing in the cornea. The cell damage was demonstrated enzymohistologically using alkaline phosphatase. RESULTS: The reaction was similar in quality with both methods and occurred at sites of simultaneous epithelial and stromal injury. Mild scarring was found around the edge of the flap after LASIK; PRK-treated corneas developed a central subepithelial haze and scarring. A hypocellular region was found in the anterior part of the ablation zone shortly after PRK. Fibroblast migration later led to hypercellularity and subsequent clinical haze formation. After LASIK this reaction was limited to the peripheral entry point of the microkeratome blade around the edge of the corneal flap, where cellular fibronectin and tenascin reactions were positive. An acellular zone was found anterior to the interface after LASIK. The keratocyte damage visualized by alkaline phosphatase was more extensive after PRK than after LASIK. CONCLUSION: The stromal reaction to surgery was more extensive after PRK than after LASIK. A cytokine-mediated interaction between the epithelium and stroma was suggested as the cause of keratocyte cell migration and scar formation.     

Synergistic effect of ethanol and mitomycin C on corneal stroma

PURPOSE: To investigate the combined effects of ethanol and mitomycin C (MMC) application on the corneal stroma of rabbits that underwent photorefractive keratectomy (PRK). METHODS: Twenty-four rabbits (24 eyes) underwent PRK to correct -9.00 diopters of myopia. Twelve eyes had ethanol application before removing the epithelium and 12 eyes had the epithelium manually removed without ethanol. Eyes in both groups had topical MMC 0.02% application for 12 seconds immediately after excimer laser ablation. Twelve rabbits were sacrificed at two time points -4 hours and 4 weeks after surgery--and immunohistochemistry was performed with TUNEL assay, alpha-smooth muscle actin (alpha-SMA), and DAPI. RESULTS: More TUNEL-positive cells were observed in the ethanol-treated group compared to the mechanical debridement group at 4 hours after surgery (P<.01). No significant difference in alpha-SMA-positive cells was detected between the two groups at 4 weeks after sugery. However, decreased keratocyte density in the anterior stroma was more pronounced in the ethanol-treated group compared to the mechanical debridement group (P<.02). CONCLUSIONS: Ethanol application for epithelial removal during PRK seems to produce a synergistic effect with MMC, resulting in fewer keratocytes in the anterior stroma of rabbit corneas treated with MMC and ethanol than in corneas treated with MMC alone after PRK.     

Frozen cultured sheets of epidermal keratinocytes in reepithelialization and repair of the cornea after photorefractive keratectomy

PURPOSE: To determine whether frozen cultured sheets of human allogeneic epidermal keratinocytes (CEAK) improved wound repair after experimental corneal ablation by photorefractive keratectomy (PRK). SETTING: Hospital "Luis Sanchez Bulnes" de la Asociación para Evitar la Ceguera en Mexico, I.A.P, and Department of Cell Biology, CINVESTAV-IPN, Mexico City, Mexico. METHODS: Transepithelial PRK was performed in the right eye of male albino rabbits to obtain a 112 microm deep and 6.0 mm diameter ablation zone. In 17 eyes, the ablations were covered with frozen CEAK; in 11 eyes, the ablations were covered with a disposable contact lens without the cultured sheets; and in the control group (13 eyes), the ablations were not covered. Subepithelial fibrosis and reepithelialization of the ablated zone were evaluated in serial paraffin-embedded tissue sections from all wounds. RESULTS: Treatment with CEAK reduced fibroblast proliferation and the inflammatory response beneath the ablated zone and produced better organization of the newly formed epithelium by eliminating significant hyperplasia or discontinuities in the periodic acid Shiff-stained basement membrane. It also led to accelerated reepithelialization. CONCLUSIONS: The use of frozen CEAK as a biologically active wound dressing improved tissue repair at 1 month in corneas ablated by transepithelial PRK in the male albino rabbit model. Treatment with CEAK could improve the outcome of PRK in humans.     

LASIK vs LASEK vs PRK: advantages and indications

The advent of the excimer laser as an instrument for use in reshaping the corneal stroma was a great step forward in refractive surgery. Laser energy can be delivered on the stromal surface in the photorefractive keratectomy (PRK) procedure or deeper on the corneal stroma by the means of a lamellar surgery in which a flap is created with the microkeratome in the laser in situ keratomileusis (LASIK) procedure. LASIK is currently the dominant procedure in refractive surgery. The main advantage of LASIK over PRK is related to maintaining the central corneal epithelium. This increases comfort during the early post-operative period, allows for rapid visual recovery, and reduces the wound healing response. Reduced wound healing correlates with less regression for high corrections and a lower rate of complications such as significant stromal opacity (haze). PRK, however, remains as an excellent option for mild to moderate corrections, particularly for cases associated with thin corneas, recurrent erosions, or a predisposition for trauma (Martial arts, military, etc.). Recently, a modification of PRK, laser subepithelial keratomileusis (LASEK), was introduced. In the LASEK procedure, an epithelial flap is created and replaced after the ablation. The benefits, if any, of the creation of an epithelial flap compared to traditional PRK are not fully appreciated. Advocates of LASEK suggest that there is less discomfort in the early postoperative period, faster visual recovery, and less haze compared to standard PRK for correction of similar levels of refractive error. Additional long-term clinical studies, along with laboratory research, will be crucial to validate these potential advantages of LASEK procedure.     

Fluorescence-guided laser removal of chemically damaged cornea

PURPOSE: To correlate the observed fluorescence spectrum with the depth of ablation during 193 nm argon-fluoride excimer laser ablation of chemically damaged corneas. SETTING: Laser facility, Cedars-Sinai Medical Center, Los Angeles, California, USA. METHODS: Three cadaver New Zealand white rabbit corneas were exposed to 1 N hydrogen chloride for 10 seconds. The resultant opaque corneas were ablated to perforation using the excimer laser. Laser-induced fluorescence was collected at 45 degrees from incidence and channeled into an ultraviolet-visible spectrometer coupled to an optical multichannel analyzer reading a diode array detector. The detector recorded single-shot fluorescence spectra. The data were examined by principal component analysis, and the evolution of eigenvectors and their weighting coefficients were used to compare data among corneas. The results were correlated with histopathological sections. RESULTS: The eigenvalues of 3 principal components corresponded to 88.9%, 10.0%, and 0.4% of the data in acid-burned corneas. Compared to that in undamaged corneas, more information was stored in the first principal component and the third eigenvector was distinctly altered. Acid-scarred tissue blue shifted the dominant fluorescence peak compared to that in normal corneal tissue. CONCLUSIONS: After severe hydrogen chloride burn to the rabbit corneal surface, monitoring the dominant peak wavelength shift of excimer-laser-induced fluorescence can detect the transition between severely acid-damaged and underlying tissue.     

Role of epithelial hyperplasia in regression following photorefractive keratectomy.

AIM--To determine the relation between epithelial hyperplasia and regression of effect after photorefractive keratectomy (PRK). METHODS--Seventy unilaterally treated patients with PRK were examined. All eyes had been treated with the Summit excimer laser 27 (SD 7) months previously with zone diameters of 4.1 to 5.0 mm. The untreated fellow eyes served as controls. Epithelial thickness was measured centrally with a thin slit optical pachometer and manifest subjective refraction was performed. RESULTS--The epithelium was 21% thicker in the treated eye (p < 0.0001). The relation between refractive regression and epithelial hyperplasia was significant (r = 0.41; p < 0.001). CONCLUSIONS--Epithelial hyperplasia after PRK correlated with the myopic shift (including hyperopia reduction) after treatment with the Summit laser. A model is proposed suggesting that both subepithelial and epithelial layers contribute to regression in the Summit treated eyes with 18 microns of epithelial hyperplasia contributing each dioptre of regression.     

In vitro human corneal model to investigate stromal epithelial interactions following refractive surgery

PURPOSE: To develop an in vitro human corneal model to evaluate stromal epithelial interactions following corneal refractive surgical procedures. SETTING: Department of Academic Ophthalmology, Rayne Institute, St. Thomas' Hospital, London, United Kingdom. METHODS: Fifty-six human donor corneas procured from the eye bank were placed in a specially designed acrylic corneal holder and were cultured using the air-interface organ culture technique for up to 4 weeks. Corneal refractive surgical procedures such as a simple epithelial defect, 4 diopter (D) and 9 D photorefractive keratectomy (PRK), 4 D and 9 D laser-assisted subepithelial keratectomy (LASEK), and 9 D laser in situ keratomileusis (LASIK) were performed on the model. Temporal events in epithelial and keratocyte cell kinetics were evaluated using digital imaging, confocal microscopy, and light microscopy. Two-way analysis of variance and Student t tests were used to assess statistical significance. RESULTS: Epithelial healing following PRK was completed by 92 hours +/- 10 (SD) at a rate of 0.58 +/- 0.45 mm2/hour. In LASEK, the epithelial flap was replaced by regenerating peripheral epithelium that showed significant delay in epithelial closure (120 +/- 5 hours) with prolonged latency (24 +/- 4 hours, P<.0001) in comparison with PRK. The magnitude of keratocyte loss corresponded to ablation depth, and keratocyte regeneration was dependent on epithelial closure. In comparison, LASIK corneas showed a lesser percentage of keratocyte loss with poor recovery of keratocyte density in the stromal flap. Epithelial viability and keratocyte density were well preserved in the in vitro human model as observed in control corneas for up to 4 weeks. CONCLUSIONS: The temporal events in stromal epithelial interactions in the in vitro human model closely mimicked in vivo observations. The human model further avoided species-specific variations and provided a suitable test bed for evaluating newer algorithms and therapeutic regimens following refractive surgery.     

Smoothing of the ablated porcine anterior corneal surface using the Technolas Keracor 217C and Nidek EC-5000 excimer lasers

PURPOSE: To demonstrate efficacy of a smoothing technique to increase regularity of the anterior corneal surface after photorefractive keratectomy (PRK), using two different excimer lasers. METHODS: Spherical ablations of -10.00 D were performed on 11 fresh porcine corneas using either the Technolas Keracor 217C scanning-spot or the Nidek EC-5000 scanning-slit beam excimer laser. Following the procedure, we performed a phototherapeutic keratectomy treatment (smoothing technique) on half of the corneal surface. The smoothing technique was performed using a viscous solution of 0.25% sodium hyaluronate, which was spread on the cornea prior to the procedure. The ablation zone was 6 mm in diameter and the transition zone extended to 3 mm. The ablation depth was set at 10 microm. Corneas were then examined with scanning electron microscopy. RESULTS: Smoother treatment zones were apparent in porcine corneas in which smoothing was performed following PRK, with both laser systems. Results from the two lasers were not directly compared. CONCLUSIONS: The smoothing procedure performed following PRK using a viscous 0.25% sodium hyaluronate masking solution and a scanning laser system rendered the porcine corneal surface more regular.     

Narrow angle light scatter in rabbit corneas after excimer laser surface ablation

Corneal haze following excimer laser ablation is an adverse after-effect of photorefractive keratectomy (PRK) and is associated with the development of subepithelial opacities. The present work pertains to the measurement of light scattering in rabbit corneas following excimer laser treatment; to the microscopic analysis of the light-scattering corneal structures; and to the development of a mathematical model of light propagation through the post-laser treatment cornea. Photorefractive keratectomy (PRK-6D, 6 mm optical zone) followed by standard postoperative pharmaceutical treatment was performed on rabbit eyes. Animals were examined clinically on a weekly basis and sacrificed after the tenth postoperative week. Confocal microscope image sequences were acquired immediately before animal sacrifice. After the scatter measurement, the corneas were prepared for histopathological evaluation. The subepithelial structures observed using the confocal microscope correspond to refractive index (and therefore optical path difference (OPD) variation. This OPD distribution can be approximated with a fractal surface, band-pass filtered in the Fourier domain. The angular distribution of scattered light is characterised by a narrow forward peak of the order of 0.5° full-width at half maximum (FWHM) in accordance with the sizes of the subepithelial structures (5–150 μm).The intensity of scattered light is correlated with the thickness of the subepithelial scar-tissue layer.     

Localization of lectin binding sites in human, cat, and rabbit corneas

Paraffin sections of human, cat, and rabbit corneas were stained with nine lectins, using an avidin-biotin-complex procedure to study glycoconjugates of the epithelium, keratocytes, and stromal matrix. Wheat germ agglutinin (WGA) stained plasma membranes of all epithelial cell layers of cat and human and superficial and wing cells of rabbit. Plasma membranes of superficial and wing cells of cat epithelium also stained with peanut agglutinin (PNA) and Ricinus communis agglutinin I (RCA-I). Human and cat keratocytes stained with WGA and RCA-I. Stromal matrices of all three species were stained with concanavalin A and lentil agglutinin. In neuraminidase-treated sections, the entire epithelium and keratocytes of all three species stained with PNA. Corneal sections from the three species did not stain with Bandeiraea simplicifolia I, Bandeiraea simplicifolia II, Ulex europeus I, and Soybean agglutinin. These data suggest the presence of oligosaccharides with: N-acetylglucosamine/sialic acid residues in cell membranes of corneal epithelium of all species studied and in the keratocytes of human and cat; terminal beta-galactose residues in cat and human keratocytes, beta-galactose-galactosamine chains in cat epithelial cell membranes; and sialic acid-beta-galactose-galactosamine chains in epithelial cell membranes and keratocytes of all three species.     

Relation between corneal haze and transforming growth factor-beta1 after photorefractive keratectomy and laser in situ keratomileusis.

PURPOSE: To investigate the relation between corneal haze formation and transforming growth factor-beta (TGF-beta) after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK). SETTING: Department of Ophthalmology, University of Tokyo Graduate School of Medicine, Tokyo, Japan. METHODS: White rabbits were divided into 4 groups, with each group receiving 1 of the following surgeries: manual epithelial abrasion, PRK, lamellar keratotomy, or LASIK. The degree of corneal haze was quantitatively analyzed by measuring the light scattering intensity of corneas before and 4 and 12 weeks after surgery. The expression of type IV collagen and TGF-beta1 in the corneas at baseline and at 4 weeks was examined immunohistochemically. RESULTS: The light scattering intensity was significantly greater 4 and 10 weeks after PRK. In contrast, epithelial abrasion, lamellar keratotomy, and LASIK did not influence the light scattering intensity of the corneas. Type IV collagen was detected in the basal lamina of the corneal epithelium and in Descement's membrane in the normal cornea. After epithelial abrasion, there was no change in the distribution of type IV collagen. Four weeks after PRK, the expression of type IV collagen was detected in the subepithelial layer of the laser-ablated area. Four weeks after lamellar keratotomy, type IV collagen was linearly and fragmentarily detected in the corneal stroma. Four weeks after LASIK, type IV collagen was linearly and continuously detected in the corneal stroma and was detected slightly in the subepithelial region of the laser-ablated area. In the normal corneas, the expression of TGF-beta1 was not detected in the keratocytes. Four weeks after PRK, the expression of TGF-beta1 increased in the keratocytes that proliferated in the subepithelial fibrous layer. In contrast, epithelial abrasion, lamellar keratotomy, and LASIK did not change the expression pattern of TGF-beta1 in the keratocytes. CONCLUSION: The multiplier effect of epithelial abrasion and excimer laser ablation in PRK may increase the expression of TGF-beta1 in keratocytes and induce corneal haze.     

Recovery of Corneal Nerve Morphology Following Laser in situ Keratomileusis

Morphological changes in the corneal nerves after laser in situ keratomileusis (LASIK) were investigated and the changes were compared with those observed after creation of the keratectomy flap without subsequent photoablation. After creating the hinged flap, a multizone excimer laser photoablation (myopic correction from 6.00 to 6.66 D; diameter 6 mm) was performed on 27 rabbit corneas. Seven of these 27 rabbits received an automated keratectomy without laser photoablation on the fellow eye. A histochemical acetylcholinesterase reaction was used to demonstrate the changes in the morphology of the corneal nerves 3 days, 2.5 and 5 months after the operations. In all specimens the deepest stromal nerve bundles showed normal morphology. Cut nerve trunks were found at the wound margins and at the level of the flap interphase in the stromal bed. At 3 days, both epithelial and basal epithelial/subepithelial nerves were found at the hinge of the flap but the rest of the flap showed a major loss of epithelial, basal epithelial/subepithelial and superficial stromal nerves. A few new regenerating thin nerve fibers were found to emerge from the cut stromal nerve trunks. They appeared to pass the wound margin into the flap area below the epithelium. At 2.5 and 5 months an increasing number of regenerating nerve leashes were observed to emerge from the cut stromal nerve trunks. They appeared to send anastomosing fibers among the neighboring stromal nerves. By this time the epithelial, basal epithelial/subepithelial and anterior stromal innervation had gained an almost normal nerve density and architecture. In the corneas with the flap only, the epithelial innervation was slightly better spared in the center of the flap, and the stromal changes were somewhat less prominent compared with the LASIK corneas.     

Intact corneal epithelium is essential for the prevention of stromal haze after laser assisted in situ keratomileusis

AIMS: To determine the effect of intact corneal epithelium on stromal haze and myofibroblast cell formation after excimer laser surgery. METHODS: Denuded epithelium alone, photorefractive keratectomy (PRK), laser in situ keratomileusis (LASIK), or LASIK with denuded epithelium was performed in rabbit eyes. Postoperative anterior stromal haze was assessed employing a standard scale. Immunohistochemical methods were used to detect alpha smooth muscle actin (alpha-SMA), a marker for myofibroblastic cells, and type III collagen in subepithelial corneal tissue. RESULTS: Three weeks after surgery, the presence of alpha-SMA positive long extended and spindle-shaped stromal cells, and synthesis of type III collagen were observed in the subepithelial stromal layer corresponding to corneal haze in PRK and LASIK with denuded epithelium, but not in denuded epithelium alone and LASIK. CONCLUSION: The intact corneal epithelium may play an important part curbing subepithelial haze and differentiation of myofibroblasts in corneal wound healing.