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Effect of cytochrome c peroxidase on corneal epithelial healing process after photorefractive keratectomy

Scalinci, Sergio Zaccharia MD; Scorolli, Lucia MD; De Martino, Liberatina MD; Corazza, Daniele MD; Morara, Mariachiara MD; Meduri, Renato Alberto MD

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Journal of Cataract & Refractive Surgery: October 2005 - Volume 31 - Issue 10 - p 1928-1931
doi: 10.1016/j.jcrs.2005.03.076
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Abstract

Photorefractive keratectomy (PRK) is the application of energy in the ultraviolet range (UV) (193 nm wavelength), generated by an argon–fluoride excimer laser to the anterior corneal stroma to reshape its curvature and thus correct a refractive error (myopia, astigmatism, and, more recently, hyperopia). The physical process of remodeling the corneal stroma by UV high-energy photons is called photoablation. Complications of PRK include undercorrection, overcorrection, and induced astigmatism, which may be caused by inadequate centration or focusing at the time of surgery. In fact, the success of PRK depends on uneventful corneal reepithelialization and healing without postoperative complications such as infection, keratitis, or haze.

The main adverse effects of PRK are direct or indirect consequences of corneal epithelial defect and ablation of the anterior stroma produced by the laser action. After PRK, the cornea becomes more susceptible to several pathogenic agents (chemical, physical, biological), with the possible appearance of infective or inflammatory events.1 This is because of corneal deepithelialization, Bowman's membrane destruction, and the exposure of the stroma, induced by photoablation. The cornea does not heal immediately but is covered by a discontinuous pseudomembrane that is insufficient to protect the underlying stroma. The release of cytokines, neuropeptides, and chemokines involved in the wound-healing cascade contribute to a vicious cycle of inflammatory events and to the onset of corneal pain.2 Twenty-four to 48 hours following refractive surgery, most patients complain of painful corneal symptoms of various intensity because of the exposure of the damaged nerve endings.3

Another undesirable complication of PRK is the development of corneal haze, with a frequency from 2.5% to 5%.4 These opacities appear to be a consequence of the destruction of Bowman's membrane and of the repair and replacement of stromal tissue post-PRK. Equilibrium of these factors in corneal wound healing is the main requirement to maintain corneal transparency. This equilibrium can be compromised by the acute injuries (chemical, inflammatory, and infective) that may occur postoperatively.5–7 Oxygen free radical–induced tissue damage following PRK is well documented.8–10 The lipid peroxidation in the superficial corneal stroma is generated by oxygen free radicals, and it induces alterations in cellular proteins and membranes. This type of acute injury is responsible in part for corneal haze because of keratocyte apoptosis.11,12 Many studies have revealed that excimer laser UV radiation decreases the detoxifying activity of glutathione peroxidase (GPx) enzyme, which is the main component of the corneal antioxidant system.13,14 The extent of tissue damage may depend on the balance between the oxygen free radicals and the local antioxidation defense system, as shown in several studies of animal models. The use of free-radical scavengers such as ubiquinone Q10 or vitamin E in preventing corneal keratocyte apoptosis and stimulating corneal reepithelialization has been proposed in animal models.15,16 This pilot study was performed to verify the applicability of the data from animal models to the human cornea using cytochrome c peroxidase and to investigate the role on corneal epithelial healing.

First described in 1940,17 cytochrome c peroxidase is an enzyme located between external and internal mitochondrial membrane of Saccharomyces cerevisiae baker's yeast. At this site, it catalyzes the oxidative reaction of ferrocytochrome c in the presence of hydrogen peroxide: H2O2 + 2 ferrocytochrome c + 2H+ → 2 ferrocytochrome c + 2H2O. Several studies have shown that this enzyme is part of the antioxidation defense system that prevents intracellular accumulation of peroxide.17–20

PATIENTS AND METHODS

This prospective study included 72 eyes of 36 patients (19 men, 17 women), mean age 33 years ± 7 (SD) (range 21 to 41 years) affected by low to moderate refractive error (myopia <8 diopters [D], astigmatism <6 D) that had been stable for more than 2 years. All the patients had bilateral simultaneous PRK. Thirty-two patients had myopia (mean defect 4.75 ± 2.50 D), and 4 had myopic astigmatism (4.25 ± 0.50 D). Exclusion criteria were diabetes mellitus, uncontrolled cardiovascular disease, rheumatoid diseases, immunosuppressive therapy, history of keloid formation, neurologic disorders, and pregnancy. Patients were also excluded if they had any of the following ocular conditions: keratoconus, glaucoma, ocular surface disorders, lens clearness alterations, uveitis, vitreoretinal diseases, optic neuropathies, and history of dry eye.

During the preoperative visit, the following examinations were performed: slitlamp biomicroscopy of the anterior and posterior segments, visual field examination (Humphrey Field Analyzer, prog. 10.2), autorefractometry (Nidek 1600 autorefractometer), determination of uncorrected (UCVA) and best corrected visual acuity (BCVA), manifest and cycloplegic refractions, corneal topography (Keratron Optikon), corneal pachymetry (Tomey TMS-2), intraocular pressure (IOP) measurement with Goldmann applanation tonometer, endothelial cell count (Topcon SP2000P), and lachrimal secretion (Schirmer test). Patients had been instructed not to wear contact lenses for 2 weeks before surgery for soft lenses and 4 weeks for hard lenses. All visits and PRK were done by a single surgeon.

After topical anesthesia and mechanical scraping of corneal superficial epithelium, photoablation, 6.0 mm diameter for myopia and 6.0 to 6.8 mm diameter for compound myopic astigmatism, was performed at the central cornea; deepness was proportional to the refractive defect. At the end of surgery, a disposable contact lens (Sky Soft) was placed on the treated eyes with the purposes of promoting epithelial regeneration and reducing painful symptoms. Postoperative therapy consisted of topical antibiotic (tobramycin 3 mg) and corticosteroid (desamethasone 1 mg) eyedrops 4 times a day for 10 days. Topical diclofenac was prescribed 4 times a day for 7 days.

For each patient, 1 eye was randomly assigned to the group that received standard therapy plus cytochrome c peroxidase; the fellow eye received standard therapy plus placebo (saline solution 0.8%) 3 times a day until the cornea had completely recovered. The drug, commercially available in Italy, was administered for 7 days after surgery or until corneal reepithelialization was complete at a dosage of 2 drops 3 times a day, corresponding to 15 000 UI. Patients were monitored daily for a week starting 24 hours following surgery to evaluate the corneal reepithelialization rate. During the biomicroscopy examinations, video slitlamp camera measurements of the corneal defect's horizontal diameter, stained by fluorescein, were done on days 1 through 7 or until complete reepithelialization. The diameter of the epithelial defect was analyzed by a computer-image analyzer (Adobe 5) and then measured with computer planimetry. The measurements of corneal wounds in the 2 groups were then compared. During follow-up, slitlamp biomicroscopy was performed to monitor corneal clarity.

RESULTS

Eyes treated with cytochrome c peroxidase showed a faster healing rate than untreated eyes. The mean defect diameter was as follows: day 1, 4.31 ± 0.39 mm in cytochrome c peroxidase eyes and 5.01 ± 0.36 mm in untreated eyes; day 2, 3.02 ± 0.38 mm in cytochrome c peroxidase eyes and 3.99 ± 0.34 mm in untreated eyes; day 3, 1.33 ± 0.33 mm in cytochrome c peroxidase eyes and 3.28 ± 0.29 mm in untreated eyes. By day 4, most cytochrome c peroxidase eyes (23 of 36, including 2 patients with compound myopic astigmatism; 64%) had healed completely; the treated eyes that had not healed were noted to have a 0.22 ± 0.12 mm residual defect. Mean defect diameter in the untreated group was 2.06 ± 0.28 mm. By day 5, all eyes (myopic and astigmatic) treated with cytochrome c peroxidase had completed the healing process; in control eyes, the mean epithelial defect was 1.32 ± 0.25 mm. By day 6, 6 control eyes (16.6%) had completed the healing process; the residual defect diameter in the other eyes in the control group was 0.44 ± 0.23 mm. By day 7, all the eyes that had not been treated with cytochrome c peroxidase had completed the reepithelialization process.

The mean corneal reepithelialization time in eyes treated with cytochrome c peroxidase was 91 ± 14 hours, compared with 154 ± 9 hours for control eyes (P<.05 Student t test), with a mean healing rate of 0.066 ± 0.007 mm/hour in the cytochrome c peroxidase group and 0.039 ± 0.006 mm/hour in eyes receiving placebo (P<.05 Student t test). Figure 1 shows the difference in the healing rates between groups.

Figure 1.
Figure 1.:
Corneal healing rate, expressed in days, in the cytochrome c peroxidase and control groups.

There were no statistically significant differences between groups in corneal haze presentation during follow-up (P = .70). The short monitoring period (7 days) did not allow study of differences between the 2 groups; however, corneal clarity grade, graded from 0 to 4 by slitlamp biomicroscopy (where 0 = clear cornea and 4 = corneal opacities), was lower in the study group than in the control group. In the study group, 25 patients (78.12%) had a corneal clarity grade between 0 and 1, 5 patients (15.62%) a grade of 1 to 2, and 2 patients (6.25%) a grade of 3 to 4. In the control group, 22 patients (68.75%) had clear corneas (grade 0 to 1), 6 patients had a grade of 1 to 2 (18.75%), and 4 patients had an opaque cornea of grade 3 to 4 (12.5%).

During the daily examinations, no patient showed ocular or systemic adverse events. No further effects of cytochrome c peroxidase have been reported.

DISCUSSION

Cytochrome c peroxidase is a mitochondrial antioxidant that catalyzes the degradation of hydrogen peroxide.21 X-ray diffraction analysis of the enzyme has allowed study of its main chemical, physical, and catalytic properties.19 It has a high affinity for 2 substrates: H2O2 (hydrogen peroxide, Km 4,5 × 10−6) and ferrocytochrome c (Km 10−5).19

The activity of cytochrome c peroxidase is comparable to GPx activity in mammals.8 Glutathione peroxidase is the major component of the defense system against oxidative damage. It is predominantly present in corneal epithelium and endothelium. A significant decrease in GPx activity and concentration after mechanical epithelial removal has been reported; photoablation of the stroma decreases GPx activity more than epithelial scraping.22

Hydrogen peroxide is physiologically present with a concentration from 20 to 50 μM in corneal tissues. After PRK, the production of free radicals is even greater.22 Local inflammatory responses following excimer laser photoablation include infiltration of the corneal stroma by polymorphonuclear cells (PMNs) and production of inflammatory mediators such as prostaglandin E2 and leukotriene B4.9 Ultraviolet radiation, PMN infiltration, and thermal increase are the probable sources of reactive oxygen species after PRK. These degrade corneal collagen and proteogycans and induce an aggressive wound-healing response.9 The disequilibrium between the concentration of the free-radical scavengers and oxygen radicals produces inappropriate corneal wound healing, which is responsible for corneal haze and refractive regression.

Identification of factors that can neutralize oxygen free-radical damage in the cornea can optimize surgical outcomes. Our hypothesis is that, as with GPx, cytochrome c peroxidase breaks the peroxidative chain reaction involving organic and fatty acid hydroperoxidase and prevents propagation of peroxide-dependent chain reactions that can lead to cell membrane lysis. Moreover, cytochrome c peroxidase has a protective action against free-radical damage even greater than that of GPx because it has the highest affinity (Km 4,5 × 10−6) for hydrogen peroxide.

We suggest that cytochrome c peroxidase is primarily involved in the healing process of corneal epithelium, where there is an immediate inflammatory response instead of stromal wound healing. Recent evidence2 has revealed the importance of the epithelial–stromal interactions. One study22 has suggested that the degree and extent of keratocyte apoptosis vary with the type of overlying epithelial injury (PRK or laser in situ keratomileusis) and are influenced by changes in surgical technique or pharmacologic therapy. This means that if rapid reepithelialization that preserves the epithelium corneal cytoarchitecture occurs, the replacement of stroma with minimal cell apoptosis is guaranteed.

In our study, cytochrome c peroxidase was efficacious in decreasing corneal reepithelialization time after PRK without significant ocular or systemic adverse events.

In conclusion, topical cytochrome c peroxidase may be useful for reducing the harmful effects of reactive oxygen radicals after PRK and accelerates the healing rate. According to this study, cytochrome c peroxidase may be an effective treatment to prevent corneal haze and refractive instability. Further investigations are required to confirm our results.

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