Effect of intraoperative mitomycin-C on healthy corneal endothelium after laser-assisted subepithelial keratectomy

ARTICLE

Effect of intraoperative mitomycin-C on healthy corneal endothelium after laser-assisted subepithelial keratectomy Li-Quan Zhao, MD, Rui-Li Wei, MD, Xiao-Ye Ma, MD, Huang Zhu, MD

PURPOSE: To evaluate the effect of mitomycin-C (MMC) on corneal endothelial cells after laserassisted subepithelial keratectomy (LASEK). SETTING: Department of Ophthalmology, Changzheng Hospital, Shanghai, China. METHODS: One hundred seventy-four eyes of 89 patients who did not previously wear contact lenses were treated with LASEK with intraoperative use of topical MMC 0.02% (15 seconds). Noncontact corneal specular microscopy was performed in all eyes preoperatively and 1, 3, and 6 months after surgery. Preoperative pachymetry and ablation depth were measured in all eyes. Repeated-measures analysis of variance was used to compare the changes in the endothelial central cell density (CCD), coefficient of variation in cell size (CV), and percentage of hexagram cells (HEX) over time. Linear regression analysis was conducted to analyze the correlation between the change in the 3 corneal endothelium indices over time and the ablation depth and residual stroma bed (RSB) thickness. RESULTS: Preoperatively, the mean CCD was 2755 cells/mm2 G 373 (SD), the mean CV was 31.45 G 8.26, and the mean HEX was 66.03% G 25.83%. After LASEK, there were no statistically significant changes in CCD, CV, or HEX (P>.05). Multiple linear regression did not identify ablation depth or RSB thickness as being predictive of a change in CCD, CV, or HEX (P>.05). CONCLUSION: The use of intraoperative topical MMC 0.02% for 15 seconds after LASEK did not affect the corneal endothelium. J Cataract Refract Surg 2008; 34:1715–1719 Q 2008 ASCRS and ESCRS

Although laser in situ keratomileusis (LASIK) is a major refractive surgery technique because of its advantages (eg, minimal pain, rapid visual rehabilitation) and its low incidence of complications, laser-assisted subepithelial keratectomy (LASEK) is also a valuable surgical technique for the treatment of eyes with high myopia, eyes with a thinner cornea, and eyes with preexisting retinal pathology.1,2 Unlike in

Accepted for publication June 6, 2008. From the Department of Ophthalmology, Changzheng Hospital Affiliated to Second Military Medical University, Shanghai, China. No author has a financial or proprietary interest in any material or method mentioned. Corresponding author: Rui-Li Wei, MD, Department of Ophthalmology, Changzheng Hospital, 415 Fengyang Road, Shanghai 200003, China. E-mail: [email protected]. Q 2008 ASCRS and ESCRS Published by Elsevier Inc.

photorefractive keratectomy (PRK), the corneal epithelium is kept intact in LASEK; however, postoperative haze remains a major problem after LASEK. During the initial application of PRK, mitomycin-C (MMC) was introduced clinically as a topical adjunctive therapy to prevent the development of corneal haze.3 In animal models, MMC has been shown to suppress the activation of keratocytes and fibroblasts and prolong apoptosis of keratocytes after PRK and after LASEK.4,5 Clinical studies show that PRK6 and LASEK7 with intraoperative use of MMC are safe and produce excellent visual outcomes with few complications, although the toxicity of MMC to endothelial cells remains controversial. Whether MMC is toxic to endothelial cells depends on its prolonged application and/or its use in higher concentrations.8 In refractive surgery, the conventional MMC concentration is 0.2 mg/mL (0.02%); however, the application time varies and is as long as 2 minutes. In this study, we measured corneal 0886-3350/08/$dsee front matter doi:10.1016/j.jcrs.2008.06.016

1715

1716

INTRAOPERATIVE MITOMYCIN-C AFTER LASEK

endothelial cells before and after LASEK with intraoperative administration of a conventional dose of MMC 0.02% but for a shorter time (15 seconds) to assess the effect of MMC on corneal endothelial cells after LASEK. To assess endothelial cell function, we used a corneal specular microscope to evaluate the endothelial cell central density (CCD) and 2 morphologic indicesdthe coefficient of variation in cell size (CV) and percentage of hexagram cells (HEX). We excluded patients who wore contact lenses to ensure that any postoperative change in corneal endothelial health was from the use of MMC alone. PATIENTS AND METHODS This prospective interventional nonrandomized observermasked study comprised 89 consecutive patients (174 eyes) who were scheduled to have LASEK to correct myopia or myopic astigmatism. All patients provided informed consent after they received a thorough explanation of the procedure and its risks according to the Declaration of Helsinki. An institutional review board approved the study. Patients aged 18 to 45 years who had unstable refraction, previous ocular surgery (refractive or other procedures), suspected keratoconus, ocular disease, and systemic disease that might alter the wound-healing process (eg, such as diabetes and connective tissue disorders) were excluded from the study. Patients who wore contact lenses were also excluded.

Preoperative Evaluation A series of ophthalmologic examinations was performed preoperatively. The evaluations included measurement of uncorrected and best spectacle-corrected visual acuity (Snellen chart, Nidek ACP 8 auto chart projector), slitlamp biomicroscopy, tonometry (CT-80, Topcon), corneal pachymetry (SP-3000 pachymeter, Tomey Laboratories, Inc.), keratometry and corneal topography (Orbscan, Bausch & Lomb), and fundoscopy. Immediately before surgery, 3 photographs of the central cornea were taken with a corneal specular microscope (Topcon SP-2000P, Topcon Corp.). The microscope provides an image of the endothelial layer, an automated endothelial cell count, and noncontact pachymetry measurements.

Surgical Technique All the LASEK procedures were performed by the same experienced surgeon (R-L.W.) using the same excimer laser (Technolas 217z, Bausch & Lomb Surgical). Topical proparacaine 0.5% was applied to each eye to anesthetize the cornea. A 20% alcohol solution mixed with distilled water in a glass syringe was applied to the cornea within an 8.5 mm corneal well. The duration of exposure to the alcohol was 15 seconds. A cellulose sponge was used to remove the alcohol, and balanced salt solution (BSS) was instilled to rinse the ocular surface. The flap edges were dried with a sponge, and the epithelial flap was peeled back with a crescent blade (Alcon Surgical), leaving a hinge at the 12 o’clock position. The stromal bed was dried with a sponge. Laser ablation was performed using the excimer laser with Planoscan software (Bausch & Lomb). The target refraction to be corrected was the manifest refractive error

to prevent overcorrection. A 7.0 mm round cellulose sponge soaked in MMC 0.02% (0.2 mg/mL) solution was then applied over the ablated surface for 15 seconds. Next, the cornea was irrigated with BSS to remove the remaining MMC. Care was taken to avoid exposure of the epithelial flap, limbus, or conjunctiva to the MMC. The epithelial flap was replaced and the undersurface gently washed with BSS. Finally, a sterile bandage soft contact lens (PureVision, Bausch & Lomb) was placed on the cornea and dexamethasone 0.1% and topical gatifloxacin 0.3% were applied. Ablation was performed using a 193 nm 217z scanningspot excimer laser system with a combined 2.0 mm and 1.0 mm spot (Zyoptix, Bausch & Lomb). Before each treatment, the laser was calibrated by a fluence test and the eye-tracking system was tested. The radiant exposure was 0.2 J/cm2 in the treatment plane, and the repetition frequency of the laser was 120 Hz.

Postoperative Follow-up Postoperative medications included tobramycin 3 mg/ mL and dexamethasone 1 mg/mL drops 4 times daily until complete reepithelialization. The bandage contact lens was removed 1 week after surgery after the epithelium completely healed. The steroid drops were tapered over the subsequent 2 months as follows: fluorometholone 0.1% drops 4 times daily for the first week; prednisolone acetate 1% ophthalmic suspension drops 18 times daily (6 drops at a time with a 5-minute interval between drops 3 times a day) for approximately the next 10 days; fluorometholone 0.1% drops 3 times daily for the following 2 weeks, twice daily for another 2 weeks, and once daily for the last 2 weeks. Carboxymethylcellulose 0.5% was given as needed during the first several months. Examinations were scheduled for 1 day, 1 week, and 1, 3, and 6 months after surgery. Observation of the corneal endothelium was performed at each visit by the same masked observer who used the images obtained with the corneal specular microscope preoperatively and postoperatively. The 3 primary outcome measures were endothelial CCD, CV, and HEX.

Statistical Analysis The SPSS statistical software package (version 13.0, SPSS, Inc.) was used for all analyses. Data were recorded as mean G SD. An analysis of variance with repeated measures (over time) was performed to determine whether there were significant changes in the endothelial CCD, CV, or HEX after surgery. A paired t test was used to compare the 1-, 3-, and 6-month postoperative values of the 3 corneal endothelium indices with the preoperative values. Multiple linear regression analysis was performed to determine whether age, preoperative refractive error, ablation depth, or residual stromal bed (RSB) thickness predicted the postoperative changes in the 3 indices over time. A P value of 0.05 or less was considered statistically significant.

RESULTS Table 1 shows the demographics of the patients and the preoperative and intraoperative measurements. The minimum postoperative follow-up was 6 months.

J CATARACT REFRACT SURG - VOL 34, OCTOBER 2008

1717

INTRAOPERATIVE MITOMYCIN-C AFTER LASEK

postoperative values of any index at any time point (PO.05) (Table 2). Table 3 shows the 3 corneal endothelial indices in relation to intraoperative ablation depth and RSB thickness. There were no statistically significant correlations between the changes in CCD corrected for physiological cell loss (preoperative versus postoperative measurements) and the intraoperative ablation depth or RSB thickness (PO.05). There were also no statistically significant correlations between changes in CV and HEX (preoperative versus postoperative) and intraoperative ablation depth or RSB thickness (PO.05). No intraoperative or postoperative complications (eg, epithelial healing defects, infection) occurred.

Table 1. Patient demographics. Parameter

Mean G SD

Range

Age (y) Sex Male Female Preop SE (D) Preop pachymetry (mm) Intraop ablation depth (mm) RSB thickness (mm)

28.38 G 6.23

18 to 44

35 54 7.68 G 2.74 528.26 G 31.70 134.44 G 35.06

d d 1.95 to 14.95 453 to 633 52 to 224

333.83 G 44.06

264 to 480

RSB Z residual stroma bed; SE Z spherical equivalent

Reepithelialization was complete in all eyes by the sixth day after LASEK. Table 2 shows the preoperative and postoperative changes in the CCD, CV, and HEX. As 75% of patients were between 23 years and 33 years of age, an analysis of the correlation between CCD and patient age was performed. Analysis of 1 eye of each patient (right eye if both eyes examined) showed no statistically significant correlation between patient age and preoperative CCD (r Z 0.187, P Z .079). There was no statistically significant correlation between the preoperative CCD and the preoperative SE (r Z 0.086, P Z .261) or the preoperative pachymetry (r Z 0.037, P Z .631). Multivariate analysis of repeated measures (over time) of the change in CCD, CV, and HEX showed that MMC treatment did not have a statistically significant effect from preoperatively to 1 month, 3 months, or 6 months postoperatively (P Z .474, P Z .153, and P Z .060, respectively). When the postoperative values were individually compared with the preoperative values without taking into account the repeatability of the instrument, there were no statistically significant differences between the preoperative values and

DISCUSSION Mitomycin-C is a bifunctional alkylating agent that inhibits DNA synthesis and cell mitosis. Topical MMC is now commonly used in several fields of ophthalmic surgery including glaucoma, pterygium excision, and ocular surface neoplasia treatment. Several clinical studies have found that MMC is effective in reducing the incidence and severity of postoperative corneal haze after PRK or LASEK because it inhibits fibroblastic proliferation in the ablated stroma.4,5 Although well tolerated in most cases, MMC has been associated with several complications such as corneal defects and delayed healing.8 These complications result from the toxicity of MMC to the corpuscular physiological function. As the human corneal endothelium is essentially nonregenerative in vivo, the effect of MMC on the endothelium can be an important index in assessing the safety of MMC. In the earliest clinical study of corneal endothelial cells, Morales et al.9 found that topical application of MMC 0.02% for 30 seconds induced loss of corneal endothelial cells 3 months after PRK in 9 eyes. The limitation of their study was that the number of eyes was too small to allow one to reach a convincing conclusion.

Table 2. Changes in corneal endothelium indices after LASEK with intraoperative MMC. Time Preop Postop 1 mo 3 mo 6 mo

Mean CCD (cells/mm2)

P Value*

Mean CV

P Value*

Mean HEX (%)

P Value*

2755 G 373

d

31.45 G 8.26

d

66.03 G 25.83

d

2761 G 407 2792 G 407 2770 G 399

.841 .155 .576

32.41 G 7.63 31.13 G 8.51 32.55 G 9.07

.158 .682 .205

64.37 G 26.17 65.48 G 24.81 70.66 G 24.31

.533 .830 .072

Means G SD CCD Z central cell density; CV Z coefficient of variation in cell size; HEX Z hexagram cells; MMC Z mitomycin-C * Postoperative versus preoperative value (P!.05 significant)

J CATARACT REFRACT SURG - VOL 34, OCTOBER 2008

1718

INTRAOPERATIVE MITOMYCIN-C AFTER LASEK

Table 3. Surgical factors related to changes in corneal endothelial indices after LASEK with intraoperative MMC. RSB Thickness Index CCD 1 mo vs preop 3 mo vs preop 6 mo vs preop CV 1 mo vs preop 3 mo vs preop 6 mo vs preop HEX 1 mo vs preop 3 mo vs preop 6 mo vs preop

r Value

P Value*

Ablation Depth r Value

P Value*

0.012 0.039 0.072

.880 .610 .345

0.016 0.102 0.105

.831 .180 .167

0.060 0.033 0.004

.434 .662 .959

0.075 0.057 0.145

.324 .452 .057

0.044 0.044 0.038

.561 .566 .616

0.027 0.013 0.023

.722 .861 .761

CCD Z central cell density; CV Z coefficient of variation in cell size; HEX Z percentage of hexagram cells; MMC Z mitomycin-C; RSB Z residual stromal bed * P!.05 significant

Another comparative study10 found no change in the number of corneal endothelial cells over 12 postoperative months in 15 eyes that had PRK with intraoperative use of topical MMC 0.02% for 15 seconds; the 15 fellow eyes had epithelial LASIK without MMC in random order. In a subsequent comparative study by de BenitoLlopis et al.,11 48 eyes were treated with intraoperative MMC 0.02% for 30 seconds over the ablated zone during LASEK and 32 eyes had LASEK without MMC. There was no significant difference between the 2 groups in corneal endothelial CCD after surgery. Both groups had a statistically significant increase in CCD 3 months after surgery. Another study12 of a larger cohort (1101 eyes) used intraoperative MMC 0.02% for longer times (range 30 seconds to 2 minutes depending on ablation depth). The authors found that the number of endothelial cells increased 6 months after PRK. Several studies13,14 report that the corneal endothelial cell count decreased as a result of cell hypoxia induced by contact lens use. The studies discussed above did not exclude patients with a long preoperative history of contact lens use; thus, their finding of an increase in the number of corneal endothelial cells is likely the result of the patients discontinuing contact lens use after surgery. With this in mind, we excluded patients who wore contact lenses preoperatively so we could observe the effects of intraoperative MMC alone on the health of the corneal endothelium after LASEK. The corneal thickness and keratometry change after LASEK or LASIK, and the image magnification of

the specular microscope changes accordingly. However, some studies report that this change is insignificant and thus can be missed in corneal endothelial cell observation by corneal specular microscopy.15 We used the same concentration of MMC (0.2 mg/mL) as in other studies but for a shorter period (15 seconds). We found no significant change in the corneal endothelial CCD within 6 months after surgery. These results are consistent with those of Diakonis et al.10 Because the HEX value reflects the change in endothelial cell morphology, it is regarded as a sensitive index for assessing endothelial damage.16 To gain a comprehensive understanding of endothelial function, we used the CV and HEX indices in our study. There was no significant change in CV and HEX between preoperatively and any postoperative time point. This agrees with a recent study by Goldsberry et al.,17 which found that the 3 endothelial indices did not change within 1 year after PRK; however, the MMC application time in that study was shorter (12 seconds). In our 6 months of follow-up of patients with healthy endothelium, use of MMC 0.02% on the ablated stroma for 15 seconds did not induce significant changes in corneal endothelial function (eg, CCD, CV, HEX) over baseline values. Early studies of LASIK18 found an acute effect on the corneal endothelium. The influence of laser enhancement may represent transient endothelial cell edema within 15 minutes and 1 day after LASIK. The most significant differences were qualitative and quantitative changes in endothelial cell morphology; that is, fewer corneal endothelial hexagram cells. Moreover, there was a correlation between the change in the corneal endothelium and the RSB thickness. An experimental study19 found that 193 nm excimer laser ablation induced loss of the corneal endothelium. Thus, adequate RSB thickness appears to be important to maintaining corneal endothelial function. With current surgical techniques, it is safe to perform excimer laser ablation when the RSB is thicker than 250 mm.20 Based on these findings, we hypothesized that MMC diffuses into the RSB to reach the corneal endothelium. The thinner the RSB, the more easily MMC diffuses deep into the corneal stroma and induces endothelial toxicity. Our study included RSB thickness and ablation depth (laser enhancement) in the analysis of correlation. In our patients, the thickest RSB was 480 mm and the thinnest, 264 mm. The largest ablation depth was 224 mm and the smallest, 52 mm. There was no significant correlation between the change in endothelial indices and the RSB thickness or ablation depth. Although a 0.02% concentration of MMC was applied for a short time in our study, a flushing dose of steroids (2 weeks postoperatively for approximately 10 days) seemed to inhibit the development of haze.

J CATARACT REFRACT SURG - VOL 34, OCTOBER 2008

INTRAOPERATIVE MITOMYCIN-C AFTER LASEK

A study of the long-term effect of steroids is underway in our laboratory. In terms of the endothelium, there were no significant changes between preoperatively and postoperatively in CCD, CV, and HEX, 3 important indices for assessing endothelial cell function. In addition, there was no significant correlation between the 3 indices and the 2 LASEK-related factors of ablation depth and RSB thickness. In summary, prophylactic intraoperative application of MMC (0.02% for 15 seconds) did not seem to affect the corneal endothelium. Therefore, its use to inhibit the development of haze in LASEK and PRK can be considered safe.

10.

11.

12.

13.

14.

REFERENCES 1. Dastjerdi MH, Soong HK. LASEK (laser subepithelial keratomileusis). Curr Opin Ophthalmol 2002; 13:261–263 2. Sekundo W, Tietjen A. Laserassistierte subepitheliale Keratek¨ bersicht u¨ber den gegenwa¨rtigen Kenntnistomie (LasEk). U stand. [Laser-assisted subepithelial keratectomy (LasEK). Review of the current state of knowledge.] Ophthalmologe 2003; 100:603–610 3. Talamo JH, Gollamudi S, Green WR, de la Cruz Z, Filatov V, Stark WJ. Modulation of corneal wound healing after excimer laser keratomileusis using topical mitomycin C and steroids. Arch Ophthalmol 1991; 109:1141–1146 4. Kim T-I, Lee SY, Pak JH, Tchah H, Kook MS. Mitomycin C. ceramide, and 5-fluorouracil inhibit corneal haze and apoptosis after PRK. Cornea 2006; 25:55–60 5. Kim T-I, Pak JH, Lee SY, Tchah H. Mitomycin C induced reduction of keratocytes and fibroblasts after photorefractive keratectomy. Invest Ophthalmol Vis Sci 2004; 45:2978–2984. Available at: http://www.iovs.org/cgi/reprint/45/9/2978. Accessed June 27, 2008 6. Bedei A, Marabotti A, Giannecchini I, Ferretti C, Montagnani M, Martinucci C, Barabesi L. Photorefractive keratectomy in high myopic defects with or without intraoperative mitomycin C: 1-year results. Eur J Ophthalmol 2006; 16:229–234 7. Thornton I, Puri A, Xu M, Krueger RR. Low-dose mitomycin C as a prophylaxis for corneal haze in myopic surface ablation. Am J Ophthalmol 2007; 144:673–681 8. Rajan MS, O’Brart DPS, Patmore A, Marshall J. Cellular effects of mitomycin-C on human corneas after photorefractive keratectomy. J Cataract Refract Surg 2006; 32:1741–1747 9. Morales AJ, Zadok D, Mora-Retana R, Martı´nez-Gama E, Robledo NE, Chayet AS. Intraoperative mitomycin and corneal

15.

16.

17.

18.

19.

20.

1719

endothelium after photorefractive keratectomy. Am J Ophthalmol 2006; 142:400–404 Diakonis VF, Pallikaris A, Kymionis GD, Markomanolakis MM. Alterations in endothelial cell density after photorefractive keratectomy with adjuvant mitomycin. Am J Ophthalmol 2007; 144:99–103 de Benito-Llopis L, Teus MA, Ortega M. Effect of mitomycin-C on the corneal endothelium during excimer laser surface ablation. J Cataract Refract Surg 2007; 33:1009–1013 Lee DH, Chung HS, Jeon YC, Boo SD, Yoon YD, Kim JG. Photorefractive keratectomy with intraoperative mitomycin-C application. J Cataract Refract Surg 2005; 31:2293–2298 Wiffen SJ, Hodge DO, Bourne WM. The effect of contact lens wear on the central and peripheral corneal endothelium. Cornea 2000; 19:47–51 Collins MJ, Carr JD, Stulting RD, Azar RG, Waring GO III, Smith RE, Thompson KP, Edelhauser HF. Effects of laser in situ keratomileusis (LASIK) on the corneal endothelium 3 years postoperatively. Am J Ophthalmol 2001; 131:1–6 Isager P, Guo S, Hjortdal JØ, Ehlers N. Endothelial cell loss after photorefractive keratectomy for myopia. Acta Ophthalmol Scand 1998; 76:304–307 Chiou AG-Y, Kaufman SC, Kaufman HE, Beuerman RW. Clinical corneal confocal microscopy. Surv Ophthalmol 2006; 51:482–500 Goldsberry DH, Epstein RJ, Majmudar PA, Epstein RH, Dennis RF, Holley G, Edelhauser HF. Effect of mitomycin C on the corneal endothelium when used for corneal subepithelial haze prophylaxis following photorefractive keratectomy. J Refract Surg 2007; 23:724–727 Kim T, Sorenson AL, Krishnasamy S, Carlson AN, Edelhauser HF. Acute corneal endothelial changes after laser in situ keratomileusis. Cornea 2001; 20:597–602 Edelhauser HF. The resiliency of the corneal endothelium to refractive and intraocular surgery; the Castroviejo Lecture. Cornea 2000; 19:263–273 Simaroj P, Kosalprapai K, Chuckpaiwong V. Effect of laser in situ keratomileusis on the corneal endothelium. J Refract Surg 2003; 19:S237–S240

J CATARACT REFRACT SURG - VOL 34, OCTOBER 2008

First author: Li-Quan Zhao, MD Department of Ophthalmology, Changzheng Hospital Affiliated to Second Military Medical University, Shanghai, China