Wound-healing response and refractive regression after conductive keratoplasty

Wound-healing response and refractive regression after conductive keratoplasty

J CATARACT REFRACT SURG - VOL 32, MARCH 2006 LABORATORY SCIENCE Wound-healing response and refractive regression after conductive keratoplasty Salom...

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J CATARACT REFRACT SURG - VOL 32, MARCH 2006

LABORATORY SCIENCE

Wound-healing response and refractive regression after conductive keratoplasty Salomon Esquenazi, MD, Jiucheng He, MD, PhD, Dooho B. Kim, MD, Nicolas G. Bazan, MD, PhD, Viet Bui, MD, Haydee E.P. Bazan, PhD

PURPOSE: To characterize the histological changes that occur after conductive keratoplasty (CK) using a rabbit model. SETTING: LSU Eye Center and Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, School of Medicine, New Orleans, Louisiana, USA. METHODS: Conductive keratoplasty was performed on 24 eyes of 12 New Zealand albino rabbits. In each eye, 24 spots were placed in a cross-corneal manner using 3 optical zones at 6.0, 7.0, and 8.0 mm. Eyes were assessed with corneal topography weekly. Rabbits were humanely killed 2, 4, 6, and 8 weeks postoperatively. The eyes were then enucleated and processed for histopathology and immunohistochemical analysis. RESULTS: All eyes showed an initial mean steepening of the corneal curvature of 2.24 diopters (D) 2 weeks postoperatively. Corneal topography revealed a 26%, 36%, and 39% regression of the refractive results at 4, 6, and 8 weeks, respectively. Immunohistochemical analysis demonstrated keratocyte apoptosis, myofibroblast appearance, and upregulation of chondroitin sulfate, MMP-1, and collagen III in the area surrounding the tip in each spot. CONCLUSION: The histological changes that occur after CK may be responsible for the well-established regression of its refractive effect. A better understanding of the wound-healing response after CK is necessary to improve the long-term stability of the procedure. J Cataract Refract Surg 2006; 32:480–486 Q 2006 ASCRS and ESCRS

Hyperopia can be treated with several surgical procedures such as photorefractive keratectomy,1,2 laser in situ keratomileusis (LASIK),3–5 noncontact laser thermal keratoplasty (LTK),6–8 contact LTK,9,10 diode laser keratoplasty,11 thermokeratoplasty,12 and conductive keratoplasty (CK).13–16 Unlike other modalities, CK uses a controlled release of radio-frequency energy that results in localized shrinkage of the stromal collagen directly related to the temperature and duration of exposure.17 This midperipheral effect results in contraction of collagen fibers with related changes in the mechanical behavior of the tissue.17–19 While some wound-healing changes after thermal procedures are desirable, others may contribute to regression of the initial refractive results.20 In the United States, CK has been shown to be safe and effective and has become a popular technique for the correction of low and moderate hyperopia. However, follow-up studies 1 and 2 years after CK show mild to Q 2006 ASCRS and ESCRS Published by Elsevier Inc.

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moderate regression rates.13–16 At 2 years, there is a loss of 20% of the initial effect.14 Another prospective study that involved 25 eyes of 14 patients with a mean preoperative hyperopia of 1.64 diopters (D) demonstrates a 29% regression from the intended plano and 43% regression from the 1-month postoperative correction 2 years after the surgery.13 The rate of regression is estimated to be C0.024 D per month between 12 and 24 months postoperatively.13 Koch et al.21 note that regression between the first and second year after noncontact LTK is approximately 0.25 D, which is identical to that reported with CK. This amount of regression is greater than that reported in the Beaver Dam Eye Study,22 which shows that the mean rate of hyperopic progression in patients over 40 years of age who had not had refractive surgery was 0.03 D per year. According to this study, the regression exclusively attributed to the refractive procedure would be 0.258 D in the second postoperative year alone. Considering that CK is used for low 0886-3350/06/$-see front matter doi:10.1016/j.jcrs.2005.12.077

LABORATORY SCIENCE: WOUND HEALING AFTER CK

hyperopic corrections, this figure may represent a 15% to 20% reduction of the refractive effect. Longer follow-up studies are underway to further characterize the refractive stability of CK. In 2002, the United States Food and Drug Administration approved the ViewPoint CK system (Refractec Inc.) for the temporary treatment of mild and moderate hyperopia between C0.75 and C3.00 D with astigmatism of 0.75 D or less. The current study attempts to characterize the histological changes that occur with time after CK using a rabbit model. MATERIALS AND METHODS Twenty-four eyes of 12 New Zealand albino rabbits weighing 1.5 to 2.0 kg were used. The use of rabbits was approved by the LSU Health Sciences Center Institutional Animal Care and Use Committee and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Each rabbit received intramuscular xylazine (10 mg/kg) and ketamine hydrochloride (50 mg/kg) anesthesia. Tetracaine eyedrops were used for topical anesthesia. Corneal topography was performed on all eyes preoperatively. The ViewPoint CK system consisting of a portable console, corneal marker, Barraquer lid speculum, a handpiece that holds a modified 350 mm by 90 mm stainless steel tip (keratoplast tip), and a foot pedal was used. The keratoplast tip was inspected under the microscope to ensure that it was not damaged or bent prior to its application. After insertion of the Barraquer speculum, the CK marker, which had been dampened with gentian violet, was used to make a corneal circular mark with 8 intersections centered on the pupil aperture. Balanced salt solution was used to irrigate and remove the excess ink. The corneal surface was then dried with a sponge. The treatment parameters, 350 kHz and 60% power (0.6 watt) for 0.6 seconds, were verified in the console. The keratoplast tip was inserted into the stroma perpendicular to the corneal surface at the location of the marked spots around the

midperipheral cornea. The foot pedal was depressed to deliver radiofrequency energy, which was applied in a sequential crosscorneal manner, first at the 7.0 mm optical zone and then at the 6.0 and 8.0 mm optical zones, to complete a total of 24 spots. The tip was kept in place at each spot until the preprogrammed treatment time was completed. Postoperative medications included a topical broad-spectrum antibiotic and a mild topical corticosteroid drop 3 times a day for 3 days.

Corneal Topography Corneal topography was performed using the Keratron portable topographer (Optikon EyeQuip) under general anesthesia. The unit’s headrest was applied gently to the rabbits’ heads, ensuring steady positioning for reproducible maps. The unit was then moved toward the eye and the corneal vertex automatically triggered the image capture. When the system detected the vertex exactly in the correct position, the image capture occurred. Four maps of the same eye were captured and automatically transferred to a desktop computer for processing. The system then performed a repeatability check, and any differences between the maps were graphed in diopters. If a map deviated too much from the others, it was automatically deleted. The mean of the simulated keratometry at a 3.0 mm optical zone from the 4 maps was recorded as the eye keratometric value. Corneal topography was performed every 2 weeks for 2 months. Tissue Preparation Three rabbits were humanely killed every 2 weeks using intravenous pentobarbital. The eyes were immediately enucleated, and entire corneas were excised, bisected, and embedded, one half vertically and the other half horizontally, in optimal cutting temperature (Miles Inc.). Cryostat sections 8 mm were prepared from each cornea. They were air-dried and then stored at ÿ80 C until further use. The sections were evaluated with hematoxylin and eosin stain and by immunohistochemical analysis. Staining with TUNEL

Accepted for publication August 15, 2005. From the LSU Eye Center (Esquenazi, Kim, Bazan, Bui, Bazan) and Neuroscience Center (Esquenazi, He, Bazan, Bazan), LSU Health Sciences Center, School of Medicine, New Orleans, Louisiana, USA. Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2005. Supported in part by USPHS grants R01EY04928 (HEPB), R01EY06635 (HEPB), LSU COBRE Grant P20RR021970 from the National Institutes of Health, Bethesda, Maryland, USA. No author has a financial or proprietary interest in any material or method mentioned. Richard Hesse, MD, and the Ochsner Clinic Foundation Department of Ophthalmology assisted and supported the study. Reprint requests to Salomon Esquenazi, MD, LSU Eye Center, Louisiana State University Health Sciences Center, 2020 Gravier Street, Suite B, New Orleans, Louisiana 70112, USA. E-mail: [email protected] lsuhsc.edu.

It is well established that an epithelial corneal injury induces rapid apoptosis of keratocytes.23 A fluorescent-based terminal deoxynucleotyl transferase-mediated UTP nick-end-labeling (TUNEL) assay was used according to the manufacturer’s recommendations to detect fragmentation of DNA associated with apoptosis. For nuclear counterstaining, the cells were treated for 30 minutes with 4’,6-diamidino-2-phenylindole (DAPI) solution. Immunohistochemistry Chondroitin sulfate proteoglycan was used as a marker of extracellular matrix (ECM) remodeling.24 To study its synthesis and distribution in the corneal stroma, a monoclonal antichondroitin sulfate clone CS-56 (Sigma Laboratories) was used. Immunofluorescence was performed as previously described.22 Briefly, the tissue sections were incubated at room temperature with a 1:150 dilution of the antibody for 1 hour. The presence of matrix metalloproteinases (MMP-1) was detected using a monoclonal mouse anti-MMP-1(1:300) antibody (Sigma Laboratories) for 2 hours at room temperature.

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Collagen III expression in the corneal stroma is a marker of scarring.25 Tissue sections were incubated with monoclonal mouse anticollagen III (1:500) (Sigma Laboratories) for 2 hours at room temperature. Alpha-smooth muscle actin (a-SMA) is a marker of stromal myofibroblasts, important in the process of wound healing. To stain for rabbit corneal myofibroblasts, tissue sections were incubated with the primary antibody (1:500) monoclonal mouse anti-a-SMA (Sigma Laboratories) for 2 hours at room temperature. In all cases, the secondary antibody, fluorescein-conjugated goat antimouse IgG (Santa Cruz Biotechnology) was applied for 1 hour at room temperature. Nuclear counterstaining with 4’,6diamidino-2-phenylindole was performed for 30 minutes at room temperature according to the manufacturer’s instructions. Cover slips were mounted with Vectashield mounting medium H: 1000 (Vector Laboratories). All tissue sections were viewed and photographed using a Nikon Eclipse TE 200 fluorescence microscope equipped with a Nikon digital camera DXM 1200. Cell Number Analysis Photographs of the tissue sections were acquired using MetaVue version 5.0 (Universal Imaging Corp.) and saved as a Tagged Image File Format file. The percentage of a-SMA–positive cells was calculated with respect to a fixed area (10.000 mm2) for each sample using the image analysis program Image Pro Plus 4.5 (Media Cybernetics). Statistical analysis was performed using the Statistical Analysis System (SAS) software version 9.0 (SAS Institute). RESULTS Corneal Topography

At the second postoperative week, there was a mean steepening of the corneal curvature of 2.24 D G 0.48 (SD) compared with the preoperative values. Regression of the refractive result was observed over time (Figure 1). At 4 weeks, the mean corneal steepening compared with the preoperative value was 1.65 D G 0.56 (SD). This represents a mean flattening of the central corneal curvature of 0.59 D compared with the initial correction observed at 2 weeks. At 6 and 8 weeks, the mean corneal steepening

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Figure 1. Mean change in the keratometric values 2, 4, 6, and 8 weeks after 24-spot CK in rabbit eyes. N represents the number of eyes at each timepoint.

compared with the preoperative values were 1.43 D G 0.48 (SD) and 1.35 D G 0.37 (SD), respectively. These changes in the mean keratometric measurements represent a regression of the refractive results of 26%, 36%, and 39% at weeks 4, 6 and 8, respectively. Histological Examination

Histological examination of the thermal lesions 2 weeks after CK showed a uniform cylinder involving the entire depth of the corneal stroma (Figure 2). Disorganization of the regular collagen pattern surrounding the thermal lesion

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Figure 2. Representative hematoxylin and eosin staining along the thermal injury induced by CK 2, 4, and 6 weeks postoperatively (arrows). Collagen coagulation was higher 2 weeks after CK and decreased with time. The tissues were processed as described (Ep Z epithelium; CK Z conductive keratoplasty treatment zone; End Z endothelium).

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was maximum at 2 weeks. The collagen fibers gradually healed, and minimal change was observed at 6 weeks. Minimal inflammatory cell infiltration was observed at all time points. A marked endothelial proliferative response was also observed in the rabbit corneas, possibly due to the deeper (100%) thickness of the treatment delivered.

of a-SMA–positive cells was compared with the total DAPIpositive cells. Two weeks after CK, the footprint produced by the keratoplast tip consisted of 6.3% G 2.15% of positive a-SMA cells compared with 4.18% G 1.76% at 4 weeks. The percentage of a-SMA–positive cells was 3.34% G 0.92% and 2.85% G 0.74% at weeks 6 and 8, respectively (Figure 6).

Keratocyte Apoptosis

Few TUNEL-positive cells were detected in and surrounding the thermal corneal stromal lesion (arrows) 2, 4, and 8 weeks after CK (Figure 3). Composition of the ECM

Up-regulation in the expression of chondroitin sulfate was observed along the stromal thermal injury induced by the keratoplast tip in all eyes. The staining was more pronounced in the posterior stroma. Similar levels of chondroitin sulfate expression were observed at all time frames (Figure 4). Increased expression of MMP-1 was observed in the first 2 weeks after the procedure compared with the fourth week (Figure 4). It was distributed throughout the thermal injury and also adjacent to the hyperplasic epithelium. Increased expression of collagen III was observed in the stroma adjacent to the thermal injury in all eyes. The expression of collagen III was more pronounced in the early postoperative time points (2 weeks) than at 4, 6, and 8 weeks (Figure 4; weeks 6 and 8 not shown in figure). Expression of a-SMA–Positive Cells

Increased expression of a-SMA cells was observed along the cylindrical stromal thermal injury in all eyes. Higher expression of a-SMA cells was observed at 2 and 4 weeks, and their expression gradually diminished over the 6-week and 8-week time period (Figures 5, A and B). An association between total stromal cells and number of a-SMA–positive cells in the area of injury was determined. Using the Image Pro Plus computer program, the percentage 2

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DISCUSSION

Several collagen shrinkage techniques used to steepen the central cornea have been proposed. A variety of laser and nonlaser devices used in thermal keratoplasty have been used to correct hyperopia. Key problems include long-term regression and asymmetric healing, which induces irregular astigmatism. Significant regression of the initial refractive results was the leading criticism of the holmium:YAG (Ho:YAG) LTK technique, which also involves temperature-induced collagen shrinkage to correct hyperopia. Using a noncontact model of LTK, Koch et al.21 report a 54% regression from 1 month to 2 years after surgery. It is speculated that the somewhat better stability of CK is due to a distinct mechanistic difference from that of LTK. Traditional thermal keratoplasty techniques use the property of thermal conductivity to transfer energy through the corneal stroma. The energy is absorbed by the tears and surrounding tissue differentially along a thermal gradient through the depth of the treatment site, resulting in a greater collagen effect in the anterior stroma than in the posterior stroma. Conductive keratoplasty generates heat in the cornea by using the electrical conductive properties of the corneal tissue. The resistance to the current flow through the corneal tissue, and not the probe itself, generates the thermal energy. The current propagates along undenatured corneal tissue, resulting in a homogeneous thermal footprint that is thought to minimize regression over time.13,14,20 Our study shows early collagen shrinkage and changes in the composition of the ECM in a homogeneous deep footprint. Greater amounts of posterior stromal and endothelial injury are observed compared with that in humans because 8

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Figure 3. TUNEL staining of keratocytes in the thermal corneal stroma injury (arrows) 2, 4, and 8 weeks postoperatively (20). The cells appear as green stain due to condensation of the nuclei. The nuclei of all the cells were counterstained with DAPI (Ep Z epithelium).

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Figure 4. Extracellular matrix components and MMP-1 expression in the area of the thermal injury 2 and 4 weeks after CK (arrows) (original magnification 20). The nuclei were counterstained with DAPI. Epithelial hyperplasia and endothelial proliferative response could be observed in the wound area (Ep Z epithelium; CK Z conductive keratoplasty treatment zone; End Z endothelium).

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Anti-Coll.III

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the rabbit cornea is approximately 350 mm thick, compared with 550 mm in a normal human cornea. These changes certainly explain the immediate corneal steepening observed after CK. However, the collagen rapidly regained its original configuration and at 4 weeks, it adopted its original appearance. These events may explain the initial regression observed after CK. However, there is some effect produced by the surgery after the collagen regains its original configuration. At this point, other structural changes are responsible for the corneal steepening observed. Matrix metalloproteinases are a group of enzymes that degrade the ECM. Collagen, which is the main component of the corneal stroma, is a substrate of MMP-1 or collagenase-1. Along with its tissue inhibitors, MMPs assist in the regulation of the overall remodeling of the ECM components. An overexpression of MMPs could degrade the corneal matrix. Koch et al.20 in a rabbit model using 10-pulse noncontact Ho:YAG laser thermal keratoplasty described a complex wound-healing response including keratocyte activation, synthesis of type I collagen, partial restoration of the stromal keratin sulfate, and type VI collagen and a retrocorneal membrane formation 3 weeks after the procedure. In our study, we observed an up-regulation of MMP-1 in the first 2 months following surgery, suggesting that

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a process of matrix degradation and remodeling is occurring in the stroma adjacent to the surgical injury site. Additionally, an increased number of TUNEL-positive cells, which indicate keratocyte apoptosis, and a-SMA cells, which indicate the appearance of myofibroblasts, were observed in the cylindrical footprint area underlying each spot. This suggests that the footprint produced by the keratoplast tip induces a full-thickness homogeneous activation of myofibroblasts. These myofibroblasts may be responsible for a continuous matrix remodeling process26 and the long-term contraction of tissue in that area. In fact, at 2 weeks, the positive collagen III staining coincides with a-SMA, suggesting that these cells could secrete this ECM component. However, there was a gradual reduction in the number of a-SMA cells, as well as collagen III, over time, which may explain the keratometric regression detected on corneal topography. At 6 and 8 weeks, there was a 50% reduction in the amount of a-SMA–positive cells compared with the second postoperative week. The denaturalization and shrinkage of collagen fibers produced by CK may be responsible for the initial and rapid modification of the corneal curvature, but the appearance of myofibroblasts may be responsible for supporting the tension exerted by these changes until they become

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Figure 5. A: Presence of myofibroblasts in the corneal stroma adjacent to the cylindrical footprint area 2, 4 (original magnification 10), 6 (original magnification 20), and 8 (original magnification 20) weeks after CK. Myofibroblast-positive cells stained with a-SMA (arrows). B: Transversal tissue section 2 weeks post-CK showing a higher expression of a-SMA–positive cells than at 6 weeks (original magnification 20). All tissue sections were counterstained with DAPI (Ep Z epithelium; CK Z conductive keratoplasty treatment zone; End Z endothelium).

permanent. In addition, these cells could secrete MMP-1 and increase the synthesis of collagen III. We postulate that both the collagen shrinkage and stromal wound healing are responsible for the regression and/or stabilization of the refractive effect produced by CK. The up-regulation of myofibroblasts may have a role in the steepening effect of the CK procedure, exerting a tensile force that maintains the collagen shrinkage induced by the radio-frequency energy. Over time, these cells are replaced by normal keratocytes that do not have the capacity to resist tension and, gradually, the effect of the surgery is lost. Therefore, contrary to LASIK surgery in which the prolonged presence of myofibroblasts are the cause of haze and reduced vision, in CK, prolonging the survival of

stromal myofibroblasts in the area of the injury may be an approach to maintain the corneal shape. Pharmacologic agents that could sustain myofibroblasts such as transforming growth factor beta27 should be investigated in future studies. In conclusion, our study provides a characterization of the histological changes that occur over time after CK. It is important, however, to recognize the limitations of the rabbit model in assessing the nature of the wound-healing response in humans. Wound healing is much more vigorous in rabbits, and many differences exist. The absence of Bowman’s layer and its influence in the healing process remains unclear. Further studies are required to further characterize the wound-healing response induced by the

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Figure 6. Percentage of a-SMA–positive cells compared with the total DAPI positive cells in the area adjacent to the keratoplast tip 2, 4, 6, and 8 weeks after CK. N represents the number of eyes at each time point.

CK procedure to optimize its effect and improve its longterm stability.

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